Cytoskeleton PDF

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CHAPTER 13
The Cytoskeleton
AB: microfilaments AB: microtubules
AB: overlay
AB: intermediate
filaments
Hepatocyte
2013 John Wiley & Sons, Inc. All rights reserved.
Keys
Traits and functions of the various cytoskeletal elements.

Techniques used to study the cytoskeleton.
Elucidate the general mechanism by which molecular motors move along
cytoskeletal elements.
Describe the structures and functions of specific molecular motors (dyneins,
kinesins, myosins).
Describe microtubule organizing centers, their structures and functions.
Explain the dynamic properties of cytoskeletal elements and how this relates to
their construction and functioning.
Elucidate the structure and function of cilia and flagella, their differences and
similarities.
Describe the mechanism of muscle contraction at cellular and molecular levels.
Elucidate the existence and importance of non-muscle motility.
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Introduction
The cytoskeleton is a network of filamentous

structures: microtubules, microfilaments, and
intermediate filaments.
Introduction
Properties of cytoskeletal components
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(13.1) Overview of the Major Functions of

the Cytoskeleton
The cytoskeleton has
many roles:
Serves as a scaffold
providing structural
support and
maintaining cell
shape.
Serves as an internal
framework to
organize organelles
within the cell.
Directs cellular
locomotion and the
movement of
materials within the
cell.
Structure and functions of the cytoskeleton
Overview of the Major Functions of the

Cytoskeleton
Role of microtubules in transporting Plasticity of the cytoskeleton: a

organelles: peroxisomes of a cell (green) mouse fibroblast that is migrating
are associated with microtubules (red). over the edge of a coverslip
Provides anchoring site for mRNA.

Serves as a signal transducer.
Ane ssent i
a lc ompone ntoft hec
ell
sdi
vi
si
onma
chi
ner
y.
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(13.2) Study of the Cytoskeleton

The Use of Live-Cell Fluorescence
Imaging
Can be used to locate fluorescently-
labeled target proteins.
Molecular processes can be observed
(live-cell imaging).
Used to reveal the location of a protein
present in very low concentrations.
Protein localization
using fluorescent
antibodies: centrin, a
protein found in the
al
g alce l
l
sfla gell
a Dynamic changes in
microtubule length in an
epithelial cell using a
rhodamine-tagged tubulin
Study of the Cytoskeleton

GFP
In vitro time lapse
Cy5
Video microscopy: follow 0.77uM/sec

activities of molecular motors
In vivo snapshot
The Use of In Vitro Single-Molecule Assays

They make possible to detect the activity of an individual protein molecule
in real time.
Can be supplement with atomic force microscopy to measure the
mechanical properties of cytoskeletal elements.
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Determining the
mechanical properties of
single cytoskeletal
filaments. A single
intermediate filament
subjected to mechanical
forces by the tip of an
atomic force microscope.
The Use of In Vitro Single-Molecule Assays

They make possible to detect the activity of an individual protein molecule in
real time.
Can be supplement with atomic force microscopy to measure the mechanical
properties of cytoskeletal elements.
Dynamic recovery of photobleached

microtubules by FRAP technique.
Cells express GFP-tubulin
Laser bleaches fluorescence
Time-lapsed recovery of fluorescence in
bleached area
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(13.3) Microtubules
Structure and
Composition
Microtubules are hollow,
cylindrical structures.
The microtubule is a set
of globular proteins
arranged in longitudinal
rows called
protofilaments.
Microtubules contain 13
protofilaments.
Each protofilament is
assembled from dimers Electron micrograph
of - and -tubulin Electron micrograph cross section through a
subunits assembled into of negatively stained microtubule revealing
tubules with plus and microtubules the 13 subunits
minus ends.
Microtubules
The structure of microtubules
Structure and
Composition
Microtubules are hollow,
cylindrical structures.
The microtubule is a set
of globular proteins
arranged in longitudinal
rows called
protofilaments.
Microtubules contain 13
protofilaments.
Each protofilament is
assembled from dimers
of - and -tubulin
subunits assembled into
tubules with plus and Ribbon model: 3D Diagram of a
minus ends. structure of the - longitudinal section
tubulin heterodimer of a microtubule
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Microtubules
Microtubule-associated proteins
Microtubule-Associated
Proteins (MAPs)
MAPs comprise a
heterogeneous group of
proteins.
MAPs attach to the surface of
microtubules to increase their
stability and promote their
assembly.
MAPs are regulated by
phosphorylation of specific
Schematic diagram of a brain amino acid residues.
MAP2 molecule bound to the
surface of a microtubule.
Microtubules
Location of microtubules
Localization of microtubules in a cultured

mouse cell shown by fluorescent anti-
tubulin antibodies. Microtubules extend
from the perinuclear region of the cell in a
radial array and curve gradually as they
conform to the shape of the cell.
Microtubules as Structural Supports and Organizers

The distribution of microtubules determines the shape of the cell.
Microtubules maintain the internal organization of cells.
Microtubules function in axonal transport.
Microtubules play a role in axonal growth during embryogenesis.
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Microtubules
Structure support and organizers
Microtubules help
maintain cell shape
by influencing
formation of the cell
wall in wheat.
Microtubules (A) and
cell wall cellulose (B)
are co-aligned
Microtubules as Structural Supports and Organizers

The distribution of microtubules determines the shape of the cell.
Microtubules maintain the internal organization of cells.
Microtubules function in axonal transport.
Microtubules play a role in axonal growth during embryogenesis.
Microtubules
Axonal transport
Movement of vesicles down
the length of an axon along
tracks of microtubules
Microtubule and
intermediate filament
organization in an axon
Microtubules as Agents of Intracellular Motility

They facilitate movement of vesicles between compartments.
Axonal transport:
Movement of neurotransmitters across the cell.
Movement away from the cell body (anterograde) and toward the cell body
(retrograde).
Mediate tracks for a variety of motor proteins.
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Microtubules
Axonal transport
Movement of vesicles down
the length of an axon along
tracks of microtubules
EM of an axon (cultured
nerve cell) showing
parallel microtubules that
function as tracks for
axonal transport

Axonal transport:
Movement away from the cell body (anterograde) and toward the cell body
(retrograde).
Microtubules
Axonal transport
Cell body
Autophagic Retrograde
vesicle movement Video micrographs
show the
progression of a
membranous
organelle along a
axon branched axon

Axonal transport:
Movement away from the cell body (anterograde) and toward the cell
body (retrograde).
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Microtubules
Motor Proteins that Traverse the Microtubular Cytoskeleton

Molecular motors convert energy from ATP into
mechanical energy.
Molecular motors move unidirectionally along their
cytoskeletal track in a stepwise manner.
Three categories of molecular motors:
Kinesin and dynein move along microtubule tracks.
Myosin moves along microfilament tracks.
Microtubules
Kinesin
Kinesins
Kinesinmember of a
superfamily called KLPs
(kinesin-like proteins).
A kinesin is a tetramer Schematic illustration
of two identical heavy of kinesin-mediated
chains and two identical and dynein-mediated
light chains. transport of vesicles
Each kinesin includes a
pair of globular heads
(motor domain),
connected to a rod-like
stalk.
Kinesin is a plus end- Force-generating
directed microtubular heads bind to the
motor based on its microtubule; tail
movement. binds to the cargo
being transported
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Microtubules
Kinesin GFP head
hand-over-hand model
microtubule
Schematic diagram of a kinesin molecule Conformational changes in the head

moving along a microtubular track (blue) and neck (red) for movement
Kinesins
They move along a single protofilament of a microtubule at a velocity
proportional to the ATP concentration.
Movement is processive, motor protein moves along an individual
microtubule for a long distance without falling off.
KLPsmov eca rgot owa rdt hec ell
splasmame mbr a ne
.
Microtubules
Kinesin-mediated organelle transport
microtubule AB microtubule AB
Alteration in the
phenotype of a cell
lacking a member of the
kinesin superfamily.
Control KO Control cells from the
extraembryonic tissues
of a normal 9.5 day
Mito AB Mito AB
mouse embryo or
embryo that lacked both
copies of gene encoding
the kinesin-like protein
KIF5B.
Control KO
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Microtubules
Cytoplasmic dynein
Cytoplasmic Dynein
Dynein responsible for the
movement of cilia and
flagella.
Structure of a cytoplasmic
Cytoplasmic dynein Huge
dynein molecule
protein with a globular,
force-generating head.
Schematic
It is a minus end-directed diagram of two
microtubular motor. vesicles moving
Requires an adaptor in opposite
(dynactin) to interact with directions along
membrane-bounded cargo. the same
microtubule
Microtubules
Cytoplasmic dynein
Cytoplasmic Dynein
Dynein responsible for the
movement of cilia and
flagella.
Cytoplasmic dynein Huge
protein with a globular,
force-generating head.
It is a minus end-directed
microtubular motor.
Requires an adaptor
(dynactin) to interact with
membrane-bounded cargo.
Schematic illustration of kinesin-mediated

and dynein-mediated transport of vesicles
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Microtubules
The centrosome
Microtubule-Organizing
Schematic diagram of
Centers (MTOCs)
a centrosome shows
MTOCs specialized the paired centrioles,
structures for the surrounding PCM, and
nucleation of microtubules. microtubules forming
Centrosome structures from the PCM
responsible for initiating
microtubules in animal cells.
It contains two barrel-
shaped centrioles
surrounded by
pericentriolar material
(PCM).
Centrioles are usually
EM cross section of a
found in pairs.
centriole showing its
pinwheel arrangement
Microtubules
The centrosome
EM showing two EM shows PCM Fluorescence micrograph

pairs of centrioles from isolated (mammalian cell) shows a
developing during centrosome centrosomal protein (Ab
the cell cycle f
or msa ma tr
ix stained yellow)
Centrosomes
Responsible for initiation and organization of the microtubular cytoskeleton.
Microtubules terminate in the PCM.
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Microtubules
The centrosome
Fluorescence micrograph of a
fibroblast exposed to colcemid with The growth of microtubules occurs by
30 min recovery before fluorescent addition of subunits at the plus end of
anti-tubulin antibody treatment the polymer away from the centrosome
Microtubule nucleation occurs perinuclear at the centrosome.

Outer microtubules in a cilia and flagella are generated at a structure known as the
Basal body.
Plant cells lack MTOCs; their microtubules are organized around the nucleus.
Microtubules
The centrosome
Fibroblast stained with EM and drawing of a centrosome

-tubulin (red) and - incubated with purified tubulin g-tubulin ring
tubulin (green) Abs then labeled with
-tubulin Abs complex model
Microtubule Nucleation: role of

-tubulin in centrosome function
MTOCs control the number of microtubules, their polarity, the number of
protofilaments, and the time and location of their assembly.
The protein
-tubulin is found in all MTOCs and is critical for MT nucleation.
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Microtubules
Four arrays of microtubules in a plant cell
The Dynamic Properties of Microtubules

There are four distinct arrays of microtubules in a dividing plant cell:
Widely distributed throughout the cortex.
Making a single transverse band.
In the form of a mitotic spindle.
As a phargmoplast assisting in the formation of the cell wall of daughter cells.
Microtubules
Micrographs (cultured tobacco cell) expressing fluorescently labeled GFP-tubulin. The

end of a newly formed microtubule (arrowhead) and branchpoint (arrow) are shown.

Newly formed microtubules branch at an angle of pre-existing microtubules.
Changes in microtubule spatial organization, a combination of 2 mechanisms:
Rearrangement of existing microtubules.
Disassembly of existing microtubules and reassembly of new ones in
different locations.
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Microtubules
EM: a microtubule with two daughter New microtubules nucleated at sites of g-

microtubules branching from its surface. tubulin present on existing microtubule

Newly formed microtubules branch at an angle of pre-existing microtubules.
Changes in microtubule spatial organization, a combination of 2 mechanisms:
Rearrangement of existing microtubules.
Disassembly of existing microtubules and reassembly of new ones in
different locations.
Microtubules
Assembly and stability
EM: microtubules
polymerized in vitro.
Middle microtubule
contains 11
protofilaments.
EM: in vitro assembly

of brain tubulin onto
plus ends of pre-
existing axoneme
The Underlying Basis of Microtubule Dynamics

Cell homogenates possess all of the macromolecules needed for assembly.
Microtubules assembled in vitro may have abnormal protofilament numbers in
the absence of a template.
Assembly is aided by MAPs and pieces of microtubules that serve as templates.
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Microtubules
The Underlying Basis of
Microtubule Dynamics
Insight into factors
that influence
microtubule
assembly and
disassembly came
from studies in vitro. Structural
cap model
GTP is required for of dynamic
microtubule instability
assembly.
Hydrolysis of GTP
leads to a
replacement of
bound GDP by new
GT Pt o recha rge
the tubulin dimer.
Microtubules
Dynamic instability: Length of a single MT in growth

Injection of tubulin (biotin) cone of a neuron over time. Reference tubulin-GFP
into fibroblast for 1 minute (yellow) and plus end (blue) are shown.
Dynamic instability
Growing and shrinking microtubules can coexist in the same region of a cell.
A given microtubule can switch back and forth between growing and
shortening phases.
It is an inherent property of the plus end of the microtubule.
Proteins called +TIPS regulate the rate of growth and shrinkage.
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Microtubules
Human lung fibroblast Dynamic instability: Length of a single MT in growth

expressing mCherry a-tubulin cone of a neuron over time. Reference tubulin-GFP
and a +TIP marker (EB3-GFP) (yellow) and plus end (blue) are shown.
Dynamic instability
Growing and shrinking microtubules can coexist in the same region of a cell.
A given microtubule can switch back and forth between growing and
shortening phases.
It is an inherent property of the plus end of the microtubule.
Proteins called +TIPS regulate the rate of growth and shrinkage.
Microtubules
Cilia and flagella
Protozoan cilia in a row are

in the same stage of the beat Cilia (red) on the
The various stages in cycle. RS: recovery stroke; ES: surface of human
the beat of a cilium effective power stroke bronchial epithelia
Cilia and Flagella: Structure and Function

Cilia and flagella are hair-like motile organelles.
They have similar structures but different motility.
Ciliat endt ooc c urinl argenumbe rsonac ell
ss ur
fac
e.
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Microtubules
Cilia and flagella
Eukaryotic flagella: The unicellular alga with Forward movement from asymmetric
labeled flagellar membrane protein (green) waveform resembling the breast stroke
Flagella exhibit a variety of different beating patterns (waveforms), depending

on the cell type.
Single-celled alga pulls itself forward by waving its two flagella in an asymmetric
manner that resembles the breaststroke of a human swimmer
Degree of asymmetry in the pattern of the beat in the algal cell is regulated by
the internal calcium ion concentration
Microtubules
The structure of the axoneme
Cilia and flagella
The structure of cilia and
flagella contains a central core
Cross section
(axoneme) consisting of
through a sperm
microtubules in a 9 + 2
axoneme
arrangement.
The basic structure of the
axoneme includes a central
sheath, connected to the A
tubules of peripheral doublets
by radial spokes.
The doublets are Schematic
interconnected to one another diagram of
by an interdoublet bridge. a protist
axoneme
A longitudinal view of the
axoneme shows continuous
nature of microtubules
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Microtubules
Longitudinal view of an axoneme
Cilia and flagella
The structure of cilia and
flagella contains a central core
(axoneme) consisting of EM: median
microtubules in a 9 + 2 longitudinal
arrangement. section of a
The basic structure of the cilium
axoneme includes a central
sheath, connected to the A
tubules of peripheral doublets
by radial spokes.
The doublets are
interconnected to one another Schematic
by an interdoublet bridge. diagram of a
longitudinal
A longitudinal view of the
section of a
axoneme shows continuous
flagellar doublet
nature of microtubules
Microtubules
Basal bodies and axonemes
EM: longitudinal section
through the basal
bodies of several cilia
Structural relationship
between basal body MTs
and the axoneme of a
cilium or flagellum
Cilia and flagella

Cilia and flagella emerge from basal bodies.
The growth of an axoneme occurs at the plus ends of microtubules.
Intraflagellar transport (IFT) is the process responsible for assembling and
maintaining flagella.
IFT depends on the activity of both plus end- and minus end-directed
microtubules.
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Microtubules
Intraflagellar transport
Cilia and flagella
EM: longitudinal
Cilia and flagella emerge section of a
from basal bodies. flagellum with
The growth of an two rows of
axoneme occurs at the protein particles
plus ends of microtubules. (arrowheads)
Intraflagellar transport between the
(IFT) is the process outer doublet
responsible for microtubules
assembling and and plasma
maintaining flagella. membrane.
IFT depends on the Inset shows
activity of both plus end- cargo moved by
and minus end-directed a direction
microtubules. specific motor
protein
Microtubules
Ciliary dynein
Sea urchin sperm (w/o cilia membrane) after
ATP
A sea urchin sperm
reactivated with
ATP following
demembranation
The Dynein Arms

The machinery for
ciliary and flagellar
motion resides in the
axoneme.
Ciliary (axonemal)
dynein is required for Chemical dissection
ATP hydrolysis, which of protozoan cilia to
supplies energy for
identify the
locomotion.
molecular motor
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Microtubules
Structure and function of ciliary dynein
EM: Platinum replica of a

rotary-shadowed, flagellar
outer-arm dynein molecule
with interpretive drawing
Dynein (from dyne, me ani

ng force,andprotein) is a huge protein (up to 2 million
daltons)
A dynein molecule consists of three heavy chains and a number of intermediate
and light chains.
Each dynein heavy chain is composed of a long stem, a wheel-shaped head, and a
stalk
Rotation of the head serves as the basic driving force for ciliary/flagellar motion
Microtubules
Structure and function of ciliary dynein
High-resolution micrographs (with interpretive model) of a

flagellar dynein heavy chain before and after its power stroke.
Dynein (from dyne, me ani

ng force,andprotein) is a huge protein (up to 2 million
daltons)
A dynein molecule consists of three heavy chains and a number of intermediate
and light chains.
Each dynein heavy chain is composed of a long stem, a wheel-shaped head, and a
stalk
Rotation of the head serves as the basic driving force for ciliary/flagellar motion
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Microtubules
Sliding-microtubule mechanism
The Mechanism of Ciliary and

Flagella Locomotion
Swinging cross-bridges
generate forces for ciliary or
flagellar movement.
Dynein arm of an A tubule
binds to a B tubule and
undergoes a conformational
change that slides tubules past
each other.
Sliding alternates from one side
of axoneme to another leading
to bending.
Forces that drive ciliary

or flagella motility
Microtubules

Flagella Locomotion
flagellar movement.
change that slides tubules past
each other.
Sliding alternates from one side
of axoneme to another leading
to bending.
The sliding-microtubule mechanism
of ciliary or flagellar motility
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Microtubules

Flagella Locomotion
flagellar movement.
change that slides tubules past Experimental demonstration of
each other. microtubule sliding. Demembranated
Sliding alternates from one side sea urchin sperm were reactivated with
of axoneme to another leading ATP and photographed by multiple
to bending. exposure. Gold beads were attached to
the exposed outer doublet MTs
The Human Perspective:

The Role of Cilia in Development and Disease
Situs inversus is a syndrome in which the

left-right body symmetry is reversed.
One cause of situs inversus is mutations in
the gene encoding ciliary proteins.
Patients with situs inversus suffer from
respiratory infections and male infertility.
Many cells have nonmotile primary cilia that
sense chemical and mechanical properties
of surrounding fluids.
Mutations in primary cilia may lead to
polycystic kidney disease.
Scanning electron micrograph
Cilia are important in developmental
showing each of these kidney
processes, and mutations lead to a range of
epithelial cells with a single,
abnormalities.
non-motile primary cilium.
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(13.4) Intermediate Filaments
Intermediate filaments
(IFs)heterogeneous group
of proteins, divided into five
major classes.
IFs classes IIV are used in
the construction of
filaments; type V (lamins)
are present in the inner
lining of the nucleus.
IFs radiate through the
cytoplasm of a wide variety
of animal cells and are
often interconnected to
other cytoskeletal filaments
by thin, wispy cross-bridges.
Intermediate Filaments
Intermediate filaments
(IFs)heterogeneous group
of proteins, divided into five
major classes.
IFs classes IIV are used in
the construction of
filaments; type V (lamins)
are present in the inner
lining of the nucleus.
IFs radiate through the
cytoplasm of a wide variety
of animal cells and are
often interconnected to
other cytoskeletal filaments Cytoskeletal elements are connected to one
by thin, wispy cross-bridges. another by protein cross-bridges (plectin).
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Assembly and Disassembly
Assembly:
Basic building block is
a rod-like tetramer
formed by tow
antiparallel dimers.
Both the tetramer and
the IF lack polarity.
IFs are less sensitive to
chemical agents than
other types of
cytoskeletal elements.
Assembly and
disassembly of IFs are
controlled by
phosphorylation and
dephosphorylation.
Models of IF assembly and architecture
Assembly:
Basic building block is Localization of
a rod-like tetramer injected
formed by tow biotinylated
antiparallel dimers. keratin during
Both the tetramer and a 20-minute
the IF lack polarity. label period
IFs are less sensitive to
chemical agents than
other types of Distribution of
cytoskeletal elements. intermediate
Assembly and filaments in the
disassembly of IFs are cell as revealed
controlled by by anti-keratin
phosphorylation and antibodies
dephosphorylation.
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Types and Functions
Organization in epithelial cells:

schematic and keratin staining
Types and Functions of IFs

IFs containing keratin form the protective barrier of the skin, and epithelial
cells of liver and pancreas.
IFs include neurofilaments, which are the major component of the network
supporting neurons.
(13.5) Microfilaments
Microfilaments are
composed of actin and are
involved in cell motility.
Using ATP, actin
polymerizes to form actin
filame nts(F-a cti
n).
The two ends of an actin
filament have different
structural characteristics
and dynamic properties.
One of the micro-filaments
appears pointed, and the
other appears barbed.
Orientation of the
EM: Determining the
arrowheads formed by
actin provides information Actin filament structure: location and polarity of
about direction of the model and EM showing its actin filaments with the
microfilament movement. double-helical architecture S1 subunit of myosin.
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Microfilaments
EM: short actin filament
labeled with S1 myosin
then used to nucleate
actin polymerization.
Addition occurs much
more rapidly at the
barbed (plus) end than
at the pointed (minus)
Actin assembly/disassembly in vitro depends

upon concentration of actin monomers.
Filament assembly leads to drop in ATP-actin.
Actin subunits are added to plus end and
removed from the minus end (steady state).
Microfilament cytoskeleton is organized by
controlling equilibrium between assembly Diagram of the kinetics of
and disassembly of microfilaments. actin-filament assembly in
vitro to achieve treadmilling
Microfilaments
Drug disruption
Actin polymerization can be a force-generating mechanism in some cells.

Dynamics of polymerization can be altered pharmacologically:
Cytoc halasinD:bl oc ks+ endsofa ctinfil
ame nt s
Phalloidin: binds to intact filaments, prevents turnover
Latrunculin: binds free monomers and prevents incorporation
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Microfilaments
Myosin as a molecular motor
Fluorescence micrograph: neurites (green) Tissue from a mouse embryo

growing out from mouse embryonic nervous l
ac kingmy osi
nIIB. Ne urit
esdon t
tissue along a coverslip laminin-coated (red). turn at the edge of the coverslip.
Myosin: The Molecular Motor of Actin Filaments

Myosins share a characteristic motor head for binding actin and hydrolyzing ATP.
The myosin tail is divergent.
Myosins can be divided into two groups:
Conventional (type II) myosins and Unconventional myosins
Microfilaments
Structure of myosin II
Conventional (Type II)
Myosins
They generate force in
muscles and some non-
muscle cells.
Each myosin II is composed
of two heavy chains, two
light chains, and two
globular heads (catalytic
sites).
All of the machinery
required for motor activity
is contained in a single head.
The tail portion plays a
structural role allowing the Electron micrograph and schematic drawing of
protein to form filaments. a myosin II molecule with one pair of heavy
chains (blue) and two pairs of light chains
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Microfilaments
Myosins
muscle cells.
Each myosin II is composed Myosin heads bound to a silicone-coated
of two heavy chains, two coverslip are incubated with actin filaments.
sites).
All of the machinery Arrowheads show the
actin filaments sliding
over the myosin
heads during the brief
structural role allowing the
protein to form filaments. period between two
exposures (1.5 sec)
Microfilaments
Myosins
muscle cells.
Each myosin II is composed
of two heavy chains, two
sites).
All of the machinery
Diagram of the staggered arrangement
of the individual myosin molecules in a
structural role allowing the
protein to form filaments. myosin II filament and EM of a bipolar
myosin filament formed in vitro
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Microfilaments
Unconventional myosins
They have only a

single head and are
unable to assembly
into filaments in vitro.
My osinI
spr eci
s e
role in cellular
activities is unclear.
Myosin V is involved
in organelle
transport.
Several of them are
associated with
cytoplasmic vesicles
and organelles.
Direct visualization of a
single myosin V molecule
Microfilaments
They have only a

single head and are
unable to assembly
into filaments in
vitro.
My os i
nI
spr eci
s e
role in cellular
activities is unclear.
Myosin V is involved
in organelle
transport.
Several of them are
associated with
cytoplasmic vesicles
and organelles. Schematic drawings of a dimeric myosin V
molecule with associated adaptor proteins
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Microfilaments
Myosin motors, such as

myosin Va, can transport
their cargo over
microfilaments, including
those present in the
peripheral regions of the cell.
The two types of motors

may act in a cooperative
manner, e.g. pigment cell in
which pigment granules
move back and forth within
cell.
Contrasting roles of microtubule-
and microfilament-based motors
in intracellular transport.
Microfilaments
Unconventional myosins and hair cells
Drawing of a hair
cell of the cochlea
Transmission EM
cross section of
a stereocilium
Fluorescence micrograph of a hair cell

(rat inner ear). Stereocilia are labeled
green from GFP-actin monomers
Hair cells are named for the bundle of stiff, hair-like stereocilia that project
from the apical surface of the cell into the fluid-filled cavity of the inner ear.
Displacement of the stereocilia by mechanical stimuli leads to the generation
of nerve impulses that we perceive as sound.
Each stereocilium is supported by a bundle of parallel actin filaments
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Microfilaments
Unconventional myosins and hair cells
Left: Myosin VIIa (green) in hair cell of Left: SEM from normal mouse cochlea hair
bullfrog. Right: Myosin XVa (green) at cells. Right: SEM from knockout mouse for
stereocilia tips in a rat auditory hair cell. myosin VIIa KO, which suffers from deafness.
Hair cells are named for the bundle of stiff, hair-like stereocilia that project
from the apical surface of the cell into the fluid-filled cavity of the inner ear.
Displacement of the stereocilia by mechanical stimuli leads to the generation
of nerve impulses that we perceive as sound.
Each stereocilium is supported by a bundle of parallel actin filaments
(13.6) Muscle Contractility
Light micrograph of a multi-

EM of a sarcomere
nucleated muscle fiber z-line
A-band: thick filament
I band: thin filament
Levels of organization M: the midline
of a skeletal muscle H: zone between A bands
A skeletal muscle fiber is a multinucleate cell as a result of fusion of myoblasts

in the embryo.
A single muscle fiber is large and highly organized.
Each muscle fiber contains hundreds of cylindrical strands called myofibrils.
33
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Muscle Contractility
The sarcomere
Sarcomere
Each myofibril showing the
consists of a overlapping
repeating array of array of thin
sarcomeres. actin-containing
(orange) and
Each sarcomere has
thick myosin-
a banding pattern
containing
that gives muscle
(purple)
fiber a striated
filaments
appearance.
Banding pattern: EM: cross section
Thin filaments through an insect
(I and A bands) flight muscle showing
Thick filaments the hexagonal
(H and A bands) arrangement of the
thin filaments around
each thick filament
The sarcomere
Schematic diagram and

electron micrographs
showing the difference
in the structure of the
sarcomere in a relaxed
and contracted muscle
The sliding Filament Model of Muscle Contraction

Skeletal muscle works by shortening fibers.
A bands remain constant in length.
H and I bands decrease in width.
Z lines on both ends of sarcomere move inward.
34
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The sarcomere
Molecular organization of the thin filament: double-helical

array of actin subunits with rod-shaped tropomyosin molecules
situated in the grooves and spaced troponin
The Composition and Organization of Thick and Thin Filaments

Thin filaments contain actin as well as tropomyosin and troponin.
Tropomyosin occupies the gap between two actin molecules.
Troponin molecules are in contact with both actin and tropomyosin.
The sarcomere
actin filaments (thin) Thick filament

The arrangement of titin molecules within the sarcomere
The third most abundant protein of skeletal muscles is titin.

Titin filaments are elastic and prevent the sarcomere from being pulled apart during
muscle stretching.
Stretch from end of sarcomere at Z line to the M band in the center.
Thene bul
i
nmol ec ulesa c tasmol ecularr ulerbyreg ulati
ngthenumbe rofa ct
in
monomers that are allowed to assemble into a thin filament.
35
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Molecular basis of contraction
A model of
the power
stroke of
the myosin
motor
Neck moves through a rotation of domain
approximately 70o producing a
movement of actin filament of 10 nm.
The Molecular Basis of Contraction

During contraction, myosin heads bend thus sliding the thin filaments over the
thick filament.
Energy from ATP hydrolysis induces a conformational change within the head.
E long a tedmy osi
nne cka ctsa sa l
evera r
m .
Attached actin filament slides a much greater distance than would be possible
byj ustmov eme ntofthe he ad.
Actinomyosin contractile cycle
The Energetics of Filament
Sliding
Energy is provided by
ATPase activity in the
myosin head.
Activated myosin attaches
to actin initiating the power
stroke.
Release of bound ADP is
followed by binding of
another ATP.
Absence of ATP prevents
dissociation of cross-
bridges causing rigor mortis.
Model of the actin-myosin contractile cycle
36
3/5/2015
Actinomyosin contractile cycle
Two cycles mechanical (interaction of
myosin with actin) and chemical
(hydrolysis of ATP) begin in
Step 1: binding of ATP to a cleft in the
myosin head, causing the detachment of
the head from the actin filament.
Step 2: hydrolysis of ATP to Pi and ADP
which energizes the head and fosters
Step 3: causing the head to bind weakly
to the actin filament
Step 4: the release of Pi which causes a
tighter attachment of the myosin head
to the thin filament and the power
stroke that moves the thin filament
toward the center of the sarcomere.
Step 5: the release of the ADP (step 5)
which sets the stage for another cycle .
Model of the actin-myosin contractile cycle
The functional anatomy of a muscle fiber
Calcium stored in internal

membranes, the sarcoplasmic
reticulum (SR). Motor neuron
impulse carried into the interior of
the fiber along the membrane of
transverse tubule to SR. The
calcium gates of the SR open,
releasing calcium into the cytosol.
Excitation-Contraction Coupling
Contact between nerve and muscle is called the neuromuscular junction.
The linking of the nerve impulse to the shortening of the sarcomere is referred
to as excitation-contraction coupling.
Action potential in muscles is propagated into the cell interior by transverse (T)
tubules.
37
3/5/2015
The role of tropomyosin in muscle contraction
Schematic diagram of the

steric hindrance model in
which the myosin-binding site
on the thin actin filaments is
controlled by the position of
Ca++
the tropomyosin molecule
Excitation-contraction coupling
T tubules terminate near the sarcoplasmic reticulum (SR), which stores Ca2+.
In a relaxed sarcomere, Ca2+ levels are low.
An action potential opens calcium channels in the SR, releasing Ca2+.
Binding of Ca2+ to troponin causes a conformational changes, shifting
tropomyosin and exposing the myosin binding site.
(13.7) Non-muscle motility
Filaments are seen to be organized into

two distinct arrays: as bundles in which
the filaments are arranged parallel to one
another (arrow) and as a cross-linked
meshwork in which the filaments are
arranged in various directions.
The study of non-muscle motility is more challenging because the critical

components tend to be present in less ordered, more labile, transient
arrangements.
They are typically restricted to a thin cortex just beneath the plasma
membrane.
Actin-binding proteins affect the localized assembly or disassembly of the
actin filaments.
38
3/5/2015
Non-muscle motility
The roles of actin-binding proteins
1) Nucleating proteins
provide a template for
adding actin monomers.
(Arp2/3 complex; formin)
branched versus
unbranched filaments
2) Monomer-sequestering
proteins bind to actin-
ATP monomers and
prevent them from
polymerizing. (thymosin
b4)
3) End-blocking (capping)
proteins regulate the
length of actin filaments
(capZ; tropomodulin).
4) Monomer-polymerizing
proteins promote the
growth of actin
filaments. (profilin)
Non-muscle motility
5) Actin filament depolymerizing
proteins bind actin-ADP
subunits for rapid turnover of
actin filaments. Example:
cofilin
6) Cross-linking proteins alter
the three-dimensional
organization of actin filaments.
Examples: filamin (X-link),
villin, fimbrin (bundling)
7) Filament-severing proteins
shorten filaments and
decrease cytoplasmic viscosity.
Example: gelsolin
8) Membrane-binding proteins
link contractile proteins to
plasma membrane. Examples:
vinculin, spectrin (dystrophin)
39
3/5/2015
Non-muscle motility
Microvilli are present on the apical

surface of epithelia that function in
absorption of solutes, such as the lining
of the intestine and wall of the kidney
tubule.
Each microvillus contains about 25 actin

filaments that are maintained in a
highly ordered arrangement by the
bundling proteins villin and fimbrin.
The role of myosin I, which is present

between the plasma membrane of the
microvillus and the peripheral actin
filaments, remains unclear. Actin filaments and actin-binding
proteins in a microvillus.
Non-muscle motility
Polymerization as a force generator
Cell infected with the EM: cell infected with

bacterium L. the same bacterium,
monocytogenes, showing the actin
which appear as red filaments that form
stained objects just behind the bacterial
in front of the green- cell and push it through
stained filamentous the cytoplasm.
actin tails.
Actin polymerization as a force-generating mechanism

Responsible for some types of motility such as cytoplasmic streaming in Listeria.
Myosin not required for motility.
ActA protein on one side of bacterium recruits Arp2/3 for actin polymerization.
40
3/5/2015
Non-muscle motility
Cell locomotion
The repetitive
sequence of
activities that
occurs as a
cell crawls
over the
Scanning electron micrograph of substratum
a mouse fibroblast crawling over
the surface of a culture dish
Cells lacking cilia or flagella move by crawling over a substrate (repetitive).

Cell locomotion is required for many activities in higher vertebrates,
including tissue and organ development, formation of blood vessels,
development of axons, wound healing, and protection against infection.
Cell locomotion also contributes to the spread of cancerous tumors.
Non-muscle motility
Cell locomotion
Step 1. Protrusion of leading edge (lamellipodium).

Step 2. Adhesion of lower surface of lamellipodium to substratum (mediated by integrins
residing in PM).
Step 3. Movement of the bulk of the cell forward over the site of attachment
(stationary). Accomplished by contractile force exerted against the substratum.
Step 4. Cell after the attachments with the substratum have been severed and the rear
of the cell has been pulled forward.
41
3/5/2015
Non-muscle motility
Motility using a lamellipodium
The leading edge of
this motile fibroblast
is fattened against
the substratum and
spread out into a veil-
like lamellipodium.
SEM: leading edge of a

cultured cell, showing the
ruffled membranes of the
lamellopodium
Cells that Crawl over the Substratum

Cultured cells crawl by forming a protrusion called a lamellipodium.
Force generation in lamellipodia occurs by adding actin monomers to
filaments, providing temporary anchorage for the cell.
Non-muscle motility
Major steps in formation of a lamellipodium
Stimulus at the cell
surface (step1) leads to
Arp2/3 complex
activation of by WASP
family (step2). Activated
complexes initiate
nucleation of actin
polymerization-
Neutrophil responding formation of actin
to a chemoattractant filaments (step 3). Once
from pipette (right) filaments have formed,
complexes attach to
Stimulus at the plasma membrane triggers localized filament side (step 4) and
polymerization of actin, causes polarization of the cell and initiate side branches
movement toward stimulus. (step 5); 70o angle
Wiskott-Aldrich syndrome: Disorder with crippled immune relative to the existing
system because white blood cells lack a functional WASP filaments. The complexes
protein and fail to respond to chemotactic signals push the PM outward
42
3/5/2015
Non-muscle motility
Lamellipodial extension
EM of mouse fibroblast: short actin-filament

branches show Arp2/3 complexes highlighted
by immunogold labeling. The Arp2/3
complexes reside at the Y-shaped junctions
where the newly polymerized filaments
have branched from preexisting filaments
Advancing lamellipodium have branched, cross-linked filamentous actin network

that resides just beneath the plasma membrane.
Tra cti
onf orc esoc c
ura tsit
e swhe ret hec ellgripst
hes ubst
rat ea ndt he
magnitude can be calculated from the dynamic patterns of substrate deformation.
Gre a t
esttrac ti
onf orce sa r
ee xertedj ustbehi ndt hecell
sleadinge dg ewhe ret he
cell adheres strongly to the underlying substratum.
Focal complexes that form near the leading edge of a motile cell exert traction
force and then typically disassemble as the cell moves forward.
Non-muscle motility
Lamellipodial extension
Migrating cell with traction
forces at highest levels (red) at
lamellipodial extension (arrow)
GFP-actin expressing fibroblast

with focal complexes shown by
rhodamine-vinculin
Advancing lamellipodium have branched, cross-linked filamentous actin network

that resides just beneath the plasma membrane.
Tra cti
onf orc esoc c
ura tsit
e swhe ret hec ellgripst
hes ubst
rat ea ndt he
magnitude can be calculated from the dynamic patterns of substrate deformation.
Gre a t
esttrac ti
onf orce sa r
ee xertedj ustbehi ndt hecell
sleadinge dg ewhe ret he
cell adheres strongly to the underlying substratum.
Focal complexes that form near the leading edge of a motile cell exert traction
force and then typically disassemble as the cell moves forward.
43
3/5/2015
Non-muscle motility
Actin and myosin in lamellipodial movement
Fluorescence micrographs
(fish keratocyte) moving
over a culture dish by
means of a broad,
flattened lamellipodium.
Actin and myosin
distribution is shown in
micrograph and drawing.
Keratocytes have been a favored system for studying locomotion because their
rapid gliding movement depends on the formation of a very broad, thin
lamellipodium.
Leading edge is filled with actin but devoid of myosin II.
Roleofmy osi
nII tog ene ratec ontrac t
il
efor cesnee dedt o
pul
l
ther
estofcell
behind the leading edge.
Non-muscle motility
Axonal outgrowth
The bulk of the axon shows little
evidence of motile activity. Video image of
The tip of the axon (growth cone) a live growth
is highly motile and shows several cone shows rod-
types of locomotor protrusions: like microspikes
(arrows) and
Microspikes- point outward to
filopodia
the edge of lamellipodium.
(arrowheads)
Filopodia - elongations that
extend and retract during
motile activity. Fluorescence
Growth cone explores its micrograph
environment and elongates its (neuron growth
axon. cone) shows actin
Lamellipodia and filopodia of filaments (green)
growth cone respond to presence and microtubules
of physical and chemical stimuli. (orange)
44
3/5/2015
Non-muscle motility
Axonal outgrowth
evidence of motile activity.
The tip of the axon (growth cone)
is highly motile and shows several
types of locomotor protrusions:
motile activity.
Growth cone explores its
environment and elongates its Directed movements of a growth
axon. cone. Video image of a live growth
Lamellipodia and filopodia of cone (Xenopus neuron) turning
growth cone respond to presence toward a diffusible protein (netrin-1)
of physical and chemical stimuli. released from a pipette (arrow).
Non-muscle motility
Axonal outgrowth
evidence of motile activity.
The tip of the axon (growth cone)
is highly motile and shows several
types of locomotor protrusions:
motile activity.
Growth cone explores its
environment and elongates its Growth cone (green) at the tip of a
axon. motor axon has made contact by
means of its filopodia with a target
Lamellipodia and filopodia of
cell that is expressing the neuronal
growth cone respond to presence
guidance factor ephrin (red).
of physical and chemical stimuli.
45
3/5/2015
Non-muscle motility
Cell shape changes during embryonic development
Ectodermal cells
elongate and form a
neural plate as
microtubules
become oriented
pa ra l
l
eltot hec e l
ls
axis.
Change in cell shape
produced by
contraction of
microfilaments.
Curvature of the
neural tube causes
outer edges to
contact one another
forming a tube Changes in cell shape: a layer of flattened ectodermal
which gives rise to cells at the mid-dorsal region of the embryo roll into a
nervous system. neural tube. Initial cell elongation likely due to
microtubules while rolling of the plate is actin-mediated.
Non-muscle motility
Cell shape changes during embryonic development
Ectodermal cells
elongate and form a
neural plate as
microtubules
become oriented
pa ra l
l
eltot hec e l
ls
axis.
Change in cell shape
produced by
contraction of
microfilaments.
Curvature of the
neural tube causes
outer edges to
Scanning electron micrograph of the dorsal
contact one another
forming a tube surface of a chick embryo as its neural
which gives rise to plate is being folded to form a tube.
nervous system.
46
3/5/2015
Synopsis
The cytoskeleton is composed of three distinct types of fibrous structures:
microtubules, intermediate filaments, and microfilaments (actin
filaments), which participate in a number of cellular activities.
Microtubules are hollow, tubular structures 25 nm in diameter that are
assembled from the protein tubulin and, in addition to the cytoskeleton,
form part of the mitotic spindle, centrioles, and the core of cilia and
flagella.
Three families of motor proteins have been identified and characterized:
kinesins and dyneins, which move along microtubules, and myosins,
which move along microfilaments.
In most cases, kinesins and cytoplasmic dynein move materials along
microtubules in opposite directions.
The nucleation of microtubules in vivo occurs in association with a
variety of MTOCs (microtubule organizing centers).
2013 John Wiley & Sons, Inc. All

rights reserved.
Synopsis
The microtubules of the cytoskeleton are dynamic polymers that are

subject to shortening, lengthening, disassembly, and reassembly.
Cilia and flagella contain a core structure, the axoneme, that is
composed of an array of microtubules that support the organelle as it
projects from the cell surface and provide the machinery for generating
the forces required for locomotion.
Intermediate filaments (IFs) are ropelike cytoskeletal structures
approximately 10 nm in diameter that, depending on the cell type, may
be composed of a variety of different protein subunits capable of
assembling into similar types of filaments.
Actin filaments (or microfilaments) are 8 nm in diameter, composed of a
double-helical polymer of the protein actin, and play a key role in
virtually all types of contractility and motility within cells.

rights reserved.
47
3/5/2015
Synopsis
The forces responsible for microfilament-dependent processes may be

generated by the assembly of the actin filament or, more often, as the
result of interaction with the motor protein myosin.
The contraction of a skeletal muscle fiber results from the sliding of
actin-containing thin filaments toward the center of the individual
sarcomeres of a myofibril, and it is driven by forces generated in the
myosin cross-bridges that extend outward from the thick filaments.
Nonmuscle motility and contractility depend on some of the same
proteins found in muscle cells, but they are arranged in less ordered,
more labile, and transient configurations.
Examples of nonmuscle motility and contractility include the crawling of
cells over a substratum and axonal outgrowth.

rights reserved.
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Traits and functions of the various cytoskeletal elements.

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The cytoskeleton is a network of filamentous

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(13.1) Overview of the Major Functions of

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Overview of the Major Functions of the

Role of microtubules in transporting Plasticity of the cytoskeleton: a

Provides anchoring site for mRNA.

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(13.2) Study of the Cytoskeleton

Study of the Cytoskeleton

Video microscopy: follow 0.77uM/sec

The Use of In Vitro Single-Molecule Assays

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Study of the Cytoskeleton

The Use of In Vitro Single-Molecule Assays

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Study of the Cytoskeleton

Dynamic recovery of photobleached

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Localization of microtubules in a cultured

Microtubules as Structural Supports and Organizers

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Microtubules as Structural Supports and Organizers

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Microtubules as Agents of Intracellular Motility

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Microtubules as Agents of Intracellular Motility

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Microtubules as Agents of Intracellular Motility

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Motor Proteins that Traverse the Microtubular Cytoskeleton

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Schematic diagram of a kinesin molecule Conformational changes in the head

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Schematic illustration of kinesin-mediated

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EM showing two EM shows PCM Fluorescence micrograph

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Microtubule nucleation occurs perinuclear at the centrosome.

Fibroblast stained with EM and drawing of a centrosome

Microtubule Nucleation: role of

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Four arrays of microtubules in a plant cell

The Dynamic Properties of Microtubules

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Micrographs (cultured tobacco cell) expressing fluorescently labeled GFP-tubulin. The

The Dynamic Properties of Microtubules

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EM: a microtubule with two daughter New microtubules nucleated at sites of g-

The Dynamic Properties of Microtubules

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EM: in vitro assembly