Rutgers Cytomechanics 2015 - Craelius

CYTOMECHANICS
William Craelius, Ph.D.

14:125:432
Spring 2015
TABLE OF CONTENTS
Preface
Chapter 1: Introduction
1.1
Background
1.2
How Cells Are Assembled
1.3
Basic Cellular Components
1.4
Tissues
1.5
Tensegrity
1.6
Forces That Hold The Cell Together
1.7
CSK Mechanoreflexes
1.8
Construction of the CSK
1.9
Applications of Cytomechanics
Exercises and Review Questions
References
Chapter 2: Materials of the Cell and Matrix

2.1
Basic Cellular Constituents
2.2 Plasma Membranes
2.3 CSK Components
2.4 Building the CSK
2.5 Cytogel
References
14
Chapter 3: Mechanics of Cell Types

3.1 A Generic Cell
3.2 Red Blood Cells
3.3 Platelets
3.4 White Blood Cells
3.5 Fibroblasts
3.6 Endothelial Cells
3.7 Myocytes
3.8 Transformed Cells
3.9 Stem Cells
References
25
Chapter 4: Material and Structural Mechanics

4.1
Background
4.2
Stress Environments of Cells
4.3
Uniaxial Stress and Strain
4.4
Two-Dimensional Stress
4.5
Types of Mechanical Loading
4.6
Measuring Cellular Material Properties
4.7
Material Stiffness and Strength
4.8
Properties of CSK Components
4.9
Flexural Moduli
4.10 Measuring Local Stiffness
4.11 Buckling
33
11
24
32

References
48
Chapter 5: Cytomechanical Tools

5.1
Background
5.2
Measuring and Manipulating Cellular Forces
5.3
Measuring Cell-Generated Traction Forces
5.4
Manipulating Cell-ECM Forces
5.5
Atomic Force Microscopy (AFM)
5.6
Magnetic Tweezers
5.7
Micropipette Aspiration
5.8
Cell Moduli from Pipet Aspiration
5.9
Nuclear Probing
5.10 Whole Cell Inflation
5.11 Optical Tweezers
5.12 Hydrostatic Loading
5.13 Shear Flow
5.14 Cell Stretching
5.15 Microelectromechanical Systems (MEMS)
5.16 Particle Tracking
References
50
Chapter 6: Thermodynamics of the Cytoskeleton

6.1
The Boltzmann Distribution: Statistical Mechanics
6.2
Diffusion
6.3
Bioelectricity
6.4
Energy Storage
6.5
State Transitions
6.6
Polymerization
References
70
Chapter 7: Kinetic Behavior

7.1
Background
7.2
Cell Modeling
7.3
Modeling the Cytoskeleton
7.4
Viscoelastic (VE) Material
References
82
Chapter 8: Micromotors
8.1
Introduction
8.2
Muscular Microstructure
8.3
The Pathway to Contraction
8.4
Generation and Regulation of Force
8.5
Skeletal Muscle Energetics
Appendix: Review of Biomechanical Terminology
References
89
68
80
87
101
102
Chapter 9: Cell Propulsion

9.1
Introduction
Cells Swimming Through Fluids
9.2
9.3
Cell Substrate Interactions
103
110
Appendix
111
9.4
9.5
Propulsive Motors
Comparative Motor Analysis
PREFACE
Cytomechanics studies how cells are influenced by the mechanical stresses and
strains that they experience continually throughout their lives. Cytomechanics
studies these pico-newton and nanometer quantities, to understand and possibly
manipulate the growth, structure and function of cells. This course emphasizes
the processes that drive tissue growth, degeneration, and regeneration, with subtopics including cellular signaling and metabolism, gene mechanics and
expression, and the biomechanical properties of cells and their components.
Projects in modeling cellular structure and behavior are done with Matlab &
Simulink.
An important feature of cell mechanics is mechanotransduction: the process
whereby cells transform mechanical energy into other forms, including chemical,
electrical, and inertial. It is based upon the ability of cells to sense the
mechanical forces always present in their environment and react to them on time
scales ranging from milliseconds to years in order to achieve biological stability at
some level. Mechanotransduction is thus central to cellular structure and
function, tissue maintenance, and ultimately, organismal fate. Understanding it
requires analysis of extremely small forces and deformations at the nanometer
level; this challenge can be met by merging engineering principles with
techniques of modern molecular biology such as immunocytochemistry, optical
imaging, and microfabrication.
Part I (the first 7 chapters) presents the basic structural components, mechanics
and energetics of cells, cellular electrophysiology and techniques for cellular
visualization and mechanical characterization. Part II (the last five chapters)
covers the role of mechanotransduction in complex cell behaviors of signaling,
moving, adhesivity, and growth. Quantitative examples and exercises based on
traditional biomechanics applied to cells are provided. Software, such as Matlab
and Excel will enhance understanding, and specific examples using them are
given. The appendices give formulae and data related to cellular mechanical
properties and the cytoskeleton, along with a brief tutorial on Simulink.
This course packet is a compilation of many textual and research papers, and
includes excerpts from same, cited in the text. Referenced or excerpted articles
are included online in the supplement. The book attempts to introduce the huge
and growing field of cell mechanics, and assumes the reader has background in
basic cell biology, biochemistry, and Physics. While reading each chapter, you
should find the Appendices useful for definitions and formulae. Companion
books in those areas are recommended as reference. For more in-depth texts
please read, The Mechanics of the Cell by David Boar, and Biomechanics by Y.C.
Fung. This book has liberally adapted ideas and material from those two texts, as
well as reviews. Please note that this book will inevitably contain mistakes, so
corrections and comments will be appreciated. Permissions for material from
research articles are pending.
CHAPTER 1: INTRODUCTION
(With acknowledgements to Ingber 2008; Eyckmans, Boudou et al. 2011)
1.1 Background
Biological cells are structures that self-assemble from basic components, adapt their
shapes, sizes and strengths to their ongoing needs, and travel to and settle in appropriate
locations using their own renewable energy. Many of these abilities result from a
particular sensory-reflex system possessed by each cell that responds to mechanical
forces within its environment. In fact, in order to properly function and grow, each cell
depends upon continual stress and strain within preferred magnitude ranges. When
force or strain magnitudes are outside the proper range, either too large or too small, cell
function and growth is adversely affected.
This inter-relationship between ambient force and cell function underlies a unique
design whose principles would be highly useful to engineers. Cytomechanics seeks to
uncover those principles using the tools of molecular biology, imaging, biomechanics,
and computer modeling. By analyzing forces and deformations on the pico-newton and
nanometer levels, cytomechanics seeks to explain and possibly manipulate the growth,
structure and function of cells.
A prominent example of the application of cytomechanics is accelerating the growth of
cells and tissues by exposing them to forces from fluid flow in bioreactors. Specifically,
growth of nerves, skin, muscles, bone, and probably all biological tissues can be
stimulated by proper application of forces. Other technology is exploiting the highly
efficient molecular motors found in bacteria to make nano-scale motors.
Although the biological effects of forces are perhaps most evident in the context of
physical activitybreathing, heart pumping, blood flow, and physical exercisesuch
forces also regulate morphogenesis, cell migration, and even cell adhesion to
extracellular matrix. Such forces can regulate a wide variety of biological processes, from
cell proliferation and differentiation to tissue mass homeostasis and complex
inflammatory cascades.
The idea that forces can regulate tissue remodeling and development was articulated
more than a century ago. In 1892, the surgeon and anatomist Julius Wolff postulated
that bone tissue adapts its structure to the mechanical environment based on the
observation that trabeculae matched the principal stress lines in bones caused by daily
physical loading (Wolff, 1892). Although the alignment of trabeculae could have arisen
strictly during prenatal development, he reported this remodeling occurred even after
healing of misaligned fractures. In the same era, mechanical forces were proposed to
shape tissues and organs during embryonic development (Roux, 1895; Thompson, 1917),
but the tools were not available to directly test such ideas experimentally. Nearly a
century passed before these concepts began to captivate the scientific community once
again.
1.2 How Cells Are Assembled
The architecture of biological cells is highly complex, and its elegance can be appreciated
in comparison with that of buildings. Building design must meet certain minimum
standards: (1) a foundation anchoring it to the correct location; (2) sufficient strength to
stand against all expected forces; (3) comfortable internal environment; (4) access
portals for incoming and outgoing traffic; and (5) food and waste processing. Using well-
established formulae from mechanical and civil engineering, the architect can select the
type and sizes of structural components, their arrangement, connections, and all the
functional components needed to satisfy the building standards. Before construction
begins, she knows every line, arc, angle, and load that the building will have.
Architecture of buildings is thus laid out as a clearly understood blueprint.
Architecture of biological cells, on the other hand, is laid out by a blueprint that is not so
clearly understood, in the form of codes on DNA molecules; structurally, cells are
squishy, wiggly, and willful. Nevertheless, cellular architecture not only must satisfy all
the same standards as buildings, it must be self-renewable. Cells solve the mechanical
and civil engineering problems they face in a myriad of elegant ways, using any and every
way to live. In fact, cells have much more intelligence than buildings: they can modify
their structure to meet changing demands and conditions. Cytomechanics seeks to learn
and apply the rules of cellular architecture. While it may never be possible for us to build
cells from scratch, we can expect to learn tricks from them that can help us solve many
technological problems.
1.3
Basic Cellular Components
Basic Plan
The basic plan of all animal cells is the same: they have a lipid membrane, skeleton, and
internal structures. Unlike most components of buildings, however, which are divided
into purely structural or purely functional categories, all cellular components can serve
both categories in elegant ways. The membrane is very weak structurally, but
nevertheless is a barrier wrap and portal to the outside, as well as a smart skin with
sensory and reflex capabilities. Mechanical strength is provided by the cell skeleton, i.e.
cytoskeleton, (CSK), that supports the membrane and maintains cellular shape. The
CSK not only is the backbone and limbs of the cell, it is also a communication network.
Within the gel-like cytosol, internal structures include the nucleus, mitochondria, and
other organelles Together the 3 components, membrane, CSK, and cytosol provide all the
structure and function of the cell.
The Lipid Membrane
All cells are enveloped by a lipid bilayer that is an aggregation of phospholipids, in a
spheroid, separating the cell interior from the exterior environment. Many components
other than lipid exist within the bilayer, and perform various functions, as will be seen in
Chapter 2. Formation of the bilayer in solution is a spontaneous event, driven by
thermal energy, as depicted below in Figure 1.1. Note that the series of time-lapse
photographs show the lipid droplet oscillating in shape, due to thermal fluctuations:
Figure 1.1.
Time-lapse views of lipid vesicle
undergoing thermal fluctuations.
See also:
http://ftp.aip.org/epaps/journ_che
m_phys/E-JCPSA6-119711337/movie1.gif
The Cytoskeleton (CSK)

The CSK is a network connecting the outside of the cell directly to points within,
including the nucleus. Two views of the CSK shown below (Figure 1.2) illustrate its
architectural complexity. On the left is a scanning electron microscope view from outside
of a cell. The dense net is composed entirely of the protein, actin. Note the random
crisscrossing of the fibers, and the nodes where 2 or more branches connect. Actin
filaments, about 10 nM in diameter, run fairly straight between nodes, or connections,
and form a thick-tangled 3-dimensional mat at this scale. At a more distant (less
magnified) view, the network resembles a gossamer spider web as shown in the cartoon
at right, showing a cross section of a segment of the cell border. Note that there is a lipid
bilayer, through which the polymer network extends. Proteinaceous channels and
integrin molecules traverse the lipid membrane. The extracellular matrix intimately
surrounds cells, and is attached at specific sites.
Figure 1.2. The Cytoskeletal Network

Three types of filaments make up the CSK, schematized below (Figure 1.3). Actin is the
thinnest of the 3 filaments, with diameter 8 nm. Actin filaments aggregate to form a
dense peripheral shell that is the primary component of the CSK of most cells.
Intermediate filaments are larger, and connect through the membrane at discrete points.
Microtubules are very straight hollow tubes with outer diameter 25nM, and can
traverse the cell spanning between the cell membrane and the nucleus. Details on the
properties of these filaments can be found in Table 2.2.
Figure 1.3: Major

Cytoskeletal Components
Actin
Intermediate
.filaments
Microtubules
8
Fundamental Principles of CSK

There are three fundamental principles of the cytoskeleton: (1) the cytoskeleton
determines cell function to a large extent, primarily through its architecture and its
interaction with the external environment. (2) It self-assembles from various
components, without a genetic blueprint. (3) It regulates cell function at long ranges of
space and time. The meaning of these principles will be elaborated upon in later
chapters.
1.4 Tissues
When cells aggregate to form tissues, things become more complicated as shown in the
schematic view of 3 skin cells below. Many different types of connections, each with their
own biochemical traffic patterns, are made among the various cells and extracellular
components.
Figure 1.4 Epithelial Tissues. BM = basement membrane.
The schematic above shows the epithelial cells connected together by weld-like
connections at the tight junctions, and connecting with the basement membrane by
adhesions, mediate by integrin, and other matrix molecules. Stromal cells can interact
with epithelial cells through mechanical forces applied to the basement membrane that
can regulate nuclear transcription via the Integrin-CSK network.
1.5
Tensegrity (Ingber 2010)
The tensegrity model states that cells, tissues, and other biological structures at smaller
and larger size scales in the hierarchy of life gain their shape stability and their ability to
exhibit integrated mechanical behavior through use of the structural principles of
tensegrity architecture. The term, tensegrity (contraction of tensional integrity) was
first created by the architect R. Buckminster Fuller, who first explored use of this form of
structural stabilization as early as 1927 in his plan for the Wichita Dymaxion house,
which minimized weight by separating compression members from tension members.
To create this cylindrical building, Fuller proposed to set a central mast in the earth as a
vertical compression strut and to suspend from it multiple circular floors (horizontal
wheels) using tension cables. Tensile guy wires that linked the mast to surrounding
anchors in the ground provided the balancing tension necessary to stabilize the entire
structure. Fuller called this special discontinuous-compression, continuous-tension
system, the Tensegrity to emphasize how it differs from conventional architectural
systems (e.g., brick-on-brick type of construction), which depend on continuous
compression for their shape stability. Fullers more formal definition in his treatise,
Synergetics, is Tensegrity describes a structural-relationship principle in which
structural shape is guaranteed by the finitely closed, comprehensively continuous,
tensional behaviors of the system and not by the discontinuous and exclusively local
compressional member behaviors. Note that there is no mention of rigid struts, elastic
strings, tensile filaments, internal vs. external members, or specific molecular
constituents in this definition. In fact, Fuller describes a balloon with non-compressible
gas molecules pushing out against a tensed rubber membrane as analogous to one of his
geodesic domes when viewed at the microstructural level (i.e., the balloon is a porous,
tensed molecular network on the microscale) and explains that both structures are
classic examples of shape stability through tensegrity.
Fuller also described hierarchical tensegrity structures in which individual struts or
tensile elements are themselves tensegrity structures on a smaller scale; key to this
concept is that smaller tensegrity units require external anchors to other tensegrity units
to maintain higher order stability. In fact, he argued that nature utilizes this universal
system of tensile structuring at all size scales and that it provides a way to mechanically
integrate part and whole.
In 1948, Fullers student, Kenneth Snelson, constructed the first stick-and-string
tensegrity sculpture, which thrilled Fuller because it visibly communicated the essence of
this novel form of shape stability to those who could not see it in more complex
structures. Snelsons sculptures contain isolated compression members that are
suspended in midair by interconnections with a continuous tensile network. Some of
these structures require anchorage to the ground to remain stable (e.g., large
cantilevered structures); however, most are entirely self-stabilizing. Similar stick-and
string tensegrity models have been used to visualize tensegrity in cells and other
biological structures for those who cannot easily visualize them. The appearance of
geodesic patterns in biological structures, including viruses, clathrin-coated vesicles, and
actin geodomes in the cytoskeleton of mammalian cells, provides additional visual
evidence of natures use of this form of architecture.
From the above discussion, it is apparent that understanding cell structure starts with a
look at its basic structural plan. At one level, it appears that the basic architecture of the
cell is the same as the geodesic dome, designed by Buckminster Fuller. Geodesic is a
highly efficient building, whose structural elements traverse the shortest distance
required to hold it up. Many non-cellular structures, including viruses, enzymes,
organelles and even small organisms, all exhibit geodesic forms. Stick models of the
structure are shown at left in Figure 1.5, and the CSK of a living cell is shown at right.
10
Figure 1.5. Living geodesic forms.
1.6
Forces that hold the cell together
Tensegrity models can be made from sticks and rubber bands. Their integrity relies on
the tension applied to the sticks by the elastic elements; hence the structure has
tensegrity. In engineering terms, the sticks represent struts, since they sustain
compression and the rubber bands represent ties (or ropes) since they hold tension.
While the geodesic dome has a characteristic spherical shape, many other shapes are
held together by tensegrity, not the least of which is standing bipeds (see below). Your
bones are struts compressed by gravity, while your skeletal muscles act as ropes,
applying tension to maintain posture. Another structure is the loaded bow, shown
below:
Figure 1.6. Biped and bow tensegrity structures.
Bow and Arrow
Compression
Muscle
Tension
mg
Tension
Cellular tensegrity describes a structural plan whereby a network of contractile

microfilaments pulls the cell membrane and all its internal constituents centrifugally
toward the nucleus at the core. Opposing this centrifugal tensile pull are two types of
compressive elements, one of which is outside and the other inside the cell. The external
component outside the cell is the extracellular matrix (ECM). Inside the cell there are
struts consisting of microtubules and/or bundles of cross-linked microfilaments that
resist compression. The third component of the CSK, the intermediate filaments, link
microtubules and contractile micro-filaments to each other as well as to the surface
membrane and the cell's nucleus. In addition, the intermediate filaments act as guy wires
or rods, stiffening the central nucleus and securing it in place. Although the CSK is
surrounded by a lipid membrane and lies within a viscous fluid resembling a gel, it is this
11
hard-wired network of molecular struts and rods that stabilize cell shape. Thus the cell
uses tension rods and compression struts to hold itself together.
In simplest terms, tensegrity structures maintain shape stability within a tensed network
of structural members by incorporating other support elements that resist compression.
The stiffness of the stick-and-string tensegrity structures, and hence their ability to resist
shape distortion, depends on the level of preexisting tension or pre-stress in the
structure before application of an external load. The distinguishing microstructural
feature accounting for this behavior is that, when placed under load, the discrete
structural elements move, changing orientation and spacing relative to one another, until
a new equilibrium configuration is attained. For this reason, a local stress can result in
global structural rearrangements and action at a distance.
To visualize tensegrity at work, think of the human body: it stabilizes its shape by
interconnecting multiple compression-resistant bones with a continuous series of tensile
muscles, tendons, and ligaments, and its stiffness can vary depending on the tone (prestress) in its muscles. If I want to fully extend my hand upward to touch the ceiling, I
have to tense muscles down to my toes, thus producing global structural rearrangements
throughout my body and, eventually, upward extension of my fingers. However, the body
is also multimodular and hierarchical: if I accidentally sever my Achilles tendon, I lose
form control in my ankle module, but I still maintain structural stability in the rest of my
body. Furthermore, every time I breath in, causing the muscles of my neck and chest to
pull out on my lattice of ribs, my lung expands, alveoli open, taught bands of elastin in
the extracellular matrix (ECM) relax, buckled bundles of cross-linked (stiffened) collagen
filaments straighten, basement membranes tighten, and the adherent cells and
cytoskeletal filaments feel the pull; however, nothing breaks and the deformation is
reversible. Tensegrity provides a structural basis to explain all these phenomena.
In the cellular tensegrity model, the stabilizing pre-stress is generated actively by the
cells contractile apparatus and passively by distension through extracellular adhesions,
by osmotic forces acting on the cells surface membrane, and, on a smaller scale, by
forces exerted by molecular filaments extending through chemical polymerization. The
model assumes that the pre-stress is carried by tensile elements in the cytoskeleton,
primarily actin microfilaments and intermediate filaments, and that the cell is both a
hierarchical and multimodular structure. This pre-stress is balanced by interconnected
structural elements that resist being compressed at different size scales, including the
cells external adhesions to the relatively inflexible ECM and internal cytoskeletal
filaments, specifically microtubules that stretch across large regions of the cytoplasm and
cross-linked bundles of cytoskeletal filaments that stabilize specialized microdomains of
the cell surface (e.g., actin microfilaments in filopodia; microtubules in cilia). In this
model, the internal cytoskeleton is surrounded by an elastic sub-membranous
cytoskeleton (e.g., actin-ankyrin-spectrin network) and its associated lipid bilayer, which
may or may not mechanically couple to the internal, tensed microfilament-microtubuleintermediate filament lattice depending on the type of adhesion complex that forms. The
entire cytoskeleton is permeated by the viscous cytosol. Most importantly, this
micromechanical model leads to specific predictions relating to the mechanical role of
distinct cellular and molecular elements in cell shape control.
Contrasting models of cell structure depicts the cell as an elastic cortex that surrounds a
viscous cytoplasm with an elastic nucleus in its center. In engineering terms, this is a
continuum model, and, by definition, it assumes that the load-bearing elements are
12
infinitesimally small relative to the size of the cell. It is essentially the balloon model
considered by Fuller, but in this case all microstructure is ignored. Because they ignore
microstructural features, continuum models cannot provide specific predictions that
relate to the functional contribution of distinct cytoskeletal filaments to cell mechanics.
Furthermore, although these models can provide empirical fits to measured mechanical
properties in cells under specific experimental conditions, they cannot predict how these
properties alter under new challenges to the cell.
How closely does the geodesic model fit the CSK? To test its validity, suppose you hit one
of the struts of the geodesic dome. This would cause the mechanical energy to quickly
travel throughout all the struts, reverberating throughout the structure at the speed of
sound. Does the cell have a corresponding behavior? The answer is yes, since when the
CSK is perturbed at a single site, either by a specific binding event, or experimental
poking, the entire structure feels it, as the energy is dissipated throughout at the speed
of sound.
Next compare dome behavior with another cellular characteristic: shape. Geodesic
domes would quickly collapse if most of its struts were removed; conversely the dome
shape cannot be radically altered by pulling on a few of its struts. Neither of these
features of domes is shared by cells. For example, the normally spherical shape of cells in
culture will persist even after all microtubules are removed either through drugs or gene
knockout. Conversely, cells can flatten when stress is applied to the cell struts by its
ECM, as depicted in Figure 1.7 below. If living cells can remain spherical without most
of its struts, and then change from spherical to flat when stressed, then their behavior
does not closely resemble that of a geodesic dome, and tensegrity must be a more
adaptable concept. In other words, the CSK must have built-in redundancy that is
provided by the microfilament network, and its structure is highly modifiable. In fact,
redundancy of the CSK is to be expected, since its construction can be characterized as
fractal, i.e. structural forms are self-similar at different scales; stated another way, the
network weight and its volume are independent of each other. Thus the simple geodesic
dome model falls short of predicting some cell capabilities.
The ability of
microfilaments to adapt to stress by either stress-stiffening or stress softening will be
further discussed in Chapter 10.
Figure 1.7.
Response of a
single cell to
stress.
1.7
CSK Mechanoreflexes
The CSK apparently can sense the forces applied to it, and adjust its size, strength, and
orientation in order to resist the forces in an efficient manner. When stresses are highly
polarized, such as along the axis of muscles or neurons, filaments of the CSK align
themselves according to principal stress directions, as shown in Figure 1.8 below.
Besides orienting along stress lines, filaments size themselves according to strength
requirements: a conservative architectural practice.
13
Figure 1.8. Alignments of CSK filaments.
1.8
Construction of the CSK
While there are many aspects of cellular architecture remaining to be discussed, lets
consider one more question: how is the CSK constructed? Surely there are blueprints, i.e.
genes, for each of the protein components, and an overall blueprint laying out their 3dimensional arrangement, but how do the ropes and rods connect themselves properly,
in the right amount and orientation to form the fantastic structures seen above? This is a
fundamental problem of biological development. While there is no simple answer, there
are two somewhat competing models or theories that provide at least scenarios of how it
could happen.
One assembly sequence for the CSK could be similar to that of the geodesic dome, with
regular sub-structures such as triangles, being welded one by one into a 3 dimensional
network. Tensegrity would hold the structure together, and would allow it to adapt and
change. The attractive concept that the CSK represents essentially a fullerene structure
was originally proposed by Ingber and subsequently supported by many studies.
While tensegrity and the geodesic dome definitely apply to cells, it is difficult to imagine
how the complex CSK structures seen above assemble and maintain their shape.
Geodesic models significantly change shape or collapse when a single strut or rope is cut,
a behavior that cells do not share. One way cell stability could occur is by percolation
[Forgacs], a process of network formation whereby individual lines grow somewhat
randomly from point to point until sufficient connectivity establishes a network.
A simple example of percolation is the growth of telephone lines linking the East and
West coasts of the U.S. There is no direct line connecting NY with Los Angeles; however,
in the development of lines between intervening cities, eventually a continuous pathway
was formed, and as lines continued to link cities, more and more pathways were formed.
Thus a large number of redundant pathways link the structure end-to-end. Figure 1.9
14
below shows a hypothetical telephone network in the U.S. many years ago. Note that
lines between cities in the Northern sector do link NY with LA. As more cities are
connected, it can be seen that more pathways will connect the 2 coastal cities. Since
removal of a single line from the network can disrupt the continuously connected
pathway from NY to LA, this network has a just critical number of elements for spanning
the distance; it is therefore at its, percolation threshold.
LA
NY
Figure 1.9. Hypothetical

telephone network in the U.S.
Similarly, Figure 1.10 shows a snapshot of a portion of the Internet web: Do you see any
resemblance to the CSK?
Figure 1.10. A
portion of the
Internet web.
The percolation model is useful in describing network signaling, as well as elasticity, as

will be seen in Chapters 7 and 10. Beginning with the model in a future assignment, you
will study the formation of spanning networks.
Thus there are at least 2 ways to think of the CSK: as a geodesic dome with regular
networks of triangular elements or as random networks of lines tied together by
percolation. The theories are not mutually exclusive, and both are useful, however
neither tells the complete story.
1.9
Applications of Cytomechanics
This topic brings us full circle to the first: why study cytomechanics? The simplest
answer is that you may find in cells valuable solutions to engineering problems. One
example of a structural solution for a strong, lightweight, and flexible material is shown
in Figure 1.11 below. Design for this material is taken directly from CSK structure, and
is in fact being developed by a company formed by Donald Ingber. Reverse engineering
15
and exploitation of biological structures can be highly profitable, since cells require no
patent royalties. The material below can absorb impact energy well, because shocks are
dissipated thoroughly throughout the structure.
Figure 1.11. Material that mimics CSK structural design.

CSK structure is finding applications in other areas. Bioactive geodesic scaffolds for
filtration and catalysis are highly efficient due to their high ratio of surface area-tovolume and low mechanical resistance to flow. One spin-off from this technology is an
improved face mask with better protection against pathogens. These filters have adapted
many features of cell structure, the CSK, and its bioactive nature. The scaffolds thus have
large pores (much larger than the pathogens) for easy air flow, but have a huge surface
area and tortuous path to trap particles. Moreover the scaffolds are coated with a
synthetic hydrogel "protoplasm" that soaks up pathogens, which are highly hydroscopic
(water seeking). The pathogens are killed using solid-state enzymes and other
bactericidal agents that are incorporated within the gel.
Such CSK- inspired materials can have unusual 3-D
characteristics, such as the structures shown in Figure
1.12 that have zero mean curvature.
Figure 1.12. CSK inspired
materials.
The CSK behavior of stress-stiffening has inspired flexible fabrics that get stiffer when
stretched, while maintaining their porosity for critical heat exchange. Development of an
"artificial gill" for oxygen production is underway, using the high surface area and
efficient solid-state catalysis offered by CSK design.
16
Biomimicry of cytomechanical design is advancing many other technologies, including
tissue engineering, wound healing, microtubular nanostructures, bioprocess
optimization, cryogenics mechanoelectrical signaling, tumor therapies, and genetic
regulation. Microtubules serve as perfectly straight templates for fabrication of
nanowires.
With the preceding cursory view of structural principals of the cell (whose mechanical
details are discussed in later chapters, we are now ready to take a closer look. In later
chapters we will ask more probing questions: What other roles does the CSK play? How
does it interact with the nucleus? How is it formed and maintained?
_______________________________________________________
Chapter 1
1. Examine Figure 1.1 (and the movie) and explain briefly the events it depicts.
2. What are the general names for structural components that resist compression &
tension? Which cellular components correspond? Compare structural properties
of microtubules, spectrin, and actin filaments, and state a different role for each
of them.
3. List three examples that could represent tensegrity structures. Sketch them and
draw their free body diagrams.
4. State three differences between cellular and building architecture.
5. Demonstrate the percolation threshold of network formation, using
connect_the_dots.m on Sakai. What conditions affect formation of a
spanning network?
6. Examine the Simulink model #1 discussed in class, and make and test your own.
Be sure to document the model and label its components.
References
Eyckmans, J., T. Boudou, et al. (2011). "A Hitchhiker's Guide to Mechanobiology."
Developmental Cell 21(1): 35-47.
More than a century ago, it was proposed that mechanical forces could drive
tissue formation. However, only recently with the advent of enabling biophysical
and molecular technologies are we beginning to understand how individual cells
transduce mechanical force into biochemical signals. In turn, this knowledge of
mechanotransduction at the cellular level is beginning to clarify the role of
mechanics in patterning processes during embryonic development. In this
perspective, we will discuss current mechanotransduction paradigms, along with
the technologies that have shaped the field of mechanobiology.
Ingber, D. E. (2008). "Tensegrity-based mechanosensing from macro to micro."
Progress in Biophysics & Molecular Biology 97(2-3): 163-179.
This article is a Summary of a lecture on cellular rnechanotransduction that was
presented at a symposium on "Cardiac Mechano-Electric Feedback and
Arrhythmias" that convened at Oxford, England in April 2007. Although critical
mechanosensitive molecules and cellular components, such as integrins, stretchactivated ion channels, and cytoskeletal filaments, have been shown to contribute
to the response by which cells convert mechanical signals into a biochemical
17
response, little is known about how they function in the structural context of
living cells, tissues and organs to produce orchestrated changes in cell behavior in
response to stress. Here, studies are reviewed that suggest our bodies use
structural hierarchies (systems within systems) composed of interconnected
extracellular matrix and cytoskeletal networks that span from the macroscale to
the nanoscale to focus stresses on specific mechanotransducer molecules. A key
feature of these networks is that they are in a state of isometric tension (i.e.,
experience a tensile prestress), which ensures that various molecular-scale
mechanochemical transduction mechanisms proceed simultaneously and
produce a concerted response. These features of living architecture are the same
principles that govern tensegrity (tensional integrity) architecture, and
mathematical models based on tensegrity are beginning to provide new and
useful descriptions of living materials, including mammalian cells. This article
reviews how the use of tensegrity at multiple size scales in our bodies guides
mechanical force transfer from the macro to the micro, as well as how it
facilitates conversion of mechanical signals into changes in ion flux, molecular
binding kinetics, signal transduction, gene transcription, cell fate switching and
developmental patterning. (c) 2008 Elsevier Ltd. All rights reserved.
Ingber, D. E. (2010). "From Cellular Mechanotransduction to Biologically Inspired
Engineering." Annals of Biomedical Engineering 38(3): 1148-1161.
This article is based on a lecture I presented as the recipient of the 2009 Pritzker
Distinguished Lecturer Award at the Biomedical Engineering Society annual
meeting in October 2009. Here, I review more than thirty years of research from
my laboratory, beginning with studies designed to test the theory that cells use
tensegrity (tensional integrity) architecture to stabilize their shape and sense
mechanical signals, which I believed to be critical for control of cell function and
tissue development. Although I was trained as a cell biologist, I found that the
tools I had at my disposal were insufficient to experimentally test these theories,
and thus I ventured into engineering to find critical solutions. This path has been
extremely fruitful as it has led to confirmation of the critical role that physical
forces play in developmental control, as well as how cells sense and respond to
mechanical signals at the molecular level through a process known as cellular
mechanotransduction. Many of the predictions of the cellular tensegrity model
relating to cell mechanical behaviors have been shown to be valid, and this vision
of cell structure led to discovery of the central role that transmembrane adhesion
receptors, such as integrins, and the cytoskeleton play in mechanosensing and
mechanochemical conversion. In addition, these fundamental studies have led to
significant unexpected technology fallout, including development of
micromagnetic actuators for non-invasive control of cellular signaling,
microfluidic systems as therapeutic extracorporeal devices for sepsis therapy, and
new DNA-based nanobiotechnology approaches that permit construction of
artificial tensegrities that mimic properties of living materials for applications in
tissue engineering and regenerative medicine.
18

CHAPTER 2: MATERIALS OF THE CELL AND MATRIX
2.1 Basic Cellular Constituents
All cellular structures are made with same building blocks: atoms of (in descending
concentrations) carbon, oxygen, hydrogen, nitrogen and phosphorus, sulphur, and several other
atoms in minute quantities. These atomic building blocks are assembled into larger blocks, i.e.
amino acids & sugars that are then strung together in even larger blocks, i.e. proteins and
carbohydrates, schematically shown in Figure 2.1 below:
Figure 2.1. Assembly of

basic structural
components of the cell
Proteins
Carbohydrates
Biomembranes
Filaments
Amino Acids
Polymeric
Structural
Components
Sugars
Phospholipids
Peptides
Atoms
Carbon
Oxygen
Hydrogen
Nitrogen
Phosphorous, Sulphur, etc.
Monomeric
Building Blocks
Size and
Complexity
As you can see, our study of cell materials is greatly simplified by the fact that all structural
components are polymers strung together with 3 types of building blocks. The structural
components are mainly proteins, with some carbohydrates. The major categories of cellular
constituents are listed in Table 2.1 below:
Compound
Water
Protein
(Polypeptide)
Lipid (Fat)
Carbohydrate
(Sugars)
Salts
(Electrolytes)
Table 2.1.
Chemical Components of Cells
Fraction in
Relative Size
Polarity of molecule
Cell (%)
of molecule
70-80
Small
Polarized
10-20
Large
Regionally polarized
2-20
1-2
Medium
Medium to large
Non-Polarized
Regionally polarized
Small
Polarized
Note from the table that electrical polarity varies among the molecules. The importance of these
differences to cell mechanics will be appreciated in our study of Energetics in Chapter 6. Note
that lipids are non-polarized, however some amino acids are also non-polarized. Carbohydrates
19

can exist as simple sugars, of medium size, or as larger complex chains, such as the backbone of
DNA.
Besides polarity and size, other important properties of molecules are their solubility, stability,
and shape. As we shall see, proteins are the molecules whose shape, an important determinant
of their function, is the most varied.
In addition to the structural components, a second category of components is the electrolytes,
and monomeric molecules dissolved within the cell soup; these include the major salts, and the
building blocks: amino acids, sugars, and cell fuels such as ATP. The major electrolytes consist
of (in order of descending concentration in the cell): Cl-, K+, Na++, Ca++, and Mg++. These salts
are all surrounded by several water molecules, attached by hydrogen bonds.
2.2 Plasma Membranes
To understand the material make-up of cells and matrix, it is entertaining to imagine how they
first sprung up in evolution, even though its details will never be known. The following
somewhat fanciful history is therefore to be pondered in that light. In the beginning there was
primordial soup: a rich sea filled with organic compounds. Over the eons, as the chemicals
began to aggregate in the slime, a critical mass of them was captured within a volume
surrounded by a barrier: a lipid membrane. Since oil and water do not mix, a lipid bubble is the
logical vessel for the aqueous contents of the cell to reside. Thus a thin hydrophobic shell
separated the hydrophilic from their surroundings.
The snapshots below show a reverse-scan of 10 s of simultaneous data from consisting of both
capacitance value and image frame. This X defines the trigger moment of the bilayer
formation.
Figure 2.1 Capacitance ~solid lines and images
~15! of a phosphatidylcholine membrane in
development. The white lines in images 1 and 4
correspond to
50 mm. The numbered closed circles represent
the capacitance values measured
simultaneously with the numbered images. In
images 3 and 4, the words 1st and 2nd
with arrows indicate the first and second
regions, respectively. The dashed lines represent
the membrane capacitance expected in case of
the first thinning continuation without the
second thinning. The 3 mark represents the
trigger moment of the bilayer formation.
20
With time lipid bubbles that were cellular

precursors, a prokaryote would have had to
develop structures to survive, since lipid
bubbles are easily squashed. Hence the CSK
was needed, and the rest is added complexity.
Now, lets take a look under the hood (Figure
2.2).
Figure 2.2. Peeling away the cell
membrane
The first thing we notice is that the biomembrane is a lipid bilayer, arranged as an amphiphile.
This means that the molecular structure has both hydrophilic and lipophilic parts, as shown in
the schematic of a cross-section below.
H2Polar
O Phosphate Heads
Figure 2.3. Lipid Bilayer

H2O
70 A
H2O
Hydrophobic core
The phosphate heads are negatively charged, so the outside layers of the phospho-lipid
sandwich quite happily dissolve in water. The core, composed of long hydrocarbon tails, i.e. fatty
acids, behaves oppositely, repelling water and all hydrophilic compounds. Thus the bilayer is
elegantly arranged to exist with both faces in water, allowing the middle to act as a barrier to
movement of water-based materials. Since the membrane separates the cell plasma from the
extra-cellular milieu, it is called the plasma membrane (PM).
An anecdote about the biomembrane is that the first estimate of its thickness is attributed to
Benjamin Franklin. The story goes that he poured a volume of cooking oil into a small pond
until it was entirely covered with oil. He then divided the volume of oil poured by the area of the
pond, finding the thickness was about 30 A. The close correspondence of this number with
present day measurements of lipid monolayers illustrates the power of simple experiments. It
also demonstrates the property of phospholipids to self-assemble into a structure, i.e. a
membrane.
Due to its lipid structure, the cell membrane is non-polar and hydrophobic. Thus the membrane
can sequester charges within the cell, since they cannot penetrate the membrane. It is this
feature that is responsible for the bioelectricity of cells, which will be discussed in Chapter 6.
21
While the PM can be considered part of the CSK, it is relatively weak. It does have measurable
stiffness, however, in shear, compression and bending, as will be seen in Chapter 4. Being
essentially a Newtonian fluid, the bilayer by itself has no tensile strength. To illustrate, look at
Figure 2.4 below, showing 2 vesicles whose walls are pure lipid bilayers. Starting from the
bottom picture, you see the vesicles just touching. The next 2 pictures on top were made as the
vesicle at right is pushed out by pressure from the pipette holding it. Note that the vesicles pass
into each other. This ghost-like behaviour is due to their liquid nature.
3. Vesicles pass
through each other
Figure 2.4.
Vesicle ghosts
2. Pipette pushes
vesicle out
1. Vesicles make
contact
2.3
CSK Components
The structural basis of force transmission in cells is the Cytoskeleton (CSK). In the cytoplasm,
the CSK is a fundamental structure for mediating force transmission (Wang et al., 1993). The
CSK is a highly dynamic cellular scaffolding structure composed of filamentous actin (6 nm in
diameter), intermediate filaments (10 nm), and microtubules (23 nm). These three cytoskeletal
elements are not single proteins, but consist of many monomers able to span large distances
within the cell. Tubulins polymerize to form hollow cylinders known as microtubules and
provide a structure for motor proteins such as kinesins and dyneins to travel between different
cell compartments. Vimentin, keratin, and lamin monomers form intermediate filaments that
connect the nucleus with the endoplasmic reticulum, mitochondria, and Golgi apparatus,
providing structural integrity to the cell. Actin monomers assemble into filamentous actin (Factin) and together with myosin filaments, form the cytoskeletal contractile apparatus. The
actomyosin CSK connects multiple parts of the cell membrane as well as the cell membrane to
the nucleus (Sims et al., 1992).
At the cell membrane, these filaments anchor into clusters of proteins that include focal
adhesions (FAs) which link the CSK through transmembrane integrin receptors with the ECM.
In the extracellular space, the ECM materializes as a mesh of cross-linked proteins and
carbohydrates, and depending on the tissue, can include different constituents including
22

collagen, laminin, elastin, and fibronectin fibers interlocked with hyaluronic acid and
proteoglycans.
From a mechanical standpoint, applying force to this cell-ECM unit leads to structural
deformations and rearrangements of the ECM, force transmission through the FA, and (given
the highly interconnected nature of the CSK) deformation of nearly every aspect of intracellular
structure, including the position of mitochondria, endoplasmic reticulum, and the nucleus (see
Figure 3.1). However, outside-in force transmission is only half the story, as cells also
generate force. Polymerization and depolymerization of microtubules drive pushing and pulling
forces, respectively, to control the position of mitotic spindles, chromosomes, and nuclei
(Dogterom et al., 2005). The head domain of myosin II pulls on actin filaments to generate
traction forces, which are transmitted to focal adhesions and deforms the ECM via an insideout transmission path. Hence, mechanical forces are experienced throughout the cell via the
integrated CSK-focal adhesion- ECM architecture.
So, underneath the membrane, and penetrating it at many points, is the CSK. The first set of
components that is encountered contains different proteins each with separate functions, as
depicted in the schematic diagram below (Figure 2.5). The cartoon represents a generic cell;
however particular cells may differ in structure. For example, the red blood cell lacks most of
the components shown below.
Figure 2.5. Perimembranous CSK
The extracellular matrix (ECM) consists of many filamentous proteins, including collagen,
fibronectin, vimentin, titin and others. The CSK near the membrane is rich in many other
filamentous and small proteins, such as tensin, vinculin, talin, -actinin, etc.
The most
abundant filamentous protein near the plasma membrane is actin. Note that the CSK connects
with ECM via integrin molecules that consist of alpha and beta subunits. These subunits have
ligands for specific receptors on matrix proteins. Integrin thus crosses the plasma membrane
(PM) to serve as the connector. The standard active ligand of integrin is the 3 amino acid
sequence RGD. Circulating cells such as white blood cells and platelets use their integrins as
antennae to locate cells and matrix where their function is needed. Figure 2.6 below
represents an Integrin of a platelet. With its 2 subunits, integrin resemble both in form and
23

function, a staple. Several other cellular proteins bind with the ECM, as will be detailed in
Chapter 11.
Figure 2.6. Integrin
The most abundant filament in most cells is filamentous actin (F-actin). Theses microfilaments
are most prominent around cell perimeters, and serve as ropes tying the network of other
proteins together. A notable exception to this cellular dependence on actin for rigidity is the red
blood cell, whose CSK is rich in spectrin. While actin (specifically F-actin) is the main
contractile generator in cells, it can also sustain some compression. Another type and
configuration of actin is globular, or G-actin. Both types exist in cells, as seen below in Figure
2.7, with the F-actin stained green, and the G-actin stained orange (different gray shades if this
is black & white rendering).
Figure 2.7.
Actin in a
fibroblast
Many other proteins link the main filamentous actin to the membrane, including tensin, talin,
and a-actinin, and vinculin, as shown in the cartoon. Although these many proteins, including
Integrin, are not abundant, they play crucial roles in mechanical signaling, as we will see in
subsequent chapters.
Adhesion of cells to the ECM takes place at focal adhesion complexes (FACs), as depicted above.
FACs are mechanical linkages that also serve as signal relay stations between the CSK and ECM.
24

Stress fibers consisting of bundles of actin filaments associated with myosin are usually attached
to the FACs. FACs can generate contractile forces during cell crawling, thus serving as
mechanical actuators.
Recalling from Chapter 1, the CSK has 3 major components: microfilaments, intermediate
filaments, and microtubules. Single strand diameter of these proteins ranges from about 8 nM
for microfilaments, 15 nM for intermediate filaments, and 25 nM for microtubules. Filaments
usually form multi-stranded threads, so large bundles of microfilaments are common.
Microtubules (MT) are seen in abundance in the cells below, differently shaded from actin. Note
that MTs tend to be straighter and run closer to the nuclei, although they extend to cell
perimeters as well. MTs are hollow tubes, whereas the other 2 filaments are like ropes and rods
(See Figure 2.8).
MT
Actin
Figure 2.8. Immunostained cells showing actin in orange and microtubules in green.
The CSK is thus structured as a porous network of struts. This plan is, in fact, no different than
that of all living materials: even bone, the densest material in the body, is composed of geodesic
structures at the meso-scale. The millimeter-size subunits are arranged in a lattice oriented to
maximize strength in the direction of principal stress. Bone, like the CSK, is a strong, shock
absorbing material that distributes mechanical energy to the network, but tends to focus large,
chronic stresses to stronger and thicker support elements.
It should be noted that CSK composition varies from cell to cell, and even region to region
within one cell depending on function as well as developmental state. For example, red cells are
rich in spectrin, but deficient in actin. Muscle cells, in contrast, have very high actin content.
Growing or healing cells may have relatively high actin in their growing portions. Some cells,
25

such as skin cells of young animals or certain animals, are rich in elastin. Material properties of
some CSK components are listed below:
Polymer
Actin
Tubulin
Intermediate
Filaments
Silk
Collagen
filament
Collagen fibril
Elastin
Cellulose Dry
Cellulose Wet
Spectrin
DNA
2.4
Table 2.2
Properties of Filaments
Typical
Persistence
Elastic
Diameter (nM) Length p
Modulus
E
(m)
(Gpa)
8
15
2
25
6000
2
10-20
Mass Density
p
(Da/nm)
110
160
1.5
10-300
0.02
0.05
0.002
80
40
0.002
1
4500
1900
Building the CSK
The long-range order of the CSK is generated by simple rules for network assembly and
disassembly. In other words, the CSK is a dynamic structure, and static pictures of it just capture
one moment of its ever-changing appearance. Examples are in Figures 2.9 below:
Figure 2.9.
a. Fluorescence micrograph of a
fish keratocyte is shown (with
the nucleus in blue). Motile cells
such as these form branched
actin-filament networks (red) at
their leading edge, and these
branched networks generate
protrusions. Together with
coordinated adhesions to a
surface (indicated by vinculin,
green) and myosin-driven
retraction, the protrusions lead
to directed movement. Scale bar,
15 m.
b. There are three basic steps
involved in the assembly of
protrusive, branched actinfilament networks: filament
elongation; nucleation and
crosslinking of new filaments
from filaments close to the
membrane; and capping of
filaments. Disassembly of the
network involves a separate set
of proteins that severs the
filaments and recycles the
subunits.
26

2.5
Cytogel
Cells are Gels.

Gels are relevant to cytomechanics because the internal composition of cells is essentially a gel.
We will investigate the mechanical properties of cells by comparing them to a gel surrounded by
a smart membrane. In particular, the concept of cell volume regulation will be studied.
Gels and Gelatinization
Gelatinization refers to the (usually) irreversible loss of the crystalline regions in suspended or
dissolved polymers that occurs suddenly when the physico-chemical environment changes. Gel
is thus a state of matter between solid and liquid. Such physical changes can be heat, pH, or
chemical composition.
Smart polymer gels actively change their size, structure, or
viscoelastic properties in response to external signals. The stimuli-responsive properties,
indicating a kind of intelligence, offer the possibility of new gel-based technology. Deformation
and the mechanism of polyelectrolyte gel behavior in electric fields are studied experimentally
and theoretically, especially, swelling and bending. Gel stiffness can be controlled by electric
fields and polarized particles.
The cytosol is a gel containing electrolytes, amino acids,
carbohydrates, metabolic fuels and products, as well as cellular organelles, such as mitochondria
and endoplasmic reticulum, and others depending on cell type. The cytoplasm, since it is a gel,
contributes some mechanical stiffness properties to the cell. Ca++ plays a major role in
maintaining the cytogel since it coordinates polymer-polymer interactions. The cytogel is
formed when the negatively charged polymers in the cytosol (proteins and nucleotides) are
trapped together, creating osmotic pressure for water influx. A schematic of a gel is shown in
Figure 2.11 below. Note that the gel can be modeled as a spring, and that swelling pressure is
a function of polymer-polymer interactions, intra-polymer interactions, and osmotic pressure.
Gel
H20
Factors
Ca++, pH, heat
Swelling pressure = osmotic pressure- elastic (compressive)

pressure
Figure 2.10. Gel Model

Chapter 2
1. Integrin acts like a staple holding cells to substrates. Draw a free body diagram showing the
types of forces involved in this interaction.
2. What does polarity have to do with solubility? Give an example.
27

3.
4.
5.
6.
7.
Calculate the capacitance of a lipid vesicle, 1 uM in diameter. Show assumptions.

Examine Figure 2.1 and explain, using drawings and formulae, what happens.
What cellular components serve as ropes or rods?
Cite a specific pathway whereby Integrin could be involved in mechanotransduction.
Describe what mechanical role the cytogel might serve. Make a Simulink model of it, using
springs. First write the equation of motion, after the gel has been subject to an osmotic
perturbation. Estimate the fractional composition of the major components of your model.
References
1.
Goldmann, W.H., Mechanical aspects of cell shape regulation and signaling. Cell
Biology International, 2002. 26(4): p. 313-317.
2.
http://www.bio.unc.edu/courses/2004spring/biol052-006/ch02final.pdf
3.
http://www.science.uwaterloo.ca/~cchieh/cact/applychem/waterbio.html
4.
Vuori, K., Integrin signaling: Tyrosine phosphorylation events in focal adhesions.

Journal of Membrane Biology, 1998. 26(3): p. 191.
28
Chapter 3: Cell Types

CHAPTER 3: MECHANICS OF CELL TYPES
(Adapted from (Bao and Suresh 2003)
3.1
A Generic Cell
There are over 200 different cell types in the human body, each with its own specialty, shape,
and mechanical properties. It is useful to recognize some basic features common to most, if not
all of them, as shown in the generic cell cartoon, Figure 3.1.a below:
Figure 3.1. Generic cell and comparative properties.

29

As seen in Figure 3.1.b, the cell membrane consists of a lipid bilayer with integral proteins.
Living cells (Figure 3.1.c) are in the micrometer size range, and have stiffnesses, as measured
by the elastic modulus, in the sub-MegaPascal range. (Note there are exceptions, i.e., algal cells
& neurons, which can be meters in size). Traditional engineering materials, such as ceramics,
metals, and plastics, are much larger and stiffer, as shown. Newer materials, i.e. nanomaterials, can have much different properties. The intracellular components perform many
functions: synthesis, sorting, storage and transport of molecules; storage and expression of
genetic information; recognition, transduction and transmission of signals; powering of
molecular motors and machines. The organelles also convert (transduce) energy from one form
into another, serving as cellular reflexes. These reflexes sense and respond to external
environments by continually altering cellular structure. Most living cells can sense and respond
to forces.
Cells in the human body are highly diverse, ranging from the red blood cell, which is little more
than a sack of hemoglobin, to nerve cells 1 meter in length, and a million times smaller in width,
with intricate bush-like branches at both ends. Despite this diversity, the mechanical plan is the
same for all cells: each is a small glop of gel held together with stiff rods and struts, and
surrounded by an oily membrane. A few types of cells are outlined here.
3.2
Red Blood Cells
Red Blood Cells (RBCs) are the simplest animal cells: they lack a nucleus and several other
organelles, they have a simple CSK, and they assume a very narrow range of sizes and shapes.
Despite their simplicity, their mechanical behavior, both in health and disease, represents the
foundation of cytomechanics. The RBC travels around the circulation for one purpose only: to
exchange gases. It must withstand large shear and deformations as it bumps into obstacles,
becomes exposed to variable osmotic pressures, and must squeeze itself, with a 7.08.5 m
diameter into tiny (< 3 m) wide capillaries (See Figure 3.2). Each RBC thus undergoes large
elastic strains many times a minute as it speeds through narrow tunnels, a feat that few manmade objects can do. This amazing malleability of RBCs has played a major role in the
development of the field of cellular mechanics, since it sparked the curiosity of its founder, Y.C.
Fung.
Two interrelated structural features of the RBC underlie its mechanical adaptability. The first is
its shape under normal conditions. The biconcave disc shape is a brilliant design, since the
shallow center has a low bending stiffness, and it can serve as an expandable reservoir to allow
swelling without increase in surface area. These properties allow the RBC to squeeze through
narrow capillaries, and to swell in hypotonic environments without breaking, as depicted below.
Since the plasma membrane of RBCs undergoes lysis at an area expansion of only 3%, the cells
would quickly explode when exposed to even slight hypotonicity if the cell did not have this
characteristic biconcave shape (See Figure 3.2).
Figure 3.2.a. A red blood cell
squeezing through a capillary
RBC in isotonic
media
RBC in
moderate
hypotonicity
Figure 3.2.b.
Shapes changes in
red blood cells.
RBC in higher
hypotonicity
30

The key to the structural adaptability of the RBC is in the composition of its CSK, which is the
simplest in the animal world. The CSK of RBCs is constructed from a single filament system
based on actin, whose polymeric structure is linked with spectrin. Thus RBCs lack the stiffer
components, intermediate filaments and microtubules, and contain the flexible polymer
spectrin to link a flexible network of actin polymers.
RBCs sometimes aggregate in the blood stream to form rouleaux (stacks of coins) as shown
below in Figure 3.3:
The elegant mechanical design of RBCs as they travel their
tortuous pathways in the vascular system can be appreciated
by observing how small defects can affect them. The first
recognized molecular disease.is sickle cell, caused by a point
mutation that converts normal adult Hemoglobin (HbA) into
sickle Hemoglobin (HbS), with a single amino acid difference.
The mechanical consequences of this defect begin with
polymerization of Hb into straight rods, leading to gelation
within the cell. When these abnormally rigid RBCs travel
through the vascular system, they initiate a cascade of
inflammatory responses that can manifest as hemolytic
anemia, vaso-occlusion, and multi-organ damage. The
gelation process and its mechanical consequences will be
further discussed in Chapter 7.
Figure 3.3. Red blood
cells.
3.3
Platelets
The second simplest cell in the body is the platelet, which, like the RBC, is an anucleate
circulating cell. Its role is to protect the vascular system by orchestrating blood clotting at sites
of injury. When they encounter an injury, platelets aggregate and transform from discoid into
filipodal structures that then spread out to form a physical barrier against blood loss. The
characteristic shapes changes, as depicted in Figure 3.4, are accomplished by active work by
the actin-myosin network, in concert with microtubules. Similar to contraction by myocytes, the
actin-myosin motions within platelets is initiated by Ca++, and is fueled by phosphorylation.
Unlike myocytes however, that have an assembled network of actin-myosin filaments ready to
work upon demand, the actin/myosin system of platelets is assembled only after an injury. In
the absence of injury stimulus, the actin and myosin in circulating platelets exist mostly in the
globular form, i.e. as monomers. Actin monomers in the resting cell are sequestered and
prevented from polymerization by a Ca++ sensitive protein. Microtubules, in contrast, are
elongated, helping to maintain the discoid shape. Upon injury, Ca++ entry causes a cascade of
events leads to profilin dissociating the actin-binding protein and commencement of
polymerization. When chains elongate to 6 or more actin monomers, myosin can bind to them,
setting up the energy-driven movements. In reciprocity to actin polymerization, microtubules
tend to de-aggregate, allowing more flexible movements of the filopodia. This remarkable
choreography occurs without a nucleus, through the interplay of just 2 filament systems.
Figure 3.4. Platelet activation
31

3.4
White Blood Cells (WBCs)
White Blood cells, or leukocytes, are members of a group of circulating cells whose job is
responding to immune needs and inflammation. As they speed through the circulatory system,
WBCs are incredibly capable of finding the trouble spot infection site, stopping, then attaching
themselves to the endothelial cells lining the vessel, and then move through them into the body
tissues to attack.
WBCs must undergo not only the same passive deformations as RBCs, but must also actively
change shape as they perform phagocytosis, i.e. envelope foreign or inflamed objects. Their
shapes, unlike RBCs, are generally spherical. How then do WBCs achieve their flexibility and
motility? In fact WBCs use an entirely different strategy to accomplish the same feats of
deformability and volume expansion without surface area change. The answer is that WBCs can
deform and change volume without undue stress on their membranes, because their plasma
membranes and CSK are extensively folded. These folds consist of CSK curvatures and
membrane microvilli; these serve the same role as the RBC concavities: they are expandable
reservoirs. WBCs expansion-shrinkage behavior is much the same as the Hoberman sphere. At
low magnification, WBCs appear smooth, but folds and microvilli can be seen at higher
magnification as depicted below in Figure 3.5.
Figure 3.5.
WBC microvilli
Outline of WBC
Microvilli
3.5 Fibroblasts
Fibroblasts serve as a glue holding tissues together and are primary manufacturers of collagen.
Shown below in Figure 3.6 are fibroblasts co-cultured with cardiac myocytes, and stained for
actin. Myocytes are brilliantly stained (white) showing an abundance of actin, while fibroblasts
(left portion) are much darker. Note that the high density of actin sometimes obscures
structural details.
Figure 3.6.
Fibroblasts and
cardiac
myocytes,
stained for actin
32
3.6
Endothelial Cells
Endothelial cells (ECs) are a good example of cytomechanical adaptability. The basic role of
ECs is to line the walls of blood vessels. They are directly exposed to flowing blood, as depicted
schematically and photographically below.
Figure 3.7.
Endothelial
Cells
ECs with black

nucleus (Fung)
Figure 3.8. Inside a blood vessel, looking at

bulging endothelial cells
ECs change shape and mechanical properties in relation to
the forces they experience from the flowing blood.
Endothelial cells not only recognize the magnitude of force,
but also distinguish between shear and normal forces, and
whether they are steady or pulsatile.
Abnormally high
forces can lead to vascular diseases including thrombosis
and atherosclerosis. Cells can 'crawl' like an inchworm by
pulling themselves forward using contractile forces.
3.7 Myocytes
The three types of muscle cells: skeletal, heart and smooth, each have different mechanical
structures and behaviors. Myocytes, or muscle cells, are the primary motor cells of the body,
and hence have the highest content of actin. Figure 3.9 below shows a single myocyte that has
been stained for actin. Myocytes tend to elongate to form log-shaped structures.
Figure 3.6
Figure 3.9. A cardiac myocyte stained for actin

33

3.8
Transformed Cells
Cells from almost any tissue can transform into tumor cells that are genetically distinct. Benign
tumors grow rapidly, but respond normally to ECM. Malignant cells have mutant actin,
disorganized CSK. They lose contact inhibition and invade ECM, and climb over other cells. Cell
shape affects malignancy, i.e. imposing a spherical shape on melanoma cells makes them more
metastatic.
3.9
Stem Cells (adapted from Lee, Knight et al. 2011)
The mechanical properties of stem cells influence their response to their mechanical
environment, their ability to migrate and ultimately their differentiation. For example, the
amorphous mass of undifferentiated mesenchymal cells, responsible for the development of the
skeleton, is susceptible to the influence of mechanical signals mediated through the extracellular
matrix. It has long been postulated that adventitious, secondary cartilage develops on cranial
membrane bones of the embryo in response to intermittent pressure and tension, associated
with movement [Hall, 1972]. The importance of intermittent loading to chondrogenic
development has been further supported by studies involving joint immobilization in the
developing chick embryo. Indeed following paralysis of skeletal muscles, abnormalities were
reported to develop, including the absence of synovial joint cavities, the fusion of long bones by
fibrous tissue cartilage or bone, the absence of adventitious and articular cartilages and the
distortion of the skeleton. An alternative approach uses theoretical models to predict the effects
of mechanical stimuli on lineage-specific stem cell differentiation. These models can predict
tissue differentiation during skeletogenesis, fracture healing, bone distraction and the
development of pseudoarthroses. As early as 1960, Pauwels proposed that tissue deformation or
stretching induces the formation of fibrous connective tissue while compression induces
cartilage formation (Figure 3.10.A). More recently researchers have used finite element
analysis to estimate the internal mechanical state within structures, to predict the influence of
hydrostatic pressure and distortional strain on tissue differentiation. These models suggest a
correlation between high levels of compressive hydrostatic stress and chondrogenesis; low
hydrostatic stress and osteogenesis; and high distortional strain associated with the formation of
fibrous connective tissue or fibro-cartilage (Figure 3.10.B).
Figure 3.10. Models of the relationship

between the mechanical environment and
differentiation of mesenchymal tissue.
34

Further adaptations of the modelling approach enable the establishment of critical values for
mechanical parameters in relation to differentiation. For example, a study proposed that local
strains lower than 5% induce intramembranous ossification, while hydrostatic pressures greater
than 0.15 MPa and local strains smaller than 15% induce endochondral ossification. However,
from a biological viewpoint, it is not correct to suggest that in all conditions these critical values
represent sharply delineated cut-off values that will predict the differentiation of tissues under
the influence of distortional strain and hydrostatic stress. Indeed fundamental changes occur
within differentiating tissues, which can drastically change the nature of loading, for example,
the generation of extracellular matrix.
In an attempt to analyze mesenchymal cell
differentiation, finite element models were developed, which incorporate the effects of the
relative velocity of fluid and solid constituents, fluid pressure and tissue deformation
[Prendergast et al., 1997]. The synthesis of extracellular matrix by differentiating MSCs may, or
may not, favour the mechanical and perfusion characteristics required for lineage specific
differentiation within that tissue, driving the progression of cell phenotype in a step-wise
manner.
A mechano-regulatory pathway (Figure 3.10.C) describes mesenchymal
differentiation in a temporal manner, where the emergence of a specific extracellular matrix
(Point XFigure 3.10.C) can favor a divergence in phenotype (red dashed line) from a steadystate condition (solid line). In the presence of significant shear strain and associated motion,
fluid velocity and shear forces are also maintained favoring differentiation to fibrous connective
tissue. However, an up-regulation or change in collagenous matrix production leads to a higher
stiffness and consequent reduction in fluid velocity and shear force leading to osteogenic
differentiation.
More recent developments on the modelling approach have incorporated other parameters that
may interact with the mechanical stimuli to direct differentiation, most notably alterations in
the oxygen environment and associated angiogenesis [Checa and Prendergast, 2009]. This
model also incorporates cell migration, proliferation and apoptosis and has been further
developed to incorporate physiological variation in cellular parameters to predict animal to
animal variation in differentiation reported in vivo within a defined bone chamber model
subjected to mechanical loading [Khayyeri et al., 2009].
__________________________________________________________________
Chapter 3
1.
Explain how red blood cells can increase volume without increasing their surface area.
Show formulae.
2. What types of forces would be experienced by ECs in their natural state? Draw a sketch
resembling a free body diagram.
3. Describe two types of transduction that a cell can perform.
4. State 2 structural adaptations of RBCs that relate to its function.
5. Compare the way in which red and white cells adapt to different environments in the
blood stream.
6. Expand your Simulink model of gel (from Chapter 2) to include damping. Show the
various degrees of damping, i.e., under, over, etc.
References
35
Bao, G. and S. Suresh (2003). "Cell and molecular mechanics of biological materials." Natural
Materials 2(11): 715-725.
Lee, D. A., et al. (2011). "Stem Cell Mechanobiology." Journal of Cellular Biochemistry 112(1):
1-9.
36
Chapter 4: Material & Structural Mechanics

CHAPTER 4: MATERIAL AND STRUCTURAL MECHANICS
4.1
Background (adapted from Ingber, 2010)
Most man-made constructions, such as Stonehenge, gain their stability from continuous
compression, as in brick-upon-brick type constructions in which gravitational forces compress
one building element down upon another. These are generally stable structures, except when
subject to side impacts, when individual building components can fall like dominoes. While the
latter phenomenon is the physical basis for a popular app (Angry Birds), it is an undesirable
structural feature. Tensegrity structures, in contrast, are composed of a network of tensed
elements linked to a subset of elements that resist being compressed, and thereby bring the
entire system into a state of isometric tension. Buckminster Fuller first coined this term, and a
graduate student, Kenneth Snelson, embodied it by constructing sculptures composed of
stainless steel beams interconnected by tension cables that hold themselves stable against the
force of gravity even though the beams never come in direct contact.
4.2
Stress Environments of Cells
Living cells are both affected by and dependent upon mechanical forces in their environment.
Cells are specialized for life in their own particular environments, whose physical stress patterns
become necessary for normal functioning of the cells. If the forces go outside the normal range,
then the cells are likely to malfunction, possibly manifesting as a disease or disability. For
example, when people spend more than a few days in zero gravity, many organs, including the
bones and heart, decline in mass,. In general, cells need continual stimulation from their usual
environmental forces to maintain their size, growth rate, and function.
Human cells live in a wide range of mechanical environments. The environments of several cell
types are outlined in the table below:
Tissue
Mechanical Environment
Normal Range
Cell Types
Bone, Cartilage
Weight bearing forces
Arterial
Endothelium
Tendon
Fluid pressure and shear
Osteocytes, Osteoblasts,
Chondrocytes
Endothelial
Skin
Organ of Corti
Muscle
(Intrafusal)
Muscle
(Extrafusal)
Mesangium
Compression and shear

Fluid shear
Tension
Continuous: 1X - 4X
body weight
Pulsatile: 60-140 mm
Hg;
Up to 560 +- 9
Kg/cm2
0-150 mmHg
100 dB
50-100 lbs
Tension; active contraction
50-100 lbs
Fluid pressure and shear
120 mm Hg
Smooth, cardiac, and

skeletal myocytes
Mesangial
Tension
Fibroblast
Epidermal, fibroblast
Hair
Nerve/specialized muscle
While we know some of the forces that are applied to cells and tissues, as listed in the table
above, we know much less about how the forces are transmitted throughout the cell.
Quantifying cellular force is difficult because they are distributed among the many complex
structures within, including the cell membrane, cytoplasm, organelles, and especially the
cytoskeleton.
37
4.3. Uniaxial Stress and Strain

Perhaps the most fundamental property we would like to know for any material is its strength.
To measure this, we can take a representative specimen of the material, with a simple shape
such as a bar or rod, and pull it until it breaks. Thus applying constant forces, F, to both sides of
the bar shown in Figure 4.1, would translate into the stress,
=
were
F/ A, assu m in g th
distributed evenly over the cross-sectional area, A.
Figure 4.1
X
During constant stress, the bar, being deformable, will elongate in the direction of the F vector,
along the X direction and would shorten in the y direction. If the initial length in the X
direction, were Lo, the deformation could be measured as the strain:
x =
L Lo
Lo
The resulting stress-strain curve would typically look like one of the following:
The curve on the right
shows that for low
strains, the behavior is
linear, but begins to
deviate from linearity at
higher strain, reaching
a
maximum
stress
before curving down,
and rupturing. The
curve maximum is the
ultimate tensile stress
(UTS) and the point at
which
the
curve
becomes non-linear is
the elastic or proportional limit. The curve on the left is derived from the one on the right, by
correcting for changes in area during the test. The elastic region of the stress-strain curve
represents the stiffness of the material, defined as Youngs modulus:
Y=
x
x ; which is sometimes called the elastic modulus, E. Stiffness can be measured in both
tensile and compressive loads.
4.4. Two-Dimensional Stress

The simplest depiction of stress is in a bar or cube, as shown below. Much of the CSK is made
from bar or rod-like structures (ropes and struts), and hence experiences these types of stress:
38

Forces on a cube in 1 or 2 dimensions are analyzed as follows. Take the case of unconstrained
isotropic object compressed in the y direction:
Before strain
After strain
y
y
(Note that for an elastic material the strain occurs almost instantaneously upon
application of the stress. Also note that to maintain constant stress, y , the applied force must
be reduced if the face area increases, but this would be a negligible change for all practical
situations). The strain in the y direction is:
direction is unconstrained:
y =
Because the transverse
x = 0 and, x = y
Now, Consider the case where the x direction is constrained from movement, i.e. transverse
movement is resisted, making:
y
x = 0
To prevent strain in the x direction, a stress x must be applied:
x = x E = y E
In other words, to prevent transverse strain, you must push on the object with x. This causes an
added stress to the y direction, due to a tendency to strain:
' y = x = 2 y
and
' y = E 2 y = x
Thus the new stress in the y direction is the original unconstrained stress plus the stress caused
by transverse constraint:
39
y = E y + x
Solving for y we have the biaxial strain equation:
y =
1
( y x )
E
4.5
Types of mechanical loading
Measuring the material properties of engineering materials such as metal, ceramics, and
polymers is a straightforward and well established procedure: specimens are carefully shaped to
fixed geometries, and subjected to forces of various types, while the resulting deformation is
measured. The classical method is to take a small piece of the object under test, place it in an
Instron machine that applies tension, or compression, and torsion or bending forces as shown
below, and measure its deformation until it breaks. This procedure yields parameters such as
).
moduli of elasticity (E) and bending (M), ultimate tensile strength (UTS), and viscosity (
Figure 4.2 Types of forces.

(a) tension/compression; (b)
bending; (c) torsion; (d) shear.
To characterize an entire object by these tests on small pieces it must be assumed that the
material being isotropic in 3 dimensions, i.e., its material properties are equivalent in all
directions and locations.
4.6 Measuring cellular material properties
Material properties of cells are much more complicated than most engineering materials.
Firstly, most cells and their components are highly anisotropic Secondly, material properties are
not constant, and are likely to change during the test procedure. Unlike inanimate objects that
possess static properties, cells behave. Thirdly, cells and their components are generally not
materials, but are structures, whose properties vary depending on the type of force applied.
Despite the complexity and changeability of cell structure, mechanical measurements can yield
important information about cell functions. The challenge of cell mechanical measurement has
been met with many ingenious testing devices. The basic principle of testing is to apply a small
deformation to the specimen while measuring as accurately as possible both the force applied
and the deformation as it evolves over time.
The most straightforward tests involve directly poking the cell with small flexible probes. A
simple tool for applying nano-Newton sized poking forces to cells is the fine-tipped
40

micropipette. These can be readily made by drawing glass pipets under heat. Cells stiffness can
be roughly estimated by the bending angle of the micropipettes tip as it pushes against the cell
wall with a known force.
Devices for applying and measuring forces of < 1 pN and deformations of < 1 nM are now
available and continuously being improved. These new techniques can illuminate the processes
responsible for operation of cellular machinery, the forces arising from molecular motors and
the interactions between cells, proteins and nucleic acids. This topic will be further detailed in
the next chapter.
A depiction of stress-strain
testing is shown. Note the
shaded cross-sectional area in
the middle of the bar indicates
that stress is assumed to be the
same in the entire cross
section of the bar:
Figure 4.3. A bar

under tension
Uniaxial stress, as in Figure 4.3, also causes deformations in the other 2 Cartesian coordinates,
Y and Z, and can be quantified by Poissons ratio:
y = y = x
y
y
41

For example, in 2 dimensions, assume the bar shown in Figure 4.3 above was originally a 1 by 1
square, and was stretched to a rectangle 2 by 1. Strain in the x direction is 1, or 100% , while
strain in the y direction is 0.3 or 30 %. Poissons ratio is then 0.3. Thus the area increased from 1
to 1.4. Since the area, and also volume, of the bar dilate, while the mass does not change, it must
be that the material density decreases during deformation. Note that since strain in the nonstressed directions are almost always opposite in sign to that in the stressed direction, the
negative sign for the strain ratios, , is usually a positive number.
For most engineering materials, < 0.3
Materials with = 0.5 are "Incompressible."
Some materials have > 1
v =-(.7-1)/1 = 0.3
Figure 4.4
Y
1
0.7
A special case for Poissons ratio is = 0.5, meaning that the material does not change crosssectional area (nor volume) when being deformed. This type of material is called
incompressible. Note that being incompressible does not mean that it is not deformable. While
most engineering materials have = 0.3 or so, the few biological materials that have been tested,
have = 0.5 or greater. Bulk muscle, for example, can have > 1. This can be seen by
contracting your biceps- a very small shortening, i.e. axial compression, can produce a much
larger expansion as the muscle bulges upward. This indicates that muscle volume drastically
increases during contraction. Note that individual muscle fibers have much closer to 0.5, so
that the physical arrangement of the bulk muscle is the cause of the large increase.
Another special case for Poissons
ratio is for a cylinder, as shown
below. For this case, we have
y = y = x
y
y
as before, and
and
so = 1
4
y=
x
E
= . (You finish.)
42

4.7
Material Stiffness & Strength
To begin mechanical analysis of the cell, we need to begin cataloging its mechanical properties.
This chapter will present material properties of selected cells and components. Comparing just
the elastic properties of cells with other materials, it is seen that cells are much more compliant,
and have a pre-stress at zero strain, as shown in Figure 4.5 below:
Comparative Mechanical
Properties
Stress
Steel
Figure 4.5
Wood
Bone
Steel Wood Bone
Cells
Strain
Cells
Cellular
pre-stress
A more quantitative look at cell (tissue) elasticity is charted in Figure 4.6:
Modulus (GPa)
Note that mechanical testing of cells is

Comparative Stiffness
much less standardized compared
with solid, engineering materials, as
10000
discussed in Chapter 5. Deformation
1200
of cells, even those as simple as the
210
human red blood cell, can be properly
100
21
analyzed only using continuum
14
models. This constitutive behavior
includes
multiaxial
stressstrain
1
relations and changes of mechanical
properties with time and/or in
0.007
response to biochemical or electrical
0.01
stimulus. However, most living cells
0.0002
are even more complex, so that
0.0001
deformation models must account for
internal structure, spatial granularity,
heterogeneity and the active features
unique to cells. For example, the Figure 4.6
Material
shape, motility and mechanical
properties of tissue cells depend largely on cell cytoskeleton, which is a dynamic system
undergoing structural changes during cell spreading and rounding or due to mechanical or
chemical signals. Thus the material properties of cells are highly variable and difficult to
quantify.
43

The resistance of single cells to
elastic
deformation,
as
quantified by an effective elastic
modulus, ranges from 102 to 105
Pa (Figure 4.6), orders of
magnitude smaller than that of
metals, ceramics and polymers.
The high degree of elasticity and
deformability
of
cells
is
illustrated by the red blood cell
in Figure 4.7, undergoing
strain > 100%: (How is this
done? See class notes).
Figure 4.7
Note from the continuum model, strain magnitudes are highly anisotropic, and are greatest at
the sites of attachment. Also note, due to the complex shape, it is difficult to calculate Youngs
modulus, but the bulk cellular modulus can be estimated assuming a nominal cross-sectional
area for the cell.
4.8 Estimating Cell Stiffness (adapted from Lee, Knight et al. 2011)
In a recent study, the mechanical properties of MSCs were assessed using micropipette
aspiration in their undifferentiated state and during osteogenic and adipogenic differentiation
[Yu et al., 2010]. The authors report values for instantaneous and equilibrium Youngs moduli
for the undifferentiated cells of approximately 450 and 100 Pa, respectively. These values rise
significantly and progressively during osteogenic differentiation over a 21-day period to reach
values approximately twice that of the undifferentiated cells. These changes are closely
associated with alterations in the cell size and morphology, and with alterations in the
cytoskeleton. Indeed a previous study demonstrated a significant reduction in the modulus of
MSCs following disruption of the actin cytoskeletal organization [Tan et al., 2008]. Embryonic
stem cells have also been studied using micropipette aspiration, with results suggesting a
marked stiffening of the nucleus during differentiation, associated with the nuclear skeletal
component Lamin A/C, which is only expressed in differentiating cells [Pajerowski et al., 2007].
These findings suggest that both embryonic stem cells and MSCs may be more deformable than
their differentiated progeny. This physical plasticity may facilitate the migration of stem cells
through solid tissues to the sites of tissue damage.
44
Wall stress in a thick sphere

Ro
To find equilibrium
forces:
Fup = Fdown
h
P
Ri
.
Ri
Spherical Stress
A stressed, thin walled
sphere experiences a wall
tension, which in the case
of a cell, is considered,
membrane tension. The
free-body diagram of a
stressed sphere is shown
below.
To find the
membrane tension, T, due
to internal pressure,
a
simple sum of forces will
lead
to
the
LaPlace
equation P = 2T/R
Figure 4.8
Ro
4.9 Mechanical Properties of CSK components

Elastic moduli
The deformability of cells is determined largely by the cytoskeleton, whose rigidity is influenced
by the mechanical and chemical environments including cellcell and cellECM interactions.
Properties of selected polymeric components of the CSK, and other polymers for comparison,
are shown below.
Polymer
Actin
Tubulin
Intermediate
Filaments
Silk
Collagen
filament
Collagen fibril
Elastin
Cellulose Dry
Cellulose Wet
Spectrin
DNA
Typical
Diameter
(nM)
8
25
10-20
Table 3.1
Persistence
Length, p
(
m )
15
6000
Elastic
Modulus, E
(Gpa)
2
2
Mass
Density, p
(Da/nm)
110
160
1.5
10-300
0.02
0.05
0.002
80
40
0.002
1
4500
1900
Note that the 2 major CSK components have equal moduli of 2 GPa, but this is 3 orders of
magnitude greater than spectrin, the major CSK component of RBCs. Since spectrin is much
more dense than either actin or tubulin, this means that its stiffness to weight ratio is very small.
45

Elastin is a major component of skin that imbues it with a high degree of deformability, as
illustrated below:
Figure 4.9.
Strain > 100%
A qualitative comparison of a highly compliant polymer like elastin with a stiff one like collagen
is shown in Figure 4.10 below. Note that stiffness of both polymers increases drastically at
large strains. Also indicated in the figure, is that the stiffness depends on the strain rate, so that
collagen can behave more like elastin when stretched at an increased rate. This viscous behavior
will be further discussed in Chapter 7.
Figure 4.10
Low elastic modulus, however, does not imply high

deformability, since RBCs lyse at strains > 3% (and
skin tissue is not directly comparable to RBCs):
Another related issue is that strength, or UTS, is not

directly related to stiffness, as seen in the chart
below:
Figure 4.11
46

Selected stiffness and strength values are presented in the table below. Note that silk is stronger
than steel:
Table 4.2
4.9
Flexural Moduli
Since the polymers that make-up the CSK are bar or rod-like structures (ropes and struts), they
can and do experiences all types of stresses as shown in Figure 4.1. For rods, like polymers,
the most important mechanical mode is bending. Suppose a thin rod of length L is initially
upright at zero temperature, but when warmed is bent under the influence of gravity into a
curvature with radius R:
bend
Figure 4.12
L
R
The rod has bent out of its natural shape, when R = , under conditions of zero temperature
and stress. In this case, we can imagine that the stiffness of the rod determines its curvature,
but it cannot be the same stiffness, Y, describing resistance to uniaxial stress. A new measure of
stiffness is therefore required, the flexural rigidity. For uniform rods, flexural rigidity is:
f = IY
I=
R 4
4
Thus the flexural rigidity is based on Youngs modulus multiplied by the geometric parameter
for rods, the area moment of inertia, I. To determine the amount of deformation of the rod, we
47

now need to know how much force is applied. Since we are dealing with molecules, not big rods,
the forces must be considered at the molecular level, and this is where the energetics of the
situation must be considered. This is where Boltzmanns relationship (Chapter 6) is needed.
Energy, Earc, expended by (or to) the rod to bend into a given arc, R, is proportional to the
flexural stiffness and to the amount of bend. For example, when R = , a straight rod, Earc will be
0. With a large bend, Earc will be large. It can be seen that the ratio, L/R, is roughly proportional
to Earc . In fact, elastic theory predicts that:
E arc = f
L
2R2
In order to derive a useful description of how bent a given rod would be at a given temperature,
Boltzmanns thermal formula, 3/2KT, the average energy per molecule, is needed. For this, a
new parameter is defined, the persistence length, which represents the length over which the
rod will be fairly straight. This length, p , is defined as the magnitude
f/ KT. To better
understand the meaning of p , consider the case when L = p and Earc=KT. In other words,
the bending energy just equals approximately the average energy of one molecule at
temperature T. In this case, we can substitute the conditions:
E arc =
k 2f
= kT = 4.1 pN nm
2 R 2 kT
Solving for R, we see that:
R=
p
R
Please note that the persistence

length, p, is identical to p
p
2
2radians = 81deg rees
Table 4.3
Thus a rod of the persistence length is
curved at 81 degrees when the thermal
energy scale reaches kT. If L << p
then the rod is relatively straight,
otherwise not. Persistence length sets
the scale of thermal fluctuations.
Filaments with contour length >>
p
are highly convoluted and can assume
many configurations.
Persistence
lengths for selected polymers are listed
in Table 4.2.
Formulae related to bending
polymers are listed in Table 4.3:
of
48

4.10 Measuring Local Stiffness
In general, the mechanical properties of homogeneous materials are determined by applying
stresses and measuring the resultant strains (or vice versa). In the regime of linear response, the
ratios between stresses and strainsthe viscoelastic moduli (or compliances)are material
properties that may show some frequency dependence. They may alternatively be determined
from the spectrum of thermal fluctuations. Macroscopic rheological methods are widely used
across many disciplines, but the softness of cytoskeletal networks, their limited availability, and
the need to resolve the mechanical properties of their local heterogeneities have motivated the
development of micro-rheological methods.
In the case of ordinary micro-rheology, observing the induced motions of suspended nonmagnetic beads yields the deformation field, and thus enables a more-detailed investigation of
local heterogeneities and stress propagation. This method (pictured), which is based on a
combination of active field-driven particles and passive tracer particles, was more recently
developed into a purely passive technique, now widely known under the name of two-point
micro-rheology. The thermal motion of single particles is determined by either video microscopy
or high-speed inter-ferometric detection. Assuming a generalized StokesEinstein relation, the
frequency-dependent moduli can be extracted. Depending on the bead radius and detection
limit, moduli from 103 to 102 Pa are measurable.
Figure 4.13
When microrheological techniques were introduced, discrepancies between the measured

network elasticity and macroscopically determined moduli were poorly understood. But it has
since been shown that the local compliances depend on the size ratio of the colloidal particle and
on some characteristic length scale of the network or of its constituents. Paradoxically, a detailed
theory of the micro-rheometry for such heterogeneous networks seems to require a complete
understanding of the network mechanics that are to be studied by this method.
Figure 4.14
49

Here is an example of testing regional elasticity in a cell:
The picture at left shows the cell with several beads inside that have been colored from blue to
red, indicating local stiffness.
At right, a close-up of the cell shows records of particle
movement over time. Note that the upper particle traversed a relatively tight area, during the
same time that the lower particle moved over a much wider area. These random motions
indicate how stiffly the beads were trapped within the cytoskeletal network, and hence indicate
local stiffness.
By measuring the mean squared displacement of a particle <Dr2 > over a period of time,
estimate of stiffness can be estimated as
G=
an
D
Dr 2
where G= Youngs modulus, D= diffusion constant of the cytoplasm, and = viscosity of the
cytoplasm .
In the case of active microrheology, magnetic forces are applied and from the resulting
displacements (in and out of phase) of magnetic particles in the system, the frequencydependent susceptibilities are determined. These may be transformed into local moduli for a
more direct comparison with the macroscopically measured moduli.
50

4.11 Buckling
Straight rods subjected to axial loads can buckle, as depicted below. The forces, P1, P2, and P3,
required to buckle each loading configuration are shown:
Figure 4.15
P1 =
2 EI
L2
P2 = 4
2 EI
L2
P3 =
4L2
2 EI
Chapter 4
1. Estimate an elastic modulus for the RBC in Figure 4.7 . State assumptions.
2. Human hair has a stiffness of 4 X 1010 dynes/cm2 . When axially strained, it undergoes
brittle breaking at a stress of 106 dynes/cm2 (there is no ductility in hair). What is the
radial strain? Assume Poissons ratio is close to that of steel, and hair length similar to
your own. Show assumptions. (Note, hair is cylindrical).
3. Calculate the energy (in terms of KT) required to bend a microtubule, 20 cm long, into a
curve with a 10cm radius? Assume the persistence length to be 2 mm.
4. Now, for the microtubule in #5, calculate the buckling force.
5. Derive Laplace law for a sphere.
6. Model 4:
References
51

Ingber, D. E. (2010). "From Cellular Mechanotransduction to Biologically Inspired
Engineering." Annals of Biomedical Engineering 38(3): 1148-1161.
This article is based on a lecture I presented as the recipient of the 2009 Pritzker
Distinguished Lecturer Award at the Biomedical Engineering Society annual meeting in
October 2009. Here, I review more than thirty years of research from my laboratory,
beginning with studies designed to test the theory that cells use tensegrity (tensional
integrity) architecture to stabilize their shape and sense mechanical signals, which I
believed to be critical for control of cell function and tissue development. Although I was
trained as a cell biologist, I found that the tools I had at my disposal were insufficient to
experimentally test these theories, and thus I ventured into engineering to find critical
solutions. This path has been extremely fruitful as it has led to confirmation of the
critical role that physical forces play in developmental control, as well as how cells sense
and respond to mechanical signals at the molecular level through a process known as
cellular mechanotransduction. Many of the predictions of the cellular tensegrity model
relating to cell mechanical behaviors have been shown to be valid, and this vision of cell
structure led to discovery of the central role that transmembrane adhesion receptors,
such as integrins, and the cytoskeleton play in mechanosensing and mechanochemical
conversion. In addition, these fundamental studies have led to significant unexpected
technology fallout, including development of micromagnetic actuators for non-invasive
control of cellular signaling, microfluidic systems as therapeutic extracorporeal devices
for sepsis therapy, and new DNA-based nanobiotechnology approaches that permit
construction of artificial tensegrities that mimic properties of living materials for
applications in tissue engineering and regenerative medicine.
Lee, D. A., M. M. Knight, et al. (2011). "Stem Cell Mechanobiology." Journal of Cellular
Biochemistry 112(1): 1-9.
Stem cells are undifferentiated cells that are capable of proliferation, self-maintenance
and differentiation towards specific cell phenotypes. These processes are controlled by a
variety of cues including physicochemical factors associated with the specific mechanical
environment in which the cells reside. The control of stem cell biology through
mechanical factors remains poorly understood and is the focus of the developing field of
mechanobiology. This review provides an insight into the current knowledge of the role
of mechanical forces in the induction of differentiation of stem cells. While the details
associated with individual studies are complex and typically associated with the stem cell
type studied and model system adopted, certain key themes emerge. First, the
differentiation process affects the mechanical properties of the cells and of specific
subcellular components. Secondly, that stem cells are able to detect and respond to
alterations in the stiffness of their surrounding microenvironment via induction of
lineage-specific differentiation. Finally, the application of external mechanical forces to
stem cells, transduced through a variety of mechanisms, can initiate and drive
differentiation processes. The coalescence of these three key concepts permit the
introduction of a new theory for the maintenance of stem cells and alternatively their
differentiation via the concept of a stem cell 'mechano-niche', defined as a specific
combination of cell mechanical properties, extracellular matrix stiffness and external
mechanical cues conducive to the maintenance of the stem cell population. J. Cell.
Biochem. 112: 1-9, 2011. (C) 2010 Wiley-Liss, Inc.
52

CHAPTER 5: CYTOMECHANICAL TOOLS
5.1
Background
(with acknowledgement to Bao and Suresh 2003; Eyckmans, Boudou et al. 2011)
Understanding how cells generate and sense forces and how the forces are sensed and
transduced into biochemical signals is vital to address fundamental questions about cell
behavior in both normal and pathological states. Accurate measurement of forces and
displacements exerted by cells both in vivo and in vitro is an essential step in this endeavor, and
a foundation of mechanobiology. The development of mechanobiology as a field has been
enabled by new visualization and measurement technologies. For example, the earliest
observations suggesting that mechanical forces drives embryogenesis and bone structure were a
natural result of newfound microscopy methods. Mechanobiology received relatively little
attention for much of the 20th century as scientists focused on developing molecular biology
tools to catalog the genetic basis for life. The recent renaissance in studying mechanics primarily
in cell culture has largely been enabled by a suite of tools to measure and manipulate such forces
in vitro. For example, in vitro application of strains that would be experienced by bone during
physical activity increases proliferation and differentiation of bone cells, and bone matrix
deposition, which are all characteristic for mechanically induced anabolic bone growth in vivo.
Mechanical stretch that mimics the effect of pulsating blood flow has been shown to trigger
many alterations in endothelial and smooth muscle cell signaling, vascular cell proliferation, and
expression of inflammatory markers.
Methods to apply shear flow on cell cultures have shown that shifts between steady and
turbulent shear flow can prevent or promote inflammatory activation, respectively, and may
explain the localization of atherosclerotic plaques to specific regions along the vascular tree.
Flow rates during development also can drive arterial versus venous phenotype. Bioreactor-type
devices have been developed also to model the compression experienced by chondrocytes in the
articular joint, tension in muscles, ligaments, and tendons, as well as impact forces associated
with trauma. In addition to such externally applied forces, non-muscle cells generate contractile
forces on their own. This was first illustrated by Harris and Stopak, showing that cells cultured
on soft polymer substrates would wrinkle the substrate surface, implication that forces could be
ever present, even in settings without an explicit mechanical stimulus, seemed heretical at the
time. However, over the past three decades, it has become clear that (1) most eukaryotic cells
can generate intracellular forces that act on the surrounding extracellular matrix (ECM) or
neighboring cells, and that (2) this contractile activity is critical for a number of biological
processes such as cell migration, mitosis, as well as stem cell differentiation and self-renewal.
For physical functions such as mitosis and migration, force is clearly essential in the same way
that oxygen is essential for life. That is, without force, mitosis and migration cannot proceed.
The role of force in genetic responses such as proliferation and differentiation, however, appears
to be regulatory in the same manner as cytokines might be. Although we have some clues to
how forces exert these regulatory functions, clarifying these mechanisms remains a central
question for mechanobiology.
5.2
Measuring and Manipulating Cellular Forces
Testing mechanical properties of engineering materials such as metal, ceramics, or polymers, is
a snap: simply place a specimen in an Instron machine and stretch or compress it in various
ways until it breaks. Testing cells is not so easy. Cells are soft, complex, non-linear, and
changeable. Unlike inanimate objects, that possess static properties, cells behave. From
previous chapters it should now be clear that the cell is not like a hard ball, but like a thin, elastic
53

shell filled with an incompressible fluid, whose fundamental properties can change with
circumstance. The challenge of cell mechanical measurement has been met with many
ingenious testing devices.
Figure 5.1. Testing cell mechanical responses
Perhaps the simplest tests involve directly poking the cell, as depicted in Figure 5.1 above. A
simple probe for applying small nano-Newton level poking forces to cells is a fine-tipped micropipet. When the tip bends as it pushes on the cell, the degree of bending indicates at least
qualitatively the cell stiffness. So the probes used are more refined than a hammer, but the
principle is the same: apply a small deformation to the specimen while measuring as accurately
as possible both the force applied and the deformation as it evolves over time.
Mechanically probing single cells and even biomolecules is becoming increasingly sophisticated.
Devices for applying and measuring forces < 1 pN and deformations < 1 nM are available now.
Combining these devices with the latest imaging, molecular biology, and bioinformatics,
including modeling and simulation has led cytomechanists to revolutionize our view of the cell.
These new techniques can illuminate like never before the processes responsible for operation of
cellular machinery, the forces arising from molecular motors and the interactions between cells,
proteins and nucleic acids.
Several methods have been developed to determine the mechanical characteristics of the cell
and to understand how those characteristics change in health and disease. An innovative
apparatus was the cell poker, which was able to measure the stiffness of living cells by
applying an axial strain via a cantilever. This apparatus was also used to demonstrate that
neutrophils may be retained in capillaries in the acute inflammatory response due to an increase
in stiffness caused by chemoattractants.
Another method that has found wide use is atomic force microscopy (AFM). Intrinsic
mechanical properties of the cell have been found by applying continuum mechanics models. .
Mathematical models have also been applied to endothelial cells to understand how force is
transmitted from their apical to basal membranes by combining the AFM with total internal
reflection fluorescence microscopy.
One of the most widely used methods for studying cellular mechanics is the micropipette
aspiration technique. An adaptation of the solution for a punch problem has been used to obtain
mechanical properties of bovine aortic endothelial cells, as well as to compare normal and
osteoarthritic human chondrocytes.
The viscoelastic behavior of cells has also been studied using magnetic bead micro-rheometry.
Creep response and relaxation curves were obtained with this technique by applying tangential
force pulses on magnetic beads fixed to the integrin receptors of the cell membrane, as shown in
section 5.3 below.
The aforementioned testing methodologies can measure cellular structural properties and help
us understand various biological processes, however, there is currently no technique that can
perform stress-controlled indentation testing on single adherent cells. The application of
54

controlled and well defined axial stresses to an anchorage-dependent cell has physiological
relevance in several cases and may prove vital in understanding how mechanical forces
influence cellular behavior. For example, articular chondrocytes are anchorage- dependent cells
that attach to their surrounding extracellular matrix and experience many forces in vivo,
including compression.
5.3
Measuring Cell-Generated Traction Forces
Cells are constantly probing, pushing, and pulling on their microenvironment. A growing body
of work is investigating what regulates the cellular generation of contractile forces. To identify
when such forces are invoked, several approaches have been developed that allow quantitative
measurement of cellular traction forces. In addition, numerous methods are being used to
manipulate cell-ECM forces, all of which have been critical in enabling the growing field of
mechanobiology. In this section, we will examine the tools used to measure and manipulate
cellular forces, and how they have contributed to our understanding of the role of mechanical
events in both physiological and pathological settings.
Owing to the evolution of biomaterials and polymer chemistry, the concept of soft substrates
linked with ECM matrices has enabled the measurement of forces generated by even single cells.
The basic concept of using an elastic substrate for force measurement was originally conceived
by Harris et al.: when adherent to a thin silicone membrane, non-muscular fibroblasts can cause
wrinkles in the substrate (Harris et al., 1981). However, due to the inherent nonlinearity of
wrinkling and the complexity of the displacement field generated by a single cell, this technique
was not applicable to accurately quantify cell forces.
The development of traction force microscopy by Dembo and Wang has been a significant
improvement to measure cellular forces. This method uses fluorescent microbeads embedded in
a polyacrylamide hydrogel as markers for tracking the deformation of the gel caused by the
adherent cell. After obtaining the displacement vector for every bead, the inverse problem is
solved to calculate the cell generated force field . With this technique, we can see that in addition
to tangential forces, cells also exert forces normal to the 2D planar adherent surface. However,
the calculation of forces is computationally intensive, because deformations propagate on these
continuous substrates. Furthermore, changing the crosslinking chemistry to manipulate the
rigidity of hydrogels may inadvertently affect surface hydration, chemistry, and adhesiveness.
To address these limitations in the design of soft substrates, microfabricated arrays of
elastomeric cantilever posts have been developed to quantify cell forces by measuring the
deflection of the posts under cell tension.
The above two force measurement techniques have demonstrated that (1) cells pull harder on
stiffer surfaces, affecting cell shape, motility, growth rate, and intracellular signaling and that
(2) adherent cells continuously apply tensile forces to substrates that are directed toward the
centroid of the cell. Similar observations have recently been reported in cells embedded in a 3D
hydrogel, suggesting that mechanotransduction mechanisms might be conserved between 2D
and 3D.
These methods whereby cells cause strains on their substrate, are critical in identifying a role for
cellular forces. For example, it had been observed that cells restricted from spreading against
extracellular matrix become growth arrested, regardless of whether that restriction is due to cell
crowding upon reaching confluence, decreased ECM ligand coating density on a surface, or
micro-patterning to define the area of spreading of a cell. These changes in cell spreading were
later found to impact cell contractility. Restricting cell spreading on micro-post arrays revealed
that decreased spreading prevented cells from generating traction forces (Tan et al., 2003).
55

Conversely, up-regulating contractility by activating RhoA rescued proliferation even in unspread cells, thus demonstrating that the mechanism by which cell shape regulates contractility
is mechanotransduction.
5.4
Manipulating Cell-ECM Forces
Intracellular forces can be modulated by using traditional molecular methods to directly target
the force-generating apparatus. Several pharmacologic agents are available for inhibiting
modulators of contractility, including the molecular motor myosin II (blebbistatin), the
upstream regulators of myosin phosphorylation Myosin Light Chain Kinase (ML-6, ML-9), and
the Rho/ROCK signaling pathway (fasudil, Y27639, C3 botulinum exotoxin), as well as the
polymerization processes of actin (latrunculin, cytochalasin D). Similarly, molecular-genetic
methods have also been used effectively to target these pathways, including the nonmuscle
myosin II isoforms themselves, and have been a mainstay in identifying force in a regulatory
pathway.
In addition to molecular methods, there are biophysical approaches to manipulate cell-ECM
mechanics. One such method is to vary the mechanical stiffness of the substrate to which cells
attach. Most commonly, substrate stiffness is manipulated by changing the degree of
crosslinking of polymeric hydrogels, the most widely available of which is polyacrylamide.
Depending on the chemistry, crosslinking is controlled by the ratio of polymer to crosslinking
agents, the duration of exposure to a light source (photopolymerization) or heat, among other
factors. Interestingly, whereas the stiffness of most substrates is determined upfront, substrate
stiffness of collagen can be altered during the course of the experiment by supplementing ribose
to the medium (Girton et al., 1999).
The role of ECM stiffness in regulating cell function is now well known. ECM stiffness has been
shown to affect migration, proliferation, and differentiation. Whereas increasing stiffness
enhances spreading and proliferation of many cell types and facilitates tumor growth, compliant
substrates appear to promote branching of neurons or adhesion of and albumin secretion by
hepatocytes.
Measuring mechanical properties of a cell or any material requires applying some kind of force
to it and recording the deformation response. Force can be applied in many modes: tension or
compression, uniaxial or biaxial, pure shear, hydrostatic or pneumatic pressure, bending,
twisting, and a combination of these. For any of these modes, varying the timing of also helps
characterize the material: for example force can be applied suddenly, slowly or rhythmically,
with sudden impulse or steps of force, ramps, or sinusoids. The common waveforms used to test
mechanical behaviors, both linear and non-linear, are depicted in Figure 5.2 below.
Figure 5.2. Types of
force application
Uniaxial
Shear
Pressure
Biaxial
Tension or
Compression
Devices that apply these forces can be conveniently

classified into three types (Figure 5.3): Type A,
whereby local probes load or stretch a portion of a
single cell; type B, mechanical loading of an entire cell;
and type C, simultaneous loading of a population of
cells.
Bending
Twisting
56
Figure 5.3. Classes of Mechanical Testing
F1
Type A
F2
P
F1
Type C
F2
Type B
Several Type A methods are available, as depicted in Figure 5.4 below:
Figure 5.4.
Type A Cell
testing
modes
Bao & Suresh
In addition to the modes of cell contact, variations in the type and timing of stimuli are possible,
as depicted in Figure 5.5 below:
57

Impulse
Step
Sinusoid
Ramp
Magnitude
TIME
Figure 5.5. Modes (top) and timing protocols (bottom) of force application
Some of the measurement techniques are further described below.
5.5
Atomic Force Microscopy (AFM)
In AFM, a sharp tip at the free end of a flexible cantilever (Figure 5.6) generates a local
deformation on the cell surface. The resulting deflection of the cantilever tip can be calibrated
to estimate the applied force. This is a high-resolution technique that traces the fine structure of
nanometer level objects. The tip can be conjugated with an antibody that binds to the cell
(functionalized) and then it can be used to pull on it with calibrated force.
Pull force
Bond to CSK
Cell surface
(Alenghat, Fabry, Tsai, Goldmann, Ingber)
Figure 5.6. Atomic

Force Microscopy
The figure at left (above) is a computer model of the mechanical deformation of a cell by the tip
of an AFM cantilever. The model is based on continuum mechanics, and it includes
contributions from the elasticity of the cell membrane as well as the interior of the cell. An
example of the type of image that AFM can produce is shown in Figure 5.7 below:
Figure 5.7.
AFM image of a
living cell. Bar=
5 microns
58

Single cells can exert large contractile forces that affect their shape. These forces exerted by cells
can be measured with AFM, as shown in Figure 5.8.
Figure 5.8. An osteosarcoma cell attached between the cantilever of an atomic force
microscope and a surface can exert contractile forces (red arrow) of more than 100 nN
(depicted from left panel to center panel and in a fluorescence micrograph, right panel). Actin
filament structures (white), including contractile stress fibres spanning the upper and lower
surfaces, are generated in the contracting osteosarcoma cell (right). Scale bar, 10 m.
5.6
Magnetic Tweezers
Magnetic beads, a few microns diameter, can be either incorporated into cells, or attached to
specific receptors. An illustration of pulling on a cell with a magnet is shown in Figure 5.9.
B Field
Magnetic
bead
Figure 5.9. Magnetic bead

stretching (Wang et al., Science)
Membrane
In this case, the bead has been incorporated into the cell, and is pulled with an external
magnetic field. Calibrated stretch of the membrane can be thereby done. A more specific force
can be applied to individual molecules using functionalized magnetic beads. These have been
coated with antibodies for specific CSK components such as Integrin. For example, when coated
with fibronectin, the beads will bind to Integrin. When the beads bind, an external magnetic
field can be applied to twist the bead, measuring the response of the CSK portion directly, as
shown in Figure 5.10.
By using a magnet to pull and twist individual molecules on a cell, this technique represents
magnetic tweezers. These magnetic-twisting studies confirmed that mechanical forces are
Figure 5.10. Pulling on CSK
(Wang et al., Science)
59

transmitted only over specific molecular paths, in particular, the Integrin hooks. When the
beads were bound to non- CSK receptors, they could not effectively convey force to the inside of
the cell. Highly specific molecular "adhesives" were attached to the cells in order to show that
tugging on particular receptors at the surface of a living cell triggers nearly instantaneous
rearrangements in the nucleus.
5.7
Micropipet Aspiration
This technique involves sucking up a small portion (patch) of a cell into a micropipet, while
measuring the deformation and pressure force. The geometry and kinematics of the situation
shown below have been worked out.
Note that the cartoon of Figure 5.11 shows a highly simplified view of what happens to the CSK
during aspiration. The pressure clearly deforms it, and by measuring the force required to pull it
up the tongue of cell a given length, a measure of stiffness can be obtained. It is also apparent
that the deformation is complex, being neither uniform nor isotropic, and applies tension,
compression, as well as shear simultaneously.
Micropipet aspiration can be either type A or B (Figure 5.3), depending on conditions. If the
micropipet is tightly sealed to the cell, there is no movement of the main body of cell into the
pipet, and hence only the patch is deformed- this is type A. If there is no seal and no friction
between the cell and the pipet, then the whole cell gets squeezed into the pipet, and it is a type B
stimulus.
Figure 5.11
Aspiration
Negative Pressure
Drawing of Micropipet
Aspiration
Magnified view
of cell patch
showing CSK
60

Another use of micropipets is to gauge the force produced
by a growing cell. At right, you see a photograph of a pipet
that has attached to the growth cone, and is being pushed
forward by it.
Figure 5.13. Pipet attached to a growth cone
5.8
Cell Moduli from Pipet Aspiration (Lee et al.)
The micropipette aspiration technique can measure stiffness of an individual cell with a known
aspiration pressure (see Fig. 5.10). The technique has been widely used to determine the
mechanical properties of a variety of cell types including stem cells [Hochmuth, 2000].
Micropipettes, with inner diameters typically between 5 and 15 mm, are used to induce
deformation of the whole cell, or alternatively discrete regions of the cell, depending on the cell
diameter. Moreover, the properties of subcellular components, most notably the nucleus and
cytoskeleton may be investigated. With the control of a hydraulic micromanipulator, the
micropipette is moved into contact with the cell surface and pressures applied and the cell
visualized using bright field or fluorescence microscopy.
Two approaches have been used for performing the aspiration technique. For the incremental
approach, pressure is applied typically in steps up to 5 cmH20 (0.49 kPa), with the equilibrium
cell aspiration length, L, recorded at each pressure. The pipette internal diameter is given by a .
The apparent Youngs modulus may then be determined using a theoretical model [Theret et al.,
1988]. In this model, the cell is assumed to be a homogeneous, elastic half-space material and
the Youngs modulus, E, is therefore given as follows, where () is defined as the wall function
with a typical value of 2.1:
(1)
Here, Youngs modulus can thus be determined from the slope of the linear regression of the
normalized length, L/a, versus the negative suction pressure P.
For the alternative approach, in which pressure is applied in a single step, the following
equation is fitted to the temporal changes in aspiration length measured experimentally (Eq. 2).
This model assumes that the cell behaves as a homogenous linear viscoelastic three-parameter
solid half-space.
(2)
The relaxation time constant t is defined as follows (Eq. 3).
(3)
61

The parameter k1, is termed the equilibrium or relaxation modulus (Er or E), k1+k2 is the
instantaneous modulus (Ei) and is the apparent viscosity. Using this viscoelastic model it is
(4)
also possible to determine the apparent equilibrium Youngs model given by:
It should be recognized that although these models benefit from being simple, they neglect
geometrical factors, such as finite cell dimensions, evolution of cell-micropipette contact region
and curvature of the micropipette edges. Thus, other models incorporating these geometric
factors into a computational form have been developed [Haider and Guilak, 2000], which can
also account for the heterogeneity in cellular properties.
5.9
Nuclear Probing
Micropipets can even be used to probe into sub-cellular structures, such as the nucleus as shown
below. This sequence of pictures shows the pipet drawing a single chromosome into the pipet
and gradually pulling it out of the nucleus. This remarkable experiment revealed the surprising,
and still unexplained, phenomenon that all the chromosomes and the nucleoli are connected
together by strings of DNA.
Micropipets have also been used to measure the strength of single chemical bonds that a cell
makes with its matrix. The pipet grabs onto the cell like a leech, and then can pull with a
calibrated force until the bond breaks (Evans). With a manually 0perated micromanipulation
device, the micropipette is moved "like a golf club" to move the beads about 10 micrometers a
second, while monitoring the cell with the video microscope.
Figure 5.14.
Pulling out the
chromosomes
5.10 Whole Cell Inflation

Micropipet aspiration can also be used to test the entire cell, as depicted in Figure 5.14. A
direct porthole connection to the entire cell made when sufficient suction is applied to break the
membrane and CSK break open. Thus the cell can be inflated with known forces, while its
diameter is measured and its surface area estimated. The whole cell swelling technique is
powerful because the micropipet can control the internal millieu, including both the cytosol
composition and the electrical potential, as depicted in Figure 5.15 below.
The total osmolarity of the cell and its external environment is important since slight imbalance
can cause large osmotic forces for swelling or shrinking. In fact, using vanHoffs formula for
osmotic pressure (See Appendix) indicates that for every 1 mOsm difference in salt
62

concentration across the cell membrane, there is a transmembrane pressure gradient of 16.7 mm
Hg.
Note that the internal and external
Figure 5.15
A. Whole Patched Cell
osmolarities are labelled, C, flows of
water are labelled Q, and pressure P. Cell
stretch can be applied by applying
Micropipette
Pp
pressure from the pipet (panel A). Cells
Qpp
can also be stretched by exposing them to
Micropipette
hypotonicity, as shown in panel B.
5.11 Optical Tweezers
Another type A or B method involves
optical tweezers or a laser trap, in which
an attractive force is created between a
dielectric bead of high refractive index
and a laser beam, pulling the bead
towards the focal point of the trap. To
deform a single cell, 2 microbeads
(typically 1 m to several micrometers in
diameter) are attached to opposite sides
of it and one or both beads are trapped by
the lasers, and then pulled apart (Figure
5.16). This technique relies on a high
power laser trapping the bead, which
involves substantial heating of it. In order
not to heat the cell also, the laser beam
must be much smaller than the size of the
bead. By functionalizing the bead, it can
bind to specific molecules, allowing the
probe to apply force to specific bonds.
Stretch
Tension
Pi
C o
Qm (K w)
Ci =constant
B. Isolated Cell
Stretch
Solutes
Pi
Solutes
C i (t)
Qm (Kw )
Trap
Bead
Actua
tor
Bead
Mesangial
Cell
C i
Mesangial
Cell
C o
Trap Bead
RNA
Magnified
View
Handle
Force
Actuator
Bead
Figure 5.16. Optical Tweezers
63
Optical tweezers offers several non-contact ways of driving cells. While early optical tweezers
simply trapped and moved particles, it is possible to spin particles or cells at high speeds and
also to orient them in separate traps and then join them together in lock-and-key assemblies. In
a levitation trap the laser beam balances the pull of gravity. To optically trap a particle in
three dimensions it is necessary to exert a longitudinal force in the same direction as the laser
beam and a transverse force at right angles to the beam. The transverse force is created by
having the maximum laser intensity at the center of the beam. If the particle is to the left, say, of
the center of the beam, it will refract more light from the right to the left, rather than vice versa.
The net effect is to transfer momentum to the beam in this direction, so, by Newtons third law,
the particle will experience an equal and opposite force back towards the center of the beam.
In this example the particle is a dielectric sphere. Similarly, if the beam is tightly focused it is
possible for the particle to experience a force that pushes back towards the laser beam. We can
also consider an energetic argument: when a polarizable particle is placed in an electric field, the
net field is reduced. The energy of the system will be a minimum when the particle moves to
wherever the field is highest, which is at the focus. Therefore, potential wells are created by
local maxima in the fields. Depiction of the apparatus for examining red blood cells being
stretched by optical tweezers is shown in Figure 5.17 below:
Figure 5.17. Apparatus for stretching RBCs with optical tweezers
64
5.12 Hydrostatic Loading

Groups of cells can be pressurized with devices
shown in Figure 5.18. In (a) a piston (platen)
pressurizes the fluid above the monolayer of cells.
When cells are grown in a 3-dimensional matrix,
pressure can be applied directly to them as in (b).
Cells can be either confined or without side
support. This type of testing is valuable to
understand behavior of bone and cartilage cells,
as well as cells that may be subjected to high
pressures under abnormal conditions.
Figure 5.18. Hydrostatic loading
5.13 Shear Flow
A variety of configurations can deliver precise amounts of shear force to groups of cells attached
to surfaces. A cone-and-plate viscometer is depicted in Figure 5.19 (a), consisting of a
stationary flat plate and a rotating inverted cone with which laminar and turbulent flows can be
applied. A parallel-plate flow chamber (b) subjects cells to laminar flow. In both cases the shear
stress applied to cells can be readily quantified. Many cellular responses can be measured as a
function of shear during and after the force application, including attachment strength, growth,
and biochemical responses. These tests are most appropriate for cells for which shear is a way
of life, such as blood cells and endothelial cells.
Figure 5.19. Shear delivery
5.14 Cell Stretching

When attached to a flexible substrate, cell populations can be stretched by pulling on the
substrate. A commercial device, Flexercell, has been extensively used for this purpose. A variety
of configurations and actuator types are available as shown below. The procedure involves
culturing cells on a thin-sheet polymer substrate, such as silicone, which is coated with ECM
molecules for cell adhesion. The substrate is then mechanically deformed while maintaining the
cell's viability in vitro. In this manner, the effects of mechanical loading on cell morphology,
phenotype and injury can be examined. Furthermore, by systematically altering the mechanical
properties of the substrate material through, for example, changing the degree of crosslink in
the polymeric gel, the individual and collective interactions of the cells with the substrate can be
studied. Such studies have been performed to investigate the propensity for migration of a group
of cells towards or away from the region of localized tension or compression in the substrate.
Various modes of cell stretching are depicted in Figure 5.20 below.
65
Figure 5.20. Cell stretching
5.15 Microelectromechanical systems (MEMS) (Rajagopalan et Al.)

MEMS sensors, because of their small size and
fine force/displacement resolution, are ideal for
force and displacement sensing at the single-cell
level. In addition, the amenability of MEMS
sensors to batch fabrication methods allows the
study of large cell populations simultaneously,
leading
to
robust
statistical
studies.
Micromechanical sensors are especially suited for
these studies because of their small size, which
allows for easy interfacing with individual cells,
and fine force/displacement resolution that
makes them capable of measuring very small
forces/displacements.
In
addition,
micromechanical platforms can be batch
fabricated cheaply using either integrated circuit
(IC) or soft lithography techniques.
Studies on mechanobiology range from the tissue
level all the way down to individual proteins and
DNA, involving a wide range of approaches and
instrumentation size scales. The commonly used
tools for probing cells and biomolecules, such as
AFM, optical and magnetic tweezers etc, have
already been the subject of many excellent
reviews.
Therefore
we
will
survey
micromechanical systems developed for cell
mechanics research and the biological insights
Figure 5.21. MEMS (Bao & Suresh)
that have resulted from these studies. Distinct
from other reviews on microengineered systems,
we highlight two recent developments in this area: (1) micromechanical devices for in vivo and
small animal studies and (2) microsystems to manipulate the physiological behavior of cellular
organisms through controlled application of forces.
66
Substrate topography can be micropatterned so that cells are induced to grow onto tiny elastic
platforms that can be moved to apply or measure force. This is a powerful technique, since the
platforms can be precisely controlled, and can be functionalized with specific cellular matrix
targets. Thus the micropattern can test and perturb cell-matrix interactions in a highly
controlled environment. For example, the strength and behavior of specific focal adhesions can
be studied. The contractile forces generated by cells during locomotion and mitosis can also
been measured with a deformable-substrate method.
Micromechanical systems used for cell mechanics studies broadly fall into two major categories.
The first category comprises hard silicon-based devices that are fabricated using standard IC
manufacturing techniques, whereas the second category comprises systems made of soft
polymers and gels.
Examples of MEMS cell testing systems are shown above. Two configurations are a cluster of
microneedles (Figure 5.21.a and b) or a cantilever beam (Figure 5.21.c). The device in
Figure 5.21.d d can apply mechanical force or deformation at several points on a cell in the
center.
The effect of various MEMS on cellular shape and behavior are shown in Figure 5.22 below.
Figure 5.22. Scanning electron micrographs of human mesenchymal stem cells

plated on PDMS micropost arrays. The diameters of the posts were the same (1.83 m)
but the lengths (L) were different, as indicated in the figure. Note that the deflection is
substantially larger for the 12.9 m micron length posts (c), which were almost 1000
times softer than the 0.97 m posts.
Figure 4.23 below shows 3D structures with sensors , for growing cells and sensing their forces.
67
Figure 5.23. A SU-8

force sensing pillar array
for cell measurements.
(a) A lateral force applied
at the tip of the pillar
bends the four cantilever
beams on which the pillar
is suspended. The
bending strain is
transduced at the base of
the cantilever using metal
strain gauges.
(b) Finite element
analysis showing that
bending induces
alternating regions of
compressive and tensile
stress in the cantilever
beams.
(c) A single device viewed
from the top.
(d) Video
An array
of finished
5.16
tracking
devices
Cell Movements
Myocytes isolated directly from hearts are log
shaped, as shown in Figure 5.24. Their speed and
magnitude of contraction can be monitored with a
video camera and imaging software that tracks
motion of the edges. Records from such an imaging
set-up are shown at right.
Figure 5.24. Myocyte contraction
5.16 Particle Tracking

Video tracking of particles injected into cells can be used to estimate regional stiffness within
cells. Figure 5.25 shows tracking traces of a particle in a gel. The first pattern (a) shows a
relatively wide travel, while the second (b), shows a very tight range of travel. Note that the bar
68

denotes a distance of 2 micrometers. The difference between a and b is that the gel was
polymerized for the b movement, making it more stiff. The mean squared distance traveled for
a, is shown in the top curves in c, and for b, shown in the bottom curves. This is an important
method, and is discussed more in Chapter 4, section 4.10.
Figure 5.25. Particle tracking

__________________________________________________________________
Chapter 5
Review Questions and Exercises
1. In testing micropipet aspiration of cell patches, what modes of force application are applied?
Use a sketch, showing at least 3 modes.
2. Calculate the cell modulus from a pipet aspiration measurement with pipet diameter 1
micrometer, and data attached in Sakai. Show work.
3. Calculate the time constant of mechanical response of a cell to a suddenly applied stress.
4. In a whole cell inflation test, how would the CSK respond to pressurization, based on what
you know of its properties?
5. In a hydrostatic loading test, describe the effects of adding side constraints to the cells.
6. Model 5: Above
69
References
Bao, G. and S. Suresh (2003). "Cell and molecular mechanics of biological materials." Nat
Mater 2(11): 715-725.
Living cells can sense mechanical forces and convert them into biological responses.
Similarly, biological and biochemical signals are known to influence the abilities of cells
to sense, generate and bear mechanical forces. Studies into the mechanics of single cells,
subcellular components and biological molecules have rapidly evolved during the past
decade with significant implications for biotechnology and human health. This progress
has been facilitated by new capabilities for measuring forces and displacements with
piconewton and nanometre resolutions, respectively, and by improvements in bioimaging. Details of mechanical, chemical and biological interactions in cells remain
elusive. However, the mechanical deformation of proteins and nucleic acids may provide
key insights for understanding the changes in cellular structure, response and function
under force, and offer new opportunities for the diagnosis and treatment of disease. This
review discusses some basic features of the deformation of single cells and biomolecules,
and examines opportunities for further research.
Eyckmans, J., T. Boudou, et al. (2011). "A Hitchhiker's Guide to Mechanobiology."
Developmental Cell 21(1): 35-47.
More than a century ago, it was proposed that mechanical forces could drive tissue
formation. However, only recently with the advent of enabling biophysical and molecular
technologies are we beginning to understand how individual cells transduce mechanical
force into biochemical signals. In turn, this knowledge of mechanotransduction at the
cellular level is beginning to clarify the role of mechanics in patterning processes during
embryonic development. In this perspective, we will discuss current
mechanotransduction paradigms, along with the technologies that have shaped the field
of mechanobiology.
70

CHAPTER 6: THERMODYNAMICS OF THE CYTOSKELETON
6.1
The Boltzmann Distribution: Statistical Mechanics
At the molecular level, mechanical behavior is statistical, and requires special parameters to
explain it. The Boltzmann distribution provides a simple explanation for the behavior of
molecules exposed to an energy gradient. The energy gradient can be forces applied by
mechanical, electrical, or chemical means. It is based on the probabilistic nature of molecules,
and has widespread applications. The basic formulation is:
F
P1
= e kT
P2
(1)
where P's are the probabilities of states or classes 1 and 2, and DF is free energy.
For example Pi can represent the concentration of particles in 2 separate compartments. Lets
say the energy in the first compartment is F1 and in the second, it is F2, and DF= F2-F1. Since
the particles must either be in compartment 1 or 2, then P1 + P2 =1. So
To illustrate this, take a system whose neutral state (P1=P2= 0.5) is an energy level of 5kT. In
other words, the neutral state is when the system can be in either state with equal probability.
1
P1( x) :=
1+ e
x 5
P2( x) :=
1+ e
( x 5)
1
P1( x)
P2( x)
Figure 6.1
0.5
10
One such system would be the particle distribution on earth surface or in the air. The dotted
line (P2) would represent the likelihood of finding the particle on the surface (for a mass of 0, P2
= 0), and the dark (red) line would represent finding it above the surface. As expected, as mass
of the particle increased, you would find more of it landing on the ground, and less of it in the
air. At some mass, represented by 5kT, there is an equal probability of finding the particle on the
ground or in the air. Many other compartmental situations can be similarly represented.
71
A mechanical behavior that is well described statistically by the Boltzmann relation is the
tendency of polymers to shift from one state to another, depending on energy levels. Examples
of these shifts are the opening and closing of ion channels and the folding and unfolding of
nucleic acids. The latter behavior can be seen in RNA strands that were pulled by optical
tweezers, as shown in Figure 6.2:
Trap
Bead
Trap Bead
RNA
Actuat
or
Magnified
View
Handle
Force
Actuator
Bead
Figure 6.2 Stretching of RNA molecule
The above set-up shows a strand of RNA held at either end by a binding site on the beads. Note
that the bead size is much larger than the RNA. When the strand is stretched, it behaves as an
ordinary spring at very low and very high levels of tension, but at intermediate ranges, it
exhibited unfolding and re-folding behavior, as depicted in Figure 6.3:
22 nM
Sudden extensions of 22 nM (unfolding)

when forces above 14 pN are applied
Figure 6.3 Length-force relationship of a strand of RNA.
As the force is slowly raised above 14 pN, the strand will suddenly unfold and then refold again,
with the unfolded state becoming more frequent as force increases, until it remains permanently
unfolded at a force level above 15 pN. In other words, the probability of unfolding is related to
force, as described by the Boltzmann equation. This behavior is quantified in Figure 6.4.
72
Unfolding upward
Folding downward
Figure 6.4.a. Opening
behavior of RNA at selected
tensions
.
Figure 6.4.b. Fraction of
hairpins folded versus force
This behavior appears to represent a balance between the applied force and the attractive forces
within the RNA fold, including base-pairing, hydration, charge shielding, and Van der Walls.
Thus the fraction of time spent in a state is equivalent to the probability of finding the strand in
that state, and is related to force applied, according to the Boltzmann relationship, where Fo is
the ground energy state, f is applied force, and z is displacement:
Popen =
1+ e
Fo fDz
kT
Within any given time period, T, the probability of being open or unfolded can be calculated as
the fraction of open time:
Popen =
t open
T
73

6.2
Diffusion
The diffusion coefficient, D, indicates how fast particles of radius a can move in a solution of
viscosity, . Units of D are
cm
sec
. There are 2 formulae to describe diffusion (in one dimension).
First the flux of particles per unit time, j, (i.e. number of particles p.u. time) passing a given
point is directly proportional to the concentration gradient, C:
j = D
dC
dx
Ficks 1st
Law
To describe how concentration changes with time, Ficks second law is used:
dC
= DD2 C
dt
where
D2 =
Ficks 2nd
Law
d2
dx 2
A special case of Ficks 2nd law can be set-up to arrive at a simple formula for estimating
diffusion time. This is the case in which an amount of substance is placed in solution at the point
x=0, and its concentration spread with time becomes uniform over x. With these initial and
boundary conditions, Ficks 2nd law is solved analytically as:
C=
1
x2
exp(
)
4pDt
4 Dt
This formula is analogous to the Gaussian probability formula:
P ( x)dx =
1
2x
exp(
x2
2x 2
)dx
Since concentration of particles is directly related to the probability of finding them in a given
distance dx, C P(x)dx. So equating the 2 formulae yields a simple estimate of the time of
diffusion for a particular solute, given its coefficient, D, as shown below:
Standard estimate of diffusion time. Please note that the
bar over the x is not a negative sign.
The right hand side of the above should read "X bar squared", and represents the spread of
concentration in the x direction. Note that it is analogous to the statistical quantity of variance.
74

6.3
Bioelectricity
6.3.a
Internal charge and electrochemical equilibrium
Cells have trapped negative charges in the form of proteins, amino acids, and nucleic acids.
These are charged mostly because of their side groups, in particular carboxyl, that is loses a
proton at neutral pH. For example, alanine is shown below:
PKa = 9
H3N-CH-COO-
pKa= 2.7
CH3
Note that the net charge of theses amphiphilic molecules can be calculated by summing the
charge of each group, using the Henderson-Hasselback equation:
AH A + H +
Kequ =
[ A ][ H + ]
[ AH ]
[ A ]
log( Kequ ) = log
+ log[ H + ]
[ AH ]
[ A ]
log
= pKa pH
[ AH ]
pH pKa +
()
log A
( HA )
- = fractional - charge =
(A )
(A
+ AH
1
- =
1+
1
alog ( pH pKa)
The above equation gives the proportion of negatively charged molecules within the group,
for a given pH. Each side chain has a particular acid-base relationship, with a specific pKa.
A corresponding relationship can be derived for the positively charged (i.e. amino) group.
Cells thus have a trapped negative charge at neutral pH, in the form of big, impermeable
solutes, is the root cause of the electrical potential of all cells. These impermeable solutes
are amino acids, nucleic acids, and other compounds with a tendency to become negatively
charged. To balance the charge, cations that are mobile and permeant through the
75

membrane, enter the cell. The tendency to neutralize charge then competes with the
tendency of chemical concentration gradients to nullify, and an electro-chemical equilibrium
results, that can be described by 2 simple formulae, the Nernst and Donnan:
Eion =
RT [ion]out
ln(
)
zF
[ion]in
and
[+ion][ion]out = [+ion][ion]in
The Nernst potential is the electrical potential that exactly balances the chemical gradient of
mobile ions that balances the trapped negative charge. The Donnan relation is a direct result
of the Nernst. You can see this by noting, if the mobile counterions are K+ and Cl- , the
Nernst potential for each ion must be the same, since at equilibrium there can be only one
potential for the cell. Thus the ions that are freely permeable are those that determine the
cell potential. For most cells these are K+ and Cl-. Note that the Donnan equilibrium applies
only to K and Cl, and not to other ion pairs, that are not freely permeable.
Deriving the Nernst equation comes directly from the Boltzmann relationship, as illustrated
below, using the distribution of particles in the atmosphere as an example:
Deriving the Basic Bioelectric Equation
First there is the distribution of particles subject to gravity:

N( h ) N o . e
w.
kT
This applies equally well to particles subject to an "E field",

since:
q .V w.h
( Joules )
and then:
N( V) N o . e
q.
V
kT
where
So for molar
quantities:
C C o .e
and
z.
V
RT
C = N particles/vol * mass/particle
RT .
z
ln
Co
C
Correction to above formula: note Faradays constant.
V =
RT C 0
ln( )
zF
Ci
76

6.3.b Ion channels
These are proteins within the cell membrane that open and close to let specific ions in or
out. The illustration below shows that ions will move through the membrane according to their
electrochemical (Nernst) gradient.
Figure 4.5
77

Methods to record the flux of ions are shown above. The flux of ions in an electrical gradient,
V, is given by Ohms Law coupled with the statistical Boltzmann relationship:
I = NgVp open
Massflux :
I
J=
F
6.4
Energy Storage
The vast majority of cellular energy is stored in the high energy phosphate bond of the ATP
molecule. One ATP molecule is equivalent to 80 pN-nM or 8 N-m/g. Note that the units of
torque and energy are equivalent.
6.5
State Transitions
The RNA unfolding example represents the general process of state transition. The general way
to represent it is:
K21
1
2
K12
With the 2 compartments or states shown, transition rate K21, represents the flow rate of
material from compartment 1 into compartment 2; K12 represents the opposite motion. This is a
2-state, closed system, since no compartment excretes material to the outside or any other
compartment. A closed system is the simplest to deal with, since the mathematics is easy (as will
be seen in the more advanced treatment at the end). Note that for the RNA example, states 1
and 2 could represent the folded and unfolded conditions, and K21 would represent the rate
constant for unfolding, and K12 the rate constant for folding.
We can represent the kinetics of the system as follows:
dX 1
= K 21 X 1 + K 12 X 2
dt
and
(1)
dX 2
= K 21 X 1 K 12 X 2
dt
Or better in matrix notation as the state variable representation:

X 1 a11
= a
X 21
2
a12 X 1
a 22
X2
(2)
78

Or more succinctly as:
X = AX
(3)
where it is assumed that X and A are matrices. Note that we are analyzing a system with only 2
compartments, but the above state equation (3) applies to systems with any number of
compartments. So if there were p components, equation (3) would represent:
X i = ai1 X 1 + ai 2 X 2 + ... + a1 p X p
(4)
Now back to our 2 state, closed system:
k12
k
A= 21
k21 k12
Once you check that this is true, you can ask, how can I analyze the system? It should be
apparent that you can solve each differential equation readily using a solver such as Simulink.
You can also solve the entire system by coupling the equations.
You first must understand what X represents. In our case, it represents probability. One way to
look at it is to recognize that the entire two-compartment system can represent many RNA
chains, that can either be in state 1 or state 2. The fraction, or state probability, P1, of being
folded and the probability of being unfolded, P2, are:
P1 =
# folded
# folded + # open
and
P2 =
# open
# folded + # open
and thus P1 + P2 = 1, and X = P.
79
Kopen = 7 sec-1
Kfold = 1.5 sec-1
Kopen = 0.9 sec-1

Figure 6.6
K fold 8.5 sec-1
6.6
Polymerization
To understand how the CSK and its functional behavior emerges from discrete components, lets
look at the kinetics of polymerization. In Figure 6.7, actin filaments were nucleated from beads
coated with the nucleation-promoting factor ActA (not shown) and were then crosslinked by
fascin inside a unilamellar lipid vesicle
Figure 6.7. Purified proteins
were loaded into a vesicle by a
microfluidic encapsulation
technique that allows the dynamics
of filament assembly to be
observed immediately after
encapsulation.
The micrograph (left) shows
labeled actin filaments (white) that
have polymerized inside the vesicle
and have assembled into a fascincrosslinked network.
The diagram (right) is a schematic
depiction of the actin filament
network present in the inset box of
the micrograph
Polymers are assembled from monomers (captured), and disassembled (released) back into
monomers, generally as a first order system as depicted below:
(capture)
dn/dt = +kon [M]
(1)
(release)
dn/dt = -koff
(2)
80

Definitions:
n = number of monomers in a single filament;
t = time;
[M] = concentration of free monomer in solution
kon = capture rate constant, with units of [concentrationtime]-1
koff = release rate of monomers;
Thus the capture rate of monomers by a single filament is proportional to the number of
monomers available for capture. The release rate does not depend on [M]. Note that the front
and back end of polymers differ. (Figure 6.8)
Figure 6.8
__________________________________________________________________
Chapter 6
First Week
1. Using the data shown in Figure 6.6 above, and the ground free energy, Fo = 79 kT, graph
the unfolding probability, using Excel or other program. Put data points from empirical
experiments for the selected forces on your theoretical curve. (Note data represent
numbers obtained from experimental measurements (not calculations); a theoretical
curve is generated by formula).
2. Make a Simulink model of the RNA unfolding kinetics. Your model should be
well documented, according to the following guidelines:
All parameter boxes should be labeled
Document boxes should be included to describe operations
Internal parameters, such as initial conditions, should be specified
Sub-systems should be used so that the entire model can be fit onto 1 page
and each sub-system can be printed separately, with documentation.
A separate description of the system and all formulae should be made.
Outputs should be the predicted, as well as measured probabilities
A reasonable noise level should be placed in the model
81

3. A proteoglycan molecule forms a gel. The pKa of its amino group = 10 , pKa of carboxyl =
3 and pKa of the R group is 7. What is its net charge at pH = 7 ?
Second Week
1. What is the origin of biopotentials in cells, and how are they calculated? Be specific.
2. Endothelial cells have the usual cell membrane that restrains movement of proteins,
but permits free flow of water and monovalent electrolytes. A Donnan equilibrium of
diffusible ions results.
a.
Based on this information, complete the table below:
Ion
Outside Concentration, mM
Inside Concentration, mM
[Na+]
150
144
[K+]
4
[Cl-]
114
[HCO3-] 28
b. What is the transmembrane biopotential?
c. What is the concentration of proteins in the cell? ( assume proteins are the only
solute other than electrolyte, and they have a charge of -1).
d. At what potential (if any) are all ions at equilibrium?
3. Model 6: Make a Simulink model of the RNA unfolding kinetics. Your model
should be well documented, according to the following guidelines:
References
1.
Liphardt, j., Science, 2001. 292.
82
83
CHAPTER 7: KINETIC BEHAVIOR

7.1
Background (Excerpts from Jamali, Azimi et al. 2010)
One of the important phenomena in cell engineering and developmental biology is the
shape of tissue and the cells organization.
Depending on the cell type and
environmental conditions, cells can create unique shapes such as flat sheets, selfenclosed monolayers, cysts, or elongated tubes. The more important question is how
these cells interact and how their local interaction causes a global geometrical
distinctive shape for tissues like the heart or kidney. The geometrical interactions and
coordinated adhesion among neighboring cells and between the cells and local
environment are critical for structure and function of epithelial tissue. Any perturbation
of these orchestrated interactions can cause abnormality in behavior and function of
tissue and often lead to initiation of tumor growth and invasion. Another interesting
subject is embryogenesis, when a stem cell with consecutive rapid divisions and
differentiation can create different tissues, wherein the interactions between cells and
environmental biochemical and biomechanical signals have critical, yet nearly unknown
roles. However, in the last two decades, improved experimental techniques and
developments have allowed for more detailed understanding of cell-cell communication
and the cells response to biochemical and biomechanical environmental signals, as
reviewed in Chapter 5. Nevertheless, biological experiments are expensive and depend
on many parameters that are mostly difficult to control and test in isolation.
7.2
Cell Modeling
Mathematical modeling and computational experiments can help explore the behavior
of the individual tissue cells along with investigating their response to environmental
cues. Due to easy isolation in in-silico computational models, incorporating the related
fundamental physical and biological parameters can explain how specific biochemical or
biomechanical parameters may affect the tissue cells and their arrangement. Such a
model can reduce the number of experiments required to obtain meaningful
observations by eliminating unlikely hypothesis while providing a better explanation of
observations. For example, to investigate how individual cells cooperate and contribute
to the overall structure and function of a particular tissue, a proper computational
84
model could define cells as individually deformable shapes, time- and space-dependent
individually regulated cell turnover, and cell-cell and cell-ECM interaction. Many
models have been developed to mimic cell behavior, such as response to external
mechanical and biochemical signals, cell-cell interaction, cell motility, and cell
morphology.
Some models have attempted to mimic collective cell behaviors such as cancer invasion
through the use of continuum and/or discrete approaches where each cell is represented
by a finite element and follows a cellular automata method. Cells can be modeled as
colloidal objects capable of interacting with their environment. In such models, cells are
capable of migrating, growing, dividing, and changing their orientation. In a model
proposed by Galle, Loeffler et al. (2005), the cells move according to the Langevin
dynamics framework and can interact based on a combination of attractive and
repulsive forces. The aim of these models is to replicate the multi-cellular growth
phenomena. By focusing on monolayer culture, Galle et al. have investigated the effect
of key factors on rate and quality of culture growth. They also analyzed the underlying
processes involved in multi-cellular spheroids, intestinal crypts, and other aspects of
developmental biology.
Models can mimic various aspects of cell population, but fail to examine the effects of
cell deformation and morphology on pattern formation and growth processes. Some
models are based on the viscoelasticity of cells, where each cell includes certain elastic
and viscous elements. Such models lend themselves to easy incorporation of the cell-cell
adhesion and repulsion, and various forces acting on individual cells in the cluster. For
example, a 3D deformable cell model with cell adhesion and signaling was developed by
Palsson and colleagues, where each cell is assumed to be ellipsoid shape, with its axis
composed of a combination of springs and viscous elements . This model was used to
investigate the role of cell signaling, cell adhesion, chemotaxis, and coordinated
differentiation in the morphology of a developed organism. Another biomechanical
approach developed by Rejniak and colleagues
represents cells as deformable
viscoelastic objects that can be arranged into tissues of various topologies. This
approach joins elastic cell dynamics with a continuous representation of a viscous
85
incompressible cytoplasm. The model covers many aspects of cellular behavior such as
cell growth, division, apoptosis and polarization. With this model it is possible to
investigate the biomechanical properties of cells and cell-cell interaction, the effects of
the microenvironment on a cellular cluster, and how individual cells work together and
contribute to the structure and function of a particular tissue. An application of this
model is tumor growth .
More recently, Coskun et al. developed a mathematical
model for amoeboid cell
movement in which a viscoelastic (springdashpot) system was used to represent the

cytoskeleton. This model was used to solve an inverse problem of amoeboid cell motility
and to find the variation of spring and dashpots parameters in time. The research shows
that the model and the solution to the inverse problem for simulated data sets are highly
accurate. In general, cell mechanics has been modeled based on non-living structures
using approaches ranging from the soft glassy material model , to the cortical shell
liquid core model, and tensegrity architecture . Few of the theoretical models that have
been proposed for analyzing the mechanical properties of adherent living cells are
capable of simultaneously incorporating (i) the discrete nature of the cytoskeleton, (ii)
cellcell and/or cellextracellular matrix (ECM) interactions, and (iii) the cellular prestress.
The complete cell behavioral model should integrate the tensegrity concept, where each
cell is capable of changing its morphology, and performing the key cellular processes of
growth, division, polarization and death. The modeled cells should interact with each
other and with their environment. Each cell in this model is an individual unit
containing several subcellular elements, such as the elastic plasma membrane,
encompassed by viscoelastic elements that perform the function of the cytoskeleton, and
the viscoelastic elements of the cell nucleus . Additionally, the cell membrane is divided
into segments where each segment (or point) incorporates the cells interaction and
communication with its environment, such as adherens junctions.
86
7.3
Modeling the Cytoskeleton.
The mechanical properties of the cytoskeleton, like elasticity and viscosity, are critical to
the validity of the model. Voigt subunits are effective for modeling a viscoelastic system;
the spring constants of the model are linear approximations to the elasticity of the inner
cell. Additionally, all springs are subjected to a damping force resulting from the
viscosity of the cytoplasm, where linear dashpots approximate the viscosity of the
cytoskeleton (Figure 7.1).
The cytoskeleton can be divided into
uniformly radial
distributed parts, each of which is represented by a Voigt subunit radiating from the
nucleus (Figure 7.1, blue subunits). Each subunit connects two points of the cell and
nuclear membrane, which are aligned in a radial direction from the center of the
nucleus. The nucleo-skeleton is represented as a viscoelastic model involving an
actomyosin system (Figure 7.1, red subunits). The model also contains N Voigt subunits
in the nucleus ( red subunits). The equation below is the sum of all possible forces on
the cell:
Figure 7.1
Cell Model
87
7.4
Viscoelastic (VE) Material
V-E analysis assumes that a complex material can be modeled as a purely viscous
material (a dashpot) combined with a purely elastic material (a spring).
It thus
mathematically separates the viscosity of a material from its elasticity. A purely viscous
component is a Newtonian fluid- it has no memory and no elasticity; it cannot deform as
a solid. In cells and tissues, material properties vary from behaving as pure fluids, to
not very pure fluids, to solids, to solid-liquid composites. Despite this diversity, V-E
tools can quantify each of their properties, since the models separate viscosity from
elasticity in a kind of finite element model.
Elastic deformations experienced by structures can be characterized by their particular
stress/strain curves. Examples of such curves for three typical behaviors are shown in
Figure 7.2:
Figure 7.2 Stress/Strain

Curves for Elastic Deformations
s
e
The above curves represent the stiffness of the structure, as measured by their slopes at
any point. The curve on the left represents rubber-type behaviour, since it is relatively
stiff for small strains, relatively compliant (un-stiff) for intermediate strains, and again
is relatively stiff for high strains. Thus there are 3 separate regions of rubber behaviour,
each of which has a molecular explanation. The middle curve is representative of
standard structural materials. The curve on the right is representative of a purely
Newtonian fluid.
An important factor that has been left out of the above analysis of elasticity is time. It
must be recognized that each curve represents a single point in time ( or as close to a
single point in time as possible). That point in time could represent either an
88
equilibrium or a non-equilibrium situation. The only way to learn the role of timedependent changes in stiffness, is to measure it at a sequence of times following each
stress or strain application. In doing this, you would be measuring viscoelastic
properties. In other words, viscoelasticity can be defined as: stiffness as a function of
time. Materials that have time dependent stress/strain behaviour are called
viscoelastic.
For example, a hydraulic shock absorber is very stiff, when measured very soon after a
displacement, but is not very stiff, later following the displacement. The key point about
a V-E material, therefore, is its time dependent behaviour, in terms of either stress or
strain.
An example of viscoelastic behavior is shown when cartilage is compressed, as in the
experiment by Grodzinski et al. (Figure 7.3):
Figure 7.3. Cartilage Compression
89
Two distinct phenomena can occur in a VE material: it can creep or it can relax. For
example, play dough, will creep (or flow) from a spherical shape, into a flat puddle,
over the course of several minutes. We could say that a constant force, or stress, of
gravity is acting upon the play dough, and the material creeps slowly. When you pull
suddenly on the material, however, it does not seem to be a liquid, and is rather stiff. So,
again you see that stiffness is relative (not in the Einsteinian sense).
Now, getting to specific ways to characterize V-E materials, there are only a few practical
ways you can do so. Testing properties of anything always requires that you perturb it
in some way, while measuring its response. There are 2 basic mechanical perturbations:
force (stress) or deformation (strain). There are generally 2 ways you can apply these
perturbations: suddenly, or slowly. The sudden method is referred to as a step input
(impulse is also possible but not practical for most material characterizations); the slow
method can take the form of a ramp, sinusoid, or other wave shape. The complete
perturbation matrix and expected material responses looks like this:
Input Shape
Type
Step (sudden)
Ramp (slow)
Stress
Sigmoidal
Strain
Creep:
Voight Complete
Relaxation:
Model
Maxwell model
Ongoing Creep
Ongoing relaxation
The table entries state the type of response to be found for each combination of
perturbation type and shape.
A complex material can be modeled as a purely viscous material combined with a purely
elastic material, thus mathematically separating the viscosity of a material from its
elasticity. A purely viscous component is a Newtonian fluid- it has no memory and no
90
elasticity; it cannot deform as a solid. Cells generally behave as solid-liquid composites.

V-E tools can quantify their behavior, since the models separate viscosity from elasticity
in a kind of finite element model.
The Voigt model is easily quantified, as shown below:
Voigt Model
since s =
and
e+
sC
de/ dt
I = (1/R) V + C dV/dt
then each of the top terms are equivalent to the bottom

terms. Then the impedance analysis proceeds:
____________________________________________________________
______
Chapter 7
1. Calculate the time taken for a microtubule to grow from the centrosome of a
typical cell to the cell boundary. How long would it take to shrink to single
heterodimer length if it undergoes rapid depolymerization? Assume: (1) it grows
from the plus end, (2) [M] = 10 uM/L, (3) rate constants as given, (4) a tubulin
heterodimer is 8 nM long.
MW of monomer = 50,000 Daltons; heterodimer is 2X monomer.
91
Tubulin
K+ on
K+ off
K- on
K- on
Monomer in
(uM-S)-
S-1
(uM-S)-1
S-1
solution
Growing
9.3
44
5.3
23
Rapid disassembly
737
915
2. Assume that and endothelial cell length is suddenly doubled. Cytoplasmic
viscosity is 500 dyne-sec-cm-2 and CSK elasticity is 1000 Pa. You may assume
the cell is cuboidal in shape.
Describe the stresses within the cell that would
happen immediately after the event, and as time evolves thereafter. What kind of
model could describe the behavior? Draw the model conceptually. What is the
time constant of response? Draw a relevant part of the response, illustrating the
time constant, and be sure to label. What components of the cell does the model
represent? Hint: the initial stress is expressed entirely in the elastic component;
another way to look at it is through the Laplace solution and transform.
3. Recognize the following models and their inputs and outputs: Explain why and
how the outputs differ.

e
s
s
e
92
10
4. Model 7. Make a Simulink model of the RNA unfolding kinetics. Your model
should be well documented, according to the following guidelines:

References
Jamali, Y., M. Azimi, et al. (2010). "A Sub-Cellular Viscoelastic Model for Cell
Population Mechanics." Plos One 5(8).
Understanding the biomechanical properties and the effect of biomechanical
force on epithelial cells is key to understanding how epithelial cells form uniquely
shaped structures in two or three-dimensional space. Nevertheless, with the
limitations and challenges posed by biological experiments at this scale, it
becomes advantageous to use mathematical and 'in silico' (computational)
models as an alternate solution. This paper introduces a single-cell-based model
representing the cross section of a typical tissue. Each cell in this model is an
individual unit containing several sub-cellular elements, such as the elastic
plasma membrane, enclosed viscoelastic elements that play the role of
cytoskeleton, and the viscoelastic elements of the cell nucleus. The cell membrane
is divided into segments where each segment (or point) incorporates the cell's
93
11
interaction and communication with other cells and its environment. The model
is capable of simulating how cells cooperate and contribute to the overall
structure and function of a particular tissue; it mimics many aspects of cellular
behavior such as cell growth, division, apoptosis and polarization. The model
allows for investigation of the biomechanical properties of cells, cell-cell
interactions, effect of environment on cellular clusters, and how individual cells
work together and contribute to the structure and function of a particular tissue.
To evaluate the current approach in modeling different topologies of growing
tissues in distinct biochemical conditions of the surrounding media, we model
several key cellular phenomena, namely monolayer cell culture, effects of
adhesion intensity, growth of epithelial cell through interaction with extracellular matrix (ECM), effects of a gap in the ECM, tensegrity and tissue
morphogenesis and formation of hollow epithelial acini. The proposed
computational model enables one to isolate the effects of biomechanical
properties of individual cells and the communication between cells and their
microenvironment while simultaneously allowing for the formation of clusters or
sheets of cells that act together as one complex tissue.
94
CHAPTER 8: SKELETAL MUSCLE
8.1
Introduction
Cellular motors come in many varieties. The most familiar motor we use every day is our
musculature, which consists of skeletal, cardiac, and smooth muscle, all of which move linearly
i.e., back-and-forth. Other types of motors operate within most of ours cells that transport
things , change cell shape, and move cells around. Most free-living cells, such as bacteria, use
rotary engines. Still other motors are used in cell migration and growth. While there is much
to study about these different motor mechanisms , our task is simplified by the fact that all the
cellular motors use the same fuel: ATP. This chapter will concentrate on the mechanical aspects
of muscle cells, and subsequent chapters will cover rotary and other types of motors.
The 630 bulk skeletal muscles that move joints in the typical human are composed of > 250
million muscle fibers (cells) in the typical human body. Force generation is accomplished by
shortening and/or parallel sliding of two protein filaments: actin and myosin within each fiber.
Muscle contraction is driven by the cycling of cross-bridges, whose unitary length change in the
nanometer range and its pico-Newton force output are fueled by conversion of chemical energy,
stored in the form of adenosine triphosphate (ATP), into a change in myosin protein
configuration. The range of force and length changes of a muscle is determined by factors such
as muscle cross section, fiber angle, tendon attachment, and lever geometry, but also by the
metabolic pathways available for ATP synthesis and by enzymes involved in cross-bridge
cycling. In addition, muscle mechanical activity is affected by the extent of actin and myosin
filament overlap. Force output can be graded by selective recruitment of motor units and/or by
variation of force output from individual units.
The most important points of this chapter are:
1. The mechanism of muscle contraction
2. Force output of muscle
3. Neural control of muscle
8.2
Muscular Microstructure
Skeletal musculature is made up of anatomically distinct units, which are attached to bones (via
tendons) or other muscles (via ligaments) to support specialized movement. In the human, there
are 630 individual muscles, which make up ~40% of total weight of the average body.
Individual muscles are composed of fasciclesassemblies of muscle fibers that are surrounded
by a connective tissue sheath that can span the entire length of a muscle [Fig. 1].
Each fiber (diameter between 10 and 100

m ) is on e sin
single membrane called the sarcolemma. Glycogen, a chief energy provider for skeletal muscle
contraction, is stored in conjunction with this membrane. The sarcolemma protrudes into the
muscle fiber at regular intervals forming transverse tubules (T-tubules). T-tubules are conduits
for electrical signals to travel from the surface to the core of the cell, where they abut the cisterns
of the sarcoplasmic reticulum (SR), the intracellular Ca store of muscle cells.
95
Figure 8.1. The
organization of skeletal
muscle, with
approximate diameters.
Note that the myosin
bundle has many heads
that contact the actin
randomly as the 2
components slide past
each other.
Fibers contain densely packed

regular bundles of myofibrils,
and
are
usually
multinucleated. Myofibrils are
the contractile units of muscle,
made up of thick (myosin,
diameter 15 nm) and thin
(actin, diameter 5 nm) protein
myofilaments.
These
myofilaments are arranged in
a hexagonal pattern and
interdigitate [Fig. 1]. In
humans,
each
myofibril
contains approximately 1500
myosin and 3000 actin
filaments. Myofibrils (and,
therefore, myofilaments) are
organized in highly structured
sarcomeres [Figure 8.1.E].
Functionally,
sarcomeres
consist of two half-sarcomeres
whose force generators have opposite polarity. Actin filaments (each 1 m long) consist of two
intertwined pearl-necklace-like chains composed of globular actin molecules (the pearls, where
myosin will bind), tropomyosin (the chain, which covers the binding sites for myosin), and
troponin (where Ca will bind to displace tropomyosin and enable actinmyosin bonding) .
Myosin filaments ( 1.6m long) consist of several hundred myosin molecules that are each
around 0.15 m long and have the appearance of a daffodil (before flowering). Individual
molecules are connected by their stems, with the bud head sticking out to the sides of the
assembled filament, as in a tightly wrapped bunch of flowers. Myofibrils also contain
mitochondria (the power station of the cell), often arranged in parallel with individual
sarcomeres.
A broader view of muscle is shown in Figures 8.2 and 8.3. The bulk muscle consists of many
fibers, which each consist of many myofibrils. The fibril contains many sarcomeres, oriented in
parallel along the length of the fibril.
96
Figure 8.2. Muscle architecture. The sliding

filaments, actin and myosin are shown making
up a single sarcomere, the fundamental unit of
contraction.
Sarcomere
Myosin head
Sarcomeres contain the sliding filaments, actin and myosin, as shown below.
Figure 8.3. A sarcomere, showing actin filaments sliding over myosin, which is
connected to the Z-discs with titin springs.
97
The dimensions of typical sarcomeres are shown in Figure 8.4:
Figure 8.4. Actin and Myosin
8.3
The Pathway to Contraction
A. Motor Units
In mammalian muscle each individual fiber is innervated by a single motor neuron. Each motor
neuron may control a range of homotypic fibers (with the number determining the precision of
movement supported). This group of fibers and its controlling neuron is called a motor unit, the
functional unit of muscle contraction. The motor unit (below), is comprised of a single alpha
motor neuron and all the fibers it enervates. This muscle fiber contracts when the action
potential (impulse) of the motor neuron that supplies it reaches a depolarization threshold. The
impulse arrives at the neuromuscular junction, which typically extends over an area of about 0.1
cm2. The depolarization generates an electromagnetic field and the potential is measured as a
voltage. The depolarization, which spreads along the membrane of the muscle, is a muscle
action potential. The motor unit action potential is the spatial and temporal summation of the
individual muscle action potentials for all the fibers of a single motor unit, as shown in Figure
8.5.
Figure 8.5
98
In this example, there are 4 motor units, each being activated at a different frequency. Their
algebraic sum yields a signal that can be registered at the surface of the bulk muscle. Since the
motor units are spatially separated, this phenomenon is known as spatial summation.
Motor neurons can connect with between 3-2,000 muscle fibers. This large range relates to the
differing functions of muscles. For example, thigh muscles are engaged in forceful, but gross,
movements, such as walking and running. Thigh muscles, i.e., quadriceps, therefore, have
relatively few motor units that each innervates a large number of muscle fibers. Since there are
roughly 300,000 muscle fibers in the quadriceps, and motor units consist of about 1000 fibers,
which translates to about 300 motor neurons controlling the muscle. Other large muscles, such
as the biceps, are similarly innervated. So when the motor neuron fires, a massive twitch of
300 muscle fibers happens. In contrast, muscles controlling fine movements, such as the eye,
have smaller numbers of muscle fibers per motor unit (usually less than 10 fibers per motor
unit), which yields much smaller twitches and hence finer control.
During a typical voluntary muscle contraction the nervous system recruits motor units in a
hierarchical arrangement as motor units with fewer and smaller muscle fibers are activated first,
followed by the motor units with larger muscle fibers. The main determinant of muscle force
output of each motor unit is their frequency of stimulation. The main determinant of the bulk
muscle force is the number of motor units recruited. Thus the biceps theoretically would
produce their maximum force (Maximum voluntary contraction , MVC) when all 300 or so of
their motor units were activating at maximum frequency.
The sequence of action of the power stroke is illustrated in Figure 8.6. In this cartoon, myosin
is assumed to be stationary, while pivoting around its bottom joint. Actin is seen to move to the
right during the power stroke.
Bottom joint
of myosin
Figure 8.6. The power stroke sequence. The myosin head captures ATP, denoted T, which is
then hydrolyzed to ADP, causing a forward rotation of the myosin, which cocks the ratchet.
Next the phosphate is released , causing binding to actin, then the ADP is released, causing the
ratchet to rotate back, in the power stroke.
B. ExcitationContraction Coupling
Contraction is initiated by an electrical impulse [somewhat paradoxically called an action
potential (AP)] of neural origin (i.e., originating in the brain or spinal cord) that reaches all
99
muscle fibers within a motor unit via the corresponding motor neuron. Subsequent to
neuromuscular transmission, the AP travels along the sarcolemma of each muscle fiber and
proceeds into the t-tubules. The t-tubular electrical signal causes release of Ca++ from the
adjacent SR. Ca binds to the actin filament, displacing the tropomyosin chain to expose the
binding sites for myosin, thereby allowing actin and myosin to interact.
The dependence of force production on Ca is depicted in Figure 8.7.A.
Figure 8.7. (A) Schematic representation of isometric force production (normalized to its
maximum value P ) as a function of cytosolic Ca++ concentration (log scale). (B) Relative
force versus relative velocity (normalized to maximum.).
In the presence of adenosine triphosphate (ATP), the key biological energy storage and transfer
molecule, actin and myosin filaments will slide past each other, driven by cross-bridge cycling
(Figure 6). This causes an increase of force and, if the external load is less than the maximal
force developed by the contractile apparatus, shortening of the sarcomere. In either case, the
mechanical event is known (again, somewhat paradoxically) as a contraction: isometric if
constant length and isotonic if constant force.
The AP-induced increase in [Ca ] is counteracted by re-uptake of Ca into the SR. This also is an
energy-consuming process that requires ATP. In the absence of maintained electrical
stimulation, [Ca ] , therefore, decreases to its resting level, which prevents further formation of
new actin-myosin bonds. A single contraction followed by relaxation is called a twitch.
8.4
Generation and Regulation of Force
A. Temporal Summation of Twitch Tension
A key mechanism controlling force in skeletal muscle is AP frequency of motor units. Since AP is
an electrical pulse that is much shorter than a mechanical muscle twitch, successive APs can
reach the fiber before it has relaxed (i.e., before [Ca ] has returned to its resting value). Restimulation of a fiber under these conditions leads to cumulative amplification of [Ca ] . This
amplification will cause a summation of force (called temporal summation). In the fiber
depicted in Fig. 5, this occurs at stimulation frequencies above 5 Hz. Note that temporal
summation combines with spatial summation, described in section 2.2 and below to produce
smooth muscle forces.
100
Figure 8.8. Temporal

summation of force
If the interval between APs is less than of the duration of a mechanical twitch ( 10-100 ms,
depending on muscle type and excitability ), the individual twitches will fuse, as seen in Figure
5. When twitches completely fuse, a tetanus resultsa maintained powerful contraction.
During a tetanic contraction, the muscle operates on the plateau region of the force-Ca
relationship [Figure 8.8]. Upon cessation of electrical stimulation (and provided that ATP is
sufficiently abundant), [Ca] is reduced by the reuptake of Ca into the SR and force declines to its
resting value.
B. Force Production
Muscle cross-sectional area provides a good indication of maximum force output. A larger crosssectional area indicates more fibers or fibers with a larger diameter, both of which translate into
larger numbers of parallel myofilaments and, hence, a bigger area for cross-bridge formation.
Maximum contractile strength is about 34 kg per cm2 of muscle cross section (300400 kPa.
This quantity is called Physiological Cross Section Area (PCSA) and is used to estimate clinical
performance of muscles, based on their area. The area of muscles is highly dependent on level
of exercise, and hypertrophy and atrophy are constantly occurring in muscles. Typical PCSA for
Biceps is 23 N/cm2 with 170 ms minimum response time. For triceps it is 13-23 N/cm2, with
100 ms response time.
The table at right summarizes some
important properties of muscle.
The PCSA for each muscle is influenced
by the arrangement of muscle fibers.
When fibers are all parallel,
fiber
direction is parallel to the main axis of
force development, so fascicle length and
muscle length are equal. In contrast,
unipennate fibers run at a pennation
angle(usually between 10 and 30 ) to
the force axis, so fascicle length and
muscle length are unequal. (Bipennate
muscles have fibers that attach to a
101
tendon in the middle of the muscle and multipennate muscles are those with multiple tendon
attachments and fiber angles.) Whereas unipennate muscles commonly have larger cross
sections, thereby generating larger total forces, the net force, contributed by their individual
fibers is reduced by the cosine of the pennation angle. The total shortening length of pinnate
muscles is reduced by the cosine of the angle. Effective force output and contractile velocity are
further affected by the position of tendon attachments to bones (relative to neighboring joints).
The mechanical gain of the resulting lever systems influences both force output and fine control
of movement.
C. LengthTension Relationship
Force output relates to the number of active cross bridges in each half-sarcomere, which is
dictated by sarcomere length, which directly relates to the amount of overlap between actin and
myosin filaments. The overlap determines the number of cross bridges that may maximally be
formed at any time. Thus the highest active tension can be generated when all myosin heads (at
either end of the myosin molecule) face actin-binding sites (Fig. 9: bc). This is the case when
sarcomere length in skeletal muscle is around 2.02.25
m in h u m an s.
When muscle fibers are stretched beyond this point, the myosin heads near the M-line will no
longer overlap with actin filaments and, therefore, cannot contribute to active force generation
(Fig. 9: d). In the extreme of excessive stretch, at around 3.5
m , actin an
will have no overlap (Fig. 9: e). At this point, cross bridges cannot form and no shortening or
tension may be actively generated. When sarcomere length is less than about 2.0
m ,
interaction of opposite actin filaments disturbs the hexagonal arrangement required for optimal
cross-bridge formation and maximum tension development is reduced [Fig. 9: ab]. At a
sarcomere length of about 1.67
m , m yosin filam en
-lines,
causing a further decrease in active tension generation.
Figure 8.9. Sarcomere
lengthtension
relationships as measured in
a single fiber. A) Piecewise
linear nature of the active
forcelength relationship.
The dashed curve denotes
the forceextension relation
of resting muscle. (B)
Sarcomere structural
interpretation of panel A.
The length of a sarcomere that optimizes force production, is thus in the midregion near 2.25
m (Fig. 9: c) an d pr esettin g m u scles to th is len gth m axim izes for ce output. A comprehensive
analysis of sarcomere length changes found that most muscles operate over a surprisingly
narrow range at 94.5% of L0.
Skeletal muscle is characterized by showing negligible resting (passive) force at sarcomere
lengths less than L0. Above L0, passive force rises quasi-exponentially with sarcomere length
[the dashed curve in Fig. 9(A)]. This behavior is largely attributable to the elastic characteristics
102
of extracellular components, especially the connective tissue that envelops individual fibers,
fascicles and the whole muscle (Fig. 1).
D. Isotonic ForceVelocity Relationship
As stated earlier, the amount of force generated also depends on the velocity of muscle
shortening. Faster movements will not permit as much force output as those with a lower
velocity. This occurs because the rates of cross-bridge attachment, detachment, and reattachment are chemically limited by the enzymatic activity of the actin-activated myosin
adenosine triphosphatase (ATPase). The number of active cross bridges at any time is, thus,
reduced during fast movements of any given muscle. In addition, there are different myosin
ATPase isoforms, which differentiate various muscle types with their individual characteristic
peak cross-bridge cycling rates .
E. Influence of Strain History
Notwithstanding the success of the sliding-filament theory of muscle contraction, there are two
related phenomena that the above theory is unable to accommodate. Both are functions of strain
history. The first phenomenon is reflected in the generation of additional force (in association
with reduced metabolic energy expenditure) during an eccentric contraction.
If a muscle is stretched during an active contraction, it responds with force in excess of that
predicted from its isometric forcelength relationship. The metabolic consequences of this
behavior were first strikingly demonstrated by use of a pair of back-to-back bicycle ergometers.
A diminutive female, pedaling backward (at constant velocity and with leg musculature
necessarily contracting eccentrically) could readily exhaust an athletic man pedaling forward
(with leg extensor musculature necessarily contracting concentrically).
The above phenomenon is attributed to the ability of skeletal muscle to generate forces in excess
of in the negative velocity region of the forcevelocity relation . At the molecular level, it is
presumed to reflect the behavior of negatively strained cross-bridges, which, under sufficient
strain, may be broken without concomitant hydrolysis of ATP. This results in diminished heat
production , thereby resulting in increased efficiency .
The second phenomenon, also strain-history dependent, confers on muscle a degree of
memory. It is most readily observable on the descending limb of the forcelength relation,
where a component of force enhancement by an eccentric contraction can be exceptionally longlived . This behavior has recently been attributed to the behavior of titin (see Figure 2). Because
titin contains a segment that is able to unfold reversibly, as the muscle is stretched, it has been
implicated as the principle source of energy storage during eccentric contractions.
F. Muscle Tone
Even in the absence of overt activity, muscles have a certain tautness. This is called muscle
tone and arises from low-frequency action potentials generated within the nervous system. The
maintenance of tone (like that of posture) is also an energy consuming process. At rest, muscle
tone accounts for a major portion of baseline energy consumption.
8.5
Skeletal Muscle Energetics
A. Efficiency of Skeletal Muscle
Efficiency is defined as the mechanical power output divided by the metabolic power input. The
peak thermodynamic efficiency of muscle is around 0.4, based on input of the high-energy fuel
ATP. This value drops to about half that level when the cost of conversion of dietary substrates
into high-energy storage compounds is taken into account. This figure further ignores the
103
anabolic costs of muscle fiber synthesis, maintenance, or repair (normal turnover rates are such
that the contractile protein of active muscle is totally replaced every 24 weeks). Such costs are
usually attributed to resting or basal metabolism.
B. Metabolic Pathways
The obligatory energy substrate that skeletal muscle utilizes is ATP, which, when broken down
into ADP and , releases up to 50 kJ per mole of chemical energy . ATP is needed to fuel crossbridge cycling, but also to pump Ca back into the SR (where one ATP is needed to transport two
Ca ions against their concentration gradient) and to maintain a suitable electrochemical
environment for normal AP propagation.
ATP concentration in skeletal muscle is about 4 mM/L, which is enough to enable contraction
for several seconds or less, since ATP turnover increases linearly with force production . To
maintain contraction and force output beyond such a limited period, ATP must be constantly
replenished. The immediate source of replenishment is creatine phosphate (CrP), which is
present in muscle cells at a concentration of about 20 mMsufficient to sustain another 1020 s
of contraction.
Glucose is the main metabolic source for ATP re-synthesis in muscle; it arises from blood
glucose, muscle glycogen, or liver glycogen. The synthesis of ATP from glucose occurs by two
main pathways: oxidative phosphorylation and glycolysis. As the name suggests, oxidative
phosphorylation requires oxygen (an aerobic process that takes place in the mitochondria),
while glycolysis does not (it takes place anaerobically in the cytoplasm). Aerobic ATP synthesis
is more efficient than anaerobic synthesis, yielding 36 ATP from one molecule of glucose,
compared to only 2 ATP via the glycolytic pathway. Force can be maintained with aerobic ATP
synthesis for an extended period of time, provided that there is an adequate supply of nutrients.
In contrast, contraction based on ATP arising solely from the glycolytic pathway can maintain
maximum force for only around one minute.
Besides its lower relative ATP production, another disadvantage of glycolysis is that it produces
lactic acid (lactate plus H ), which is not efficiently metabolized by skeletal muscle . Excess
production of lactic acid leads to hydrogen ion accumulation within the muscle cell and causes a
reduction in intracellular pH. The pathophysiological consequences of this acidification are still
under investigation. Recent evidence suggests that lactoacidosis may play a less-prominent role
than previously assumed in the impairment of muscle force production upon repetitive
activation (e.g., muscle fatigue). Oxidative phosphorylation, therefore, supports the majority of
sustained locomotor activities, while anaerobic glycolysis is used for activities that require brief
periods of large power output.
C. Fiber Types
Muscle fibers can be categorized by their dominant metabolic pathway for ATP synthesis. There
are three main types (easily identified by their color): slow oxidative fibers, type I (red); fast
oxidative/glycolytic, type IIA (pink); and fast glycolytic, type IIB (white).
The different fiber colors are related to their content of myoglobin, the oxygen store of muscle
cells. Myoglobin, which is red in appearance, is present in particularly high concentration in
slow oxidative fibers (which also contain more mitochondria). Most of these fibers tend to be in
small motor units and are recruited early during muscle activity. Fast glycolytic cells, in
contrast, have very low levels of myoglobin and mitochondria, and large motor unit size. Fast
oxidative/glycolytic fibers have mixed properties.
104
Fiber types are consistent within a motor unit, but there is usually a mix of types within a given
bulk muscle. An exception to this rule is fish, which have distinct and well-defined bands of
white or red fibers.
To summarize fiber types,
Fibers are a long cylindrical cell with hundreds of nuclei
10-100 mm in diameter
1-30 cm in length
The contractile component is a myofibril
The non-contractile component is endomysium
slow twitch fiber (type I) are red in color because of abundant blood supply and
myoglobin ; they are slower to the peak when contracted and fatigue resistant
fast twitch fiber (type IIB) are pale in color because of less blood supply; they rapidly
peak in force when contracted, but are easy fatigue
intermediate fibers (type IIA) are pink, in between I and II
D. Fatigue
Fatigue (characterized by a decrease of force output) is the consequence of prolonged
contraction. In the exercising human, fatigue can be subdivided into peripheral and central
components . Central fatigue is of neuronal origin, with factors such as decreased motivation,
alertness, or neuronal excitability causing a reduction in AP frequency of motor neurons
(although the extent and detailed mechanisms of these effects are not very well understood).
Peripheral fatigue is caused primarily by biochemical processes within the muscle fiber itself.
Depletion of metabolic substrates (e.g., glycogen) has been shown to reduce muscle-force
production. In addition, accumulation of metabolic by-products (e.g., glycolytic intermediates
and inorganic phosphate), increased osmotic pressure (lacto-acidosis), and elevated muscle
temperature all contribute to peripheral fatigue. Secondary to the above biochemical changes,
there are intrinsic alterations in either cross bridge cycling and/or SR Ca handling (i.e., release
and/or re-uptake) as mechanisms that contribute directly to the reduced force generation in
fatigued muscle.
E. Muscle Sensory Cells

All skeletal muscles have both feed-forward control, through the 1A alpha motor neurons, but
feedback control, through muscle spindles, as shown below. Muscle spindles are attached to
afferent (sensory) neurons that act essentially as strain gauges, telling the CNS about the state of
stretch of muscles. The job of muscle spindles is to report to the central nervous system the
state of muscle length. The muscle spindle is composed of intrafusal type muscle and its
neuronal connections. Ordinary bulk muscle is called extrafusal, and this type is much more
abundant than the intrafusal type. Each bulk muscle needs only a few muscle spindles as
sensors. The cartoon below shows 2 muscle spindles with their innervations. The muscle
spindles lie in parallel with bulk muscle, and thus their length changes as the muscle contracts
and lengthens. As the spindles are stretched, they send signals, coded as action potentials, to
the central nervous system (CNS) , via Ia and II afferents. In addition to the afferent (sensory)
nerve fibers attached to the muscle spindle, note that efferent fibers (
effer en ts)
with the muscles also.
The picture below is a muscle spindle whose nerves are stained ,
showing them wrap around the muscle.
105
Figure 8.10. The top

image shows a muscle
spindle with its
innervations. Bottom is a
stained micrograph of a
muscle spindle.
Sensory responses from a muscle spindle are depicted in Figure 8.11. Note that as the muscle
is stretched, the rate of nerve impulses increases in synchrony. Ia afferents can powerfully
excite the ALPHA MOTOR NEURONS of the muscle containing the spindle. This is the basis of
the classical STRETCH REFLEX in which extension of the muscle (and thus its spindles) cause a
reflex contraction. Golgi tendon organs (not shown) are also involved in reflexes.
Figure 8.11. Sensory output from
a muscle spindle
The gain of these muscle spindle stretch receptors is regulated by the gamma motor neurons,
which activate the intrafusal muscle, causing contraction. This can be understood as automatic
gain control, (AGC) present in many amplifiers. This AGC works by the following mechanism:
the muscle spindle length can be changed in 2 ways: (1) it changes its length in sequence with
the bulk muscle, and (2) it changes its length when the CNS activates the motor neurons,
which contract the intrafusal muscle. In case (1), output of the spindle resembles that of Figure
7. Here, the motor neurons are off, and the sensory gain is maximal. In case (2) the motor
neurons are active, and the intrafusal fiber actively contracts during the stretch and hence its
output is reduced, as shown below in Figure 8. So for a given stretch of the bulk muscle, the
106
output of the spindle can be reduced. The reason for this control has to do with reflexes, and is
beyond the scope of this chapter.
Figure 8.12. Gain control of a muscle
stretch receptor. In each panel (a to e) ,
the top trace represents afferent neuron
pulses, originating from the sensory
organ , subjected to constant stretch; the
bottom trace represents activation of the
control neuronal influence, similar to
activation of motor neurons. Activation
of causes slowing the rate of sensory
pulses, to a degree proportional to the
magnitude of activation.
E. Summary
In comparison with artificial actuators, muscle surpasses them only in the fuel delivery and
renewability. Muscles are linear actuators that can bend around joints. They are adapted for
intermittent duty and stiffness (compliance) control. Knowledge of Biological force actuators is
crucial to the development of novel actuator technologies. To best mimic the biology, clues
derived from skeletal muscle actuator design should drive the design process.
APPENDIX: REVIEW OF BIOMECHANICAL TERMINOLOGY
Stress-strain Relations in Muscle (from Madden et Al.[1])
Stress is the typical force per cross-sectional area of the actuator material, in this case, muscle.
Peak stress is the maximum force per cross-sectional area that a material is able to maintain
position (also known as blocking stress). Thus force developed by actuators scales linearly with
cross-sectional area (whose surface normal is parallel to the direction in which actuation is
occurring).
Strain represents the displacement normalized by the original material length in the direction
of actuation. Typical strain is the strain that is often used in working devices, whereas peak or
107
max strain is the maximum strain reported. The peak strain generally cannot be obtained when
operating at peak stress. Strain rate is the average change in strain per unit time during an
actuator stroke. The maximum strain rate is usually observed at high frequencies when strains
are small. Bandwidth is relevant to some actuators, and is the frequency at which strain drops
to half of its low frequency amplitude. Limitations on bandwidth and strain rate can result from
a range of factors including speed of delivery of the input energy, rates of diffusion or heating,
internal dissipation, inertia related effects including speed of sound, and kinetics associated
with the energy transduction method .
Work density is the amount of work generated in one actuator cycle normalized by actuator
volume. It is very important to note that this does not include the volume occupied by
electrolytes, counter electrodes, power supplies, or packaging, unless otherwise stated. These
additional contributions to actuator volume do not always scale linearly with work output and
therefore need to be considered separately. Also it is emphasized that the work density is not
simply the product of peak stress and peak strain. Specific power or power to mass ratio is the
power output per unit mass of actuator material. Typically only the mass of the material itself is
considered. Peak power density can be found from the maximum product of simultaneously
measured stress and strain rate divided by density. Because of the interdependence of load and
rate, peak power is in general less than the product of peak stress and peak strain rate
normalized by density.
Efficiency is the ratio of work generated to input energy expended. Stored electrical energy and
even thermal energy can in principle
be recovered in order to improve efficiency.
Electromechanical coupling in muscle refers to the degree of ratio of electrical activation to
mechanical force output.
.
Elastic modulus is the material stiffness and generally represents the instantaneous value,
before any creep is induced. Note that mammalian muscle stiffness at rest is 40 MPa, but this
value increased 50X during contraction. Each myosin bundle can produce 5.3 pN of force
during its power stroke. There are 100 molecules per actin filament. Each filament has c.s.a. of
1.8 X 10 15 m2 in the relaxed muscle. Cycle life is the number of useful strokes that the material
is known to be able to undergo. Cycle life is often highly strain and stress dependent.
Chapter 8
1. How much force does a single myosin-actin bridge attain? How much energy does it take
and how for a single cycle of actin-myosin interaction. ? Calculate the energy efficiency
of skeletal muscle.
2. Describe the different roles of IA and II afferents. State what principles and pathways are
involved.
3. Workshop: With the gamma model, simulate the operation of a muscle spindle subject
to stretch, similar to the traces of Figure 11. Test the effect of gamma motor neuron
activity and compare results to Figure 12. Describe the model, and how it works.
108
References
1.
Madden, J.D.W., N.A. Vandesteeg, P.A. Anquetil, P.G.A. Madden, A. Takshi, R.Z. Pytel,
S.R. Lafontaine, P.A. Wieringa, and I.W. Hunter, Artificial muscle technology: Physical
principles and naval prospects. Ieee Journal of Oceanic Engineering, 2004. 29(3): p.
706-728.
109

CHAPTER 9: CELL PROPULSION
9.1
Introduction
Cells explore and sense their environment in many ways. Eukaryotic cells extending actin
protrusions and form integrin adhesions with their surroundings. These movements are
powered by a combination of protrusion generation, adhesion formation and cellular
contraction. Directed migration of cells is driven both by chemical signals, i.e. chemotropism,
and mechanical signals, i.e., mechanotropism. Movement and migration of cells is governed by
the milieu in which cells exist- this is composed of various fluids as well as solid surfaces
comprising tissues. Here we will review principles of fluid mechanics, and propulsion
mechanisms in various environments.
9.2 Cells swimming through fluids
Knowing the forces involved as cells move though fluids or when fluids flow past cells is an
important part of Cytomechanics, and requires knowledge of basic fluid mechanics. Here, we
outline just the bare minimum of such knowledge, and refer the reader to texts, such as, Life in
Moving Fluids, by S. Vogel, for more complete treatment.
Whether we consider cells migrating through their milieu (i.e. blood cells), or their milieu
flowing around them (i.e., Endothelial cells in arteries), we are interested both in the forces
involved, and any propulsive or adaptive mechanisms. To estimate these forces we need to know
the properties of the surrounding milieu, i.e. blood or plasma, sea water, etc., the shape and size
of the cell or cellular component, and relative speeds. Here, we will outline these topics.
Viscosity is the internal resistance to flow, and relates to the thickness, or stickiness of
the fluid. The concept of viscosity, or lack of slipperiness, was introduced by Newton. The
greater the viscosity, the less easily the moving body slides past the fluid. In other words,
viscosity acts as a drag on objects moving through it. Viscosity, , is measured in units of Pa-s,
known as Poise. The shear stress in a fluid represents the force needed for one lamina or layer of
fluid to slip over another; shear rate is the measure of how fast one lamina slips over another.
This model is represented below:
A
(1)
f = vo A / d
f crit = 2 /
(2)
Figure 9.1
The top plate, area, A, is being dragged over the bottom plate, separated by distance , d,
with fluid of density, , separating them, with force, f, and velocity, Vo. Note that the force (1 &
2) is proportional to the velocity, and inversely proportional to the separation distance.
Drag can be defined as the rate of removal of momentum from a body moving in a fluid.
So, a moving body experiencing a large drag will stop quickly. For example, imagine yourself
110

diving into a pool of molasses- you would glide relatively little, being stopped by the high viscous
forces. But if there were a large force pulling you, such that it exceeded the value in equation (2),
then only inertial forces would dominate. So for small forces, flow is laminar, dominant force is
friction due to viscosity; with larger forces, above the critical force, turbulence occurs, and the
dominant force is inertial.
To calculate the drag force on a particle or cellular object, we use the drag equation:
1
= 2
(3)
2
Where CD is the drag coefficient, dependent on Reynolds number (Re), as shown below:
Figure 9.2 Reynolds number for flow

around a sphere
Figure 9.3 Reynolds number for flow

around a cylinder
Now we need to know the relevant Reynolds numbers, that are dependent on object
shape, size and velocity, and on the fluid properties. Reynolds number quantifies the relative
magnitude of inertial and frictional forces in a body moving through a fluid.
Some representative Re numbers, and fluid properties are :
Fluid
m (kg m-3) (Pa-S) fcrit (N)
Air
Water
Olive Oil
Glycerine
Corn Syrup
1
1000
900
1300
1000
2 X 10-5
0.0009
0.08
1
5
4 X 10-10
8 X 10-10
7 X 10-6
0.0008
0.03
From these data and equation (3) , we can estimate an example: To pull a 5 m cell
(sphere) at a speed of 1 /sec, we can estimate f ~ 10-5 pN , based on a CD = 104 , the media
being plasma, with density near that of water =1000 .
111
Viscosity is constant for most fluids, but

varies greatly with temperature. Blood is an
exceptional fluid, in that its viscosity depends on
the nature of the flow, which depends on blood
velocity and diameter of the vessel. Typical
behavior of blood is shown at right:
Figure 9.4
Blood is much more viscous than

water because it contains formed
elements & plasma proteins, hence it
flows more slowly under the same
conditions . Water viscosity at room
temperature= 0.01 centipoise [1
centipoise (cp)] ; plasma is ~ 1.8 cp;
Blood viscosity = 3-10 cp .
In summary, the motion of a body depends on the ratio of viscous and inertial effects: Reynolds
number. Re is small for cells, and large for almost all animals. The cellular world is ruled by
friction.
9.2.1 Cell Substrate Interactions
It has been shown that the elasticity of the cellular substrate (characterized by Youngs modulus,
E) in contact with the cell influences the direction of cell migration. This process has been
termed durotaxis. The stiffness of the cellular environment is not only important for cell
migration but also appears to influence the metastatic process of cancer cells in vivo. Therefore,
a mechanistic understanding of how environmental mechanical signals influence cellular
movement and cell dynamics is an important question in cell biophysics.
Mechanical properties of the environment not only influence cell migration but also can
influence many aspects of the cell life cycle. When cells are plated onto a planar substrate, the
morphology and behavior of the cell depends on the stiffness of this substrate. If the substrate is
soft, aggregates of adhesion molecules remain small and transitory. In the opposite limit, when
the substrate is stiff, a clear network of contractile stress fibers (bundles of F actin) develops,
and strong focal adhesions anchoring the cell to the substrate are seen. Substrate stiffness can
also influence the long-term fate of cellular development. It has been shown that differentiation
of mesenchymal stem cells to more specialized cells is influenced by the stiffness of the cellular
substrate. The mechanisms behind this dependence are complex but appear to involve three
basic systems: the actin cytoskeleton, cell-surface adhesions and motor-based contraction.
Indeed, a simplified mechanically-based model showed that when the time scales of adhesion
movement and the cytoskeleton interaction are considered, cells on stiff substrates will form
numerous actin filament bundles with many filaments, while cells on soft substrates will have
fewer bundles with a smaller number of filaments. This stiffness-dependent organization of the
cellular cytoplasm could be an important feature in cellular mechanosensation.
112
It is useful to recognize that cells can move within substrates of pure liquids, semi-solids
and solids. These 3 types of substrates are modeled below. In (A), a pure liquid is represented
as a dashpot, with drag force proportional to viscosity times velocity. Applying force, F, leads to
a gradual acceleration to a constant velocity. In semi-solid (B), an object can be moved by the
force, but will eventually come to rest. In solid (C), the object is trapped, but will oscillate due to
the elasticity of the substrate.
Figure 9.5
9.4
Propulsive motors
The motor we use every day is our skeletal muscle (Chapter 8, which moves linearly i.e., backand-forth. Most free-living cells, such as bacteria, use rotary engines. Most cells contain motors
that crawl or hop. Still other motors are used in cell migration and growth. While there is much
to study about these different motor mechanisms, our task is simplified by the fact that all the
cellular motors use the same fuel: ATP. The several known motor types are outlined below:
9.4.1 Rotary Motors
The f1-ATPase motor is the best-studied rotary motor, and is found in thermophilic bacteria.
The basic plan is shown in the cartoons below. In Figure 9.1, at left, the filament that drives the
motion (the tail of the cell) is attached to the cell membrane with a kind of bearing composed of
proteins.
The f1-ATP-ase motor can either make or break ATP, and hence is reversible . It can generate a
torque of 40 pN-nM. It can perform work of 80 pn-nM (40 * 2p/3) for each 1/3 revolution.
This is equivalent to free energy from ATP hydrolysis.
113

The rotations can be visualized by attaching a large actin
filament. The key to the high efficiency of this motor is its
mechanism of fuel injection.
The ATP synthase (top right) not only is natures smallest rotary motor, but also has an
important role in producing most of the chemical energy that aerobic and photosynthetic
organisms need to stay alive. Operation of this motor was studied by using electromagnets to
force it to rotate and generate chemical energy (adenosine triphosphate, ATP). In the process,
the direction of its rotation during ATP synthesis was determined.
ATP synthase is composed of two linked multi-subunit complexes, called F0 and F1. F0 is
embedded in cellular membranes and conducts protons, whereas F1 is a peripheral complex and
contains the catalytic sites. Together they couple the flow of protons down an electrochemical
gradient to the synthesis of ATP from ADP (adenosine diphosphate) and inorganic phosphate.
114
Figure 9.7 The binding-change model for F0F1 ATP synthase.

a. Looking up at F1 from the membrane. Each blue or green area represents a pair of and -subunits, in which the catalytic sites are interfacial but mostly on the -subunit.
In step 1, the -subunit rotates through 120, driving conformational changes in the
three surrounding catalytic sites that alter their affinities (O, L or T, for open, loose or
tight) for substrates and product. In step 2, ATP forms spontaneously from tightly
bound ADP and inorganic phosphate (Pi).
b. View from the side of F0F1. The -subunit contains two partial channels. To traverse
the membrane, a proton must move through one channel to the center, bind to one of
the c subunits, and then be carried to the other partial channel by rotation of the c-ring.
The c-subunits are anchored to the -subunit (part of the rotor), whereas the a-subunit
is anchored through b2 (the stator) to the 33 hexamer. Hence, rotation of the c-ring
relative to the a-subunit in F0 will drive the rotation of the -subunit relative to the
33 hexamer in F1. New results show that the -subunit rotates in a clockwise
direction when the engine generates ATP.
9.4.2 Transport Motors
Transport of materials within eukaryotic cells takes place constantly along microtubules (MTs).
Materials such as vesicles can be carried by proteins called kinesin or dynein.
MTs have 13
protofilaments running parallel to the axis. The MT does not rotate when gliding over kinesincoatings. The kinesin takes 8 nm steps (or 7 & 9 for waddling), as shown in the figure below.
The walking needs high ATPase rate due to large duty cycle. MTs can be fabricated artificially to
demonstrate their operation .
The figure below shows cartoons of the carrier molecule walking along. At lower right,
real-time snap shots of the motion of a labelled carrier along an MT is shown.
115
Figure
9.4.3. Walking Motors

The actin-myosin (A-M) ratcheting process is shown from the point of view of actin, which is
assumed to be stationary, while the myosin walks along it (Figure 9.4).
Figure 9.8. The A-M process.
116

9.4.4 Cell Migration Motors
Figure 9.5. How neurites propel

themselves along a substrate
Figure 9.6. Forward protrusion in concert with contraction of the rear cell body
overcomes ECM resistive adhesive tractions to allow forward cell migration.
Bacterial pathogens have a cunning way of moving about inside the cells they infect they
harness components of the hosts cytoskeletal machinery, in particular, the protein actin. One
such pathogen, Rickettsia conorii, is transmitted to humans through tick bites, and causes
Mediterranean spotted fever. This bacterium assembles an elaborate tail made of actin
filaments (Figure 9.7) to propel itself through the cytoplasm of the infected host cell and to
invade neighboring cells. Rickettsia tails consist of parallel, unbranched filaments that closely
resemble those present in thread-like cellular filopodia.
Figure 9.7
Figure 9.8
117

9.3
Comparative Motor Analysis
As seen in the Figure, there are three modes of linear cellular motion. Characteristics of each
type are shown in the Table.
Figure 9.8. The

difference between
walking and hopping
propulsion
Figure 9.9
Motor
Step Size
Max Force
Max Efficiency (%)
Processivity
Mode
A-M
Variable
5 pN
20
None
Hops
Kinesin
8 nm
5 pN
50
Good
Walks
F1ATPase
120
40 pN-nm
~100
Good
Crawls
118
Chapter 9
Review Exercises and Questions
1. The motor protein, kinesin, can generate a force of 6 pN. How fast could it move a
bacterium through a typical cell?.
2. Model the rotary motor of F1 ATPase, showing the behaviors described in class.
119
Appendix
Appendix 1
Numerical Modelling with Simulink
Rules for analog simulation

1Write diff-Q with highest order on left
2. Assume you know it, and integrate to get x. (Check integration limits)
3. Perform required operations on lower derivatives.
4. Check and simulate.
5. Scale magnitudes and timing.
Dimensional Analysis
This is a technique for deriving mathematical descriptions of physical systems,
using educated guessing. The key is using standard letters to describe the 4
fundamental physical dimension, and its corresponding SI unit:
Quantity
Dimension
Units
Length
Mass
Time
Charge
1.1
M
T
Q
(m)
(kg)
(s)
(coul)
Most, if not all, other quantities used in cytomechanics are simple combinations of
these dimensions, such as, velocity = LT-1 , acceleration is
LT-2 , and so on.
As an example of dimensional analysis is finding the period of a simple pendulum,
that we know has units of T. Looking at the pendulum, we see that it is a mass,
dimension M, at the end of a string, dimension L. The only force acting on the
mass is gravity, g, with dimension , LT-2 . So now to derive the formula, we need
to write the dimensional relationship as:
T = f ( M , L, LT 2 )
We can see that there is no way that there is no way that the fundamental
dimension, M, can be transformed into T, so we can eliminate M, and realize that
the period must be independent of mass. So, to get T from L and LT-2 , i.e., g, we
can manipulate them as follows:
L
)
T= f(
LT 2
or, simplifying:
L
T= f(
)
g
Note that there will be other dimensionless parameters, in this case, 2. Other
physical systems may not be so easy to decipher, however, it is always useful to
120
Appendix
check dimensions and units.
Appendix 2
Fundamental Constants and Relations

Parameter
Symbol
Value
Boltzmann constant
1.38 10-23 J/ K
Thermal energy at room temperature
kT ( T = 295K)
Energetic Equivalents
aKT
4.1 pN-nM
4.1 10-21 J
4.1 10-24 erg
2.5 kJ/mole
0.59 kcal/mole
0.025 eV
Average kinetic energy
per molecule = 3/2 KT
Persistence Length p =
Kf/KT
Thermal Voltage = KT/q
Diffusion coefficient =
D=
KT
6r
Particles in gravity =
mgh/KT
Viscosity of water
0.001 Pa-s
A.3. Generalized Boltzman Free energy formula:

F
P1
= e kT
P2
where P's are the probabilities of states or classes 1 and 2, and F is free energy. For
example Pi can represent the concentration of particles in 2 separate compartments.
121
Appendix
A.4. Mechanical Formulae

A.4.1 Bending energy of a rod:
Hence:
Earc =
fL
2R2
= YI
p =
f
kT
SpecialCase :
when L = p andE arc = kT
E arc =
R=
p
R
f2
2kTR
= kT
p
2
= 2
Hence,
p
L
=
= 2 ...radians = 81
R p / 2
122
Appendix
A.5 Diffusion
Diffusion coefficient indicates how fast particles of radius a can move in
2
cm
a solution of viscosity, . Units are
P( x )dx =
1
2x
sec
exp(
x2
2x
)dx
x2
1
C=
exp(
)
4Dt
4 Dt
2 Dt = x
So
Water flow
Q m( t ) d t
V c ( t)
V c( 0)
0
Q m K w. A c (
P)
123
Appendix
A.7 Joule-Helmoltz Formula:

(Heat produced) = (energy input) X (0.24 cal/joule).
A.8
Entropic Spring Constant:
K sp =
3 kT
2 Lc p
where
Lc = ContourLengthofthePolymer
p = persistenceLength
A.9 Properties of Polymers

The table below gives Young's modulus, persistence length and mass per
unit length of the selected molecules. Note that Tubulin is the protein
from which microtubules are made.
Polymer
Typical
Diameter (nM)
Actin
Tubulin
Intermediate
Filaments
Silk
Collagen
filament
Collagen fibril
Elastin
Cellulose Dry
Cellulose Wet
Spectrin
DNA
8
25
10-20
Persistence
Length p
(m)
15
6000
Elastic
Modulus E
(Gpa)
2
2
Mass Density
p
(Da/nm)
110
160
5
1.5
10-300
0.02
0.05
0.002
80
40
0.002
1
4500
1900
124
Appendix
A.10. Units and Conversions

1 N=105 dynes
2
1 cal=4.186 joules
1 watt = 1joule/sec
1 N=1 Kg-m/sec2
1watt-sec= 1 joule
1 dyne=1 g-cm/sec2
1 atm=1.013 * 106 dynes/cm2
1 atm=76 cm Hg
1 hp (horse power) = 745.7 W

1 joule = 6.242 * 1018 ev
1 amp (current)= 1 coulomb/sec
1 bar=106 dynes/cm2
1 volt = joule/coulomb
1 pound=4.448 N
1 pound/inch2 (1 psi)=6894 N/m2
1 dyne/cm2=0.1 Pa
Boltzmanns constant(k)= 1.38 * 10-23

joules/oK
1 g(standard gravity)=980.665 c/sec2
1 Pa = 1 N/m2
gas constant (R)=8.31 joule/mole-oK
1 dyne/cm2=0.1 N/m2
log RT/F = 58 mV @ 25 C
1 torr =1 mmHg at 0oC
1 mole= 6.023 *1023 Avogadros
1 torr =133.32 N/m2

1 cm H2O at 4 o C= 98 N/m2
number
1 molar (M)= 1 mol/L
1 poise (viscosity)=0.1 N-sec/m2
1 molal= 1 mole(solute)/Kg(solvent)
1 joule=107 ergs
1 Faraday=964800 coulombs/mole
1 joule=1 N-m
Charge of 1 photon= 1.6 *10-19ev
A.11 List of Symbols

Symbol
C, Co, Ci
D
Da
E
e
Definition
Concentration, outside and inside cell (Moles/Liter);
also refers to curvature C= 1/R, where R= radius.
Diffusion Constant ( cm2/sec)
Dalton- the mass of 1/12 of a C-12 atom
Youngs elastic Modulus (Pa); Also used for energy (J)
Elementary unit of charge
125
Appendix
F
G
G
H
II
J
Ka, Kv
f
M
kb
[M]
M
P
Q,q
R
ree
S
S
T
V
Y
or p
Force (Newtons); also Faradays constant = 96,500 coul/mole

Gibbs free energy; also shear modulus
Gravity
Enthalpy
Moment of inertia of cross-section
Mass flux rate
Area and volume expansion moduli
3 Flexural bending modulus
Bending moment (N-m)
Boltzmanns constant
Concentration of monomer
Molecular mass
Pressure (Pa)
Electric charge
Faraday gas constant; principal radii of curvature
End-to-end length of a polymer
Entropy
Arc length (m)
Temperature (K)
Volume (m3)
Youngs Modulus
Area
Surface tension
Strain; also permittivity
Poissons ratio
Extension
Time constant
Angular frequency
Shear modulus
Viscosity
Density (g/m3)
Osmotic Pressure
Reflection coefficient
Stress (Pa)
Persistence length (m)
126

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CYTOMECHANICS

William Craelius, Ph.D.

Chapter 2: Materials of the Cell and Matrix

Chapter 3: Mechanics of Cell Types

Chapter 4: Material and Structural Mechanics

Exercises and Review Questions

Chapter 5: Cytomechanical Tools

Chapter 6: Thermodynamics of the Cytoskeleton

Chapter 7: Kinetic Behavior

Chapter 9: Cell Propulsion

Exercises and Review Questions

The Cytoskeleton (CSK)

Figure 1.2. The Cytoskeletal Network

Figure 1.3: Major

Fundamental Principles of CSK

Figure 1.5. Living geodesic forms.

Cellular tensegrity describes a structural plan whereby a network of contractile

Figure 1.8. Alignments of CSK filaments.

Figure 1.9. Hypothetical

The percolation model is useful in describing network signaling, as well as elasticity, as

Figure 1.11. Material that mimics CSK structural design.

Chapter 2: Materials of the Cell and Matrix

Figure 2.1. Assembly of

Chapter 2: Materials of the Cell and Matrix

Chapter 2: Materials of the Cell and Matrix

With time lipid bubbles that were cellular

Figure 2.3. Lipid Bilayer

Chapter 2: Materials of the Cell and Matrix

Chapter 2: Materials of the Cell and Matrix

Chapter 2: Materials of the Cell and Matrix

Figure 2.6. Integrin

Chapter 2: Materials of the Cell and Matrix

Chapter 2: Materials of the Cell and Matrix

Building the CSK

Chapter 2: Materials of the Cell and Matrix

Cells are Gels.

Swelling pressure = osmotic pressure- elastic (compressive)

Figure 2.10. Gel Model

Chapter 2: Materials of the Cell and Matrix

Calculate the capacitance of a lipid vesicle, 1 uM in diameter. Show assumptions.

Vuori, K., Integrin signaling: Tyrosine phosphorylation events in focal adhesions.

Chapter 3: Cell Types

Figure 3.1. Generic cell and comparative properties.

Chapter 3: Cell Types

Chapter 3: Cell Types

Figure 3.4. Platelet activation

Chapter 3: Cell Types

Chapter 3: Cell Types

ECs with black

Figure 3.8. Inside a blood vessel, looking at

Figure 3.9. A cardiac myocyte stained for actin

Chapter 3: Cell Types

Figure 3.10. Models of the relationship

Chapter 3: Cell Types

Chapter 3: Cell Types

Chapter 4: Material & Structural Mechanics

Weight bearing forces

Fluid pressure and shear

Compression and shear

Tension; active contraction

Fluid pressure and shear

Smooth, cardiac, and

Chapter 4: Material & Structural Mechanics

4.3. Uniaxial Stress and Strain