CYTOSKELETON MICROSCOPIC ANATOMY

CYTOSKELETON
Required Reading: Ross, 7th ed. pp.55-69
Suggested Supplementary Reading: Alberts, Chap. 16

LEARNING OBJECTIVES

1. Describe the structure and means of formation of the three types of cytoskeletal filaments –
microtubules, microfilaments (actin filaments) and intermediate filaments
2. List the properties (e.g. stability, polarity, dynamic nature) of each of the cytoskeletal components.
3. Explain how accessory proteins organize and modify the cytoskeleton.
4. Describe the role of the cytoskeleton in cell morphology, intracellular transport, cell motility, ability
to resist mechanical forces, and cell division.
5. Describe when the identification of selected components of the cytoskeleton has clinical utility.
6. Explain how the presence of an abnormal cytoskeleton underlies selected clinical conditions

I. INTRODUCTION

Cytoskeleton:
Forms the skeleton and the muscles of the cell. Forms the internal scaffolding of the cell.
Is a system of filamentous protein polymers that provide for the architecture, shape and motility of the
cell and for the directed movement of organelles and macromolecules inside the cell
The three major components of the cytoskeleton
1. Microtubules (MTs)
2. Microfilaments (actin filaments)
3. Intermediate filaments (IFs)
Plus accessory and regulatory proteins
Accounts for up to 25% of total cell protein in a non-muscle cell
Together the 3 components of the cytoskeleton resist deformation and transmit mechanical forces.

II. MICROTUBULES (MTs)

Composition (Ross, p55, Fig. 2.39)
• present in virtually all eukaryotic cells
• composed of tubulin protein heterodimers and microtubule-associated proteins (MAPs)
• are polymers of tubulin proteins made up of one a-tubulin and one b-tubulin dimer, which are
coded for by different genes
• microtubules are stiff hollow cylinders with an outer diameter of 25nm
• MAPs perform numerous functions including stabilizing MTs, spacing them out one from the
other, and regulating their interaction with other cytoskeletal elements
• g-tubulin also exists. It is found in the centrosome (also called the microtubule organizing center
or MTOC) where it helps to initiate the formation of a MT.

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MT

Properties
• are highly dynamic if not stabilized, which makes them sensitive to some antimitotic drugs
(e.g., taxol, vincristine)
• can undergo rapid bouts of assembly and disassembly
• act as a scaffolding for microtubule-based motor proteins to transport cargo (e.g., organelles)
within the cell

MT Structure
• are polarized polymers, i.e., they have a plus end and a minus end
• plus end is more dynamic (it grows and shrinks more); minus end is relatively more stable
• motor proteins read the polarity and move toward one end or the other of the MT
• some motor proteins move only toward the plus end and others move only toward the minus end

Molecular model
• composed of 13± protofilaments that associate laterally to form a hollow cylinder
• a protofilament is a linear stack of a-/b-tubulin heterodimers

Functions of microtubules include

• serve as railways along which intracellular transport can occur
• are key determinants of cell shape and polarity
o important in extended cells such as neurons for cell shape and axonal transport
o important in polarized epithelial cells and extended muscle cells
• make up the central core (axoneme) of cilia and flagella
• major components of centrioles and basal bodies
• make up the mitotic spindle
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Microtubule polarity and dynamic instability (Ross, p55, Fig. 2.39)

• dimers are added to and removed from the plus end much more rapidly than at the minus end
• the plus end can be capped by capping proteins for increased stability
• filaments show dynamic instability, i.e., they can undergo rapid cycles of growth and
shrinkage
• b-tubulin of the dimer must be loaded with GTP to participate in MT assembly. The GTP is
hydrolyzed to GDP as a function of time after the dimer is incorporated into a MT.
• a cycle of growth ends when the growing tip undergoes what is known as a catastrophe
o effect (i.e., growth stops and disassembly begins)
• in disassembled dimers the GTP has been hydrolyzed to GDP; they must be recharged with GTP
before they can participate in growth again
• when shrinkage stops and growth starts again after a catastrophe, the tubule is in a phase known
as rescue
• taxol stabilizes MTs, while colchicine prevents tubulin polymerization, and vinblastine and
vincristine depolymerize MTs

Some MAPs are Motor Proteins (ATPases) that move cargo along microtubules (Ross, p58, Fig.
2.44)
• MTs are the highways of the cell along which organelles and macromolecules such as proteins
and mRNAs can be moved within the cell (think long distance transport)
• associated motor proteins are enzymes (ATPases) that link the MT and the cargo, and move
along the MT, thus transporting the cargo
• motor proteins have a head domain that binds to the MT and determines the direction of
movement, and a tail or base that binds to the cargo and determines the specificity of which
cargo the motor protein can transport
• there are two major classes of MT motor proteins: kinesins (all but one move only toward the
plus end), and dynein (moves only toward the minus end)

Other MAPs are non-motor proteins that have other functions

• non-motor MAPS include the proteins Tau (implicated in Alzheimer’s disease), MAP1A,B,C, &
D; MAP2; & MAP4
• some organize MTs into either bundles of parallel filaments or irregular meshworks
• others regulate MT stability and dynamics (growth & disassembly)

The microtubule organizing and nucleation center: The centrosome (Ross, p58, Fig. 2.43 and
p67, Fig. 2.51)
• nucleation is the formation of a new cytoskeletal polyme
• polymerization is the elongation of an existing cytoskeletal polymer after initial nucleation has
occurred
• nucleation is slow, but elongation is rapid
• nucleation occurs at a microtubule-organizing center (MTOC) such as the centrosome
• the centrosome is found near the nucleus and is the main microtubule organizing center
• consists of a centriole, accessory proteins, and a pericentriolar matrix
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• functions of centrosomal proteins include: anchoring the MTs, severing and releasing MTs
from the centrosome, providing scaffolds & adaptors for other proteins that link to the
centrosome
• minus end of MT is anchored in the centrosome, plus end radiates out toward cell periphery
• g-tubulin (only found in centrosome) is required for nucleation; remains at the minus end of the
MT

Microtubules in cell division (Ross, p66, Fig. 2.55)

• cell division depends on MTs and on their dynamic instability
• MTs move the chromosomes during mitosis
• in mitosis, the centrioles first replicate and form 2 centrosomes that migrate to poles of the
mitotic spindle to organize astral microtubules
• centrosomes separate and align through interactions of astral MTs with cell cortex and between
opposing spindle microtubules
• the kinetochores of chromosomes are captured by spindle MTs
• chromosomes move to opposite poles as a result of microtubule dynamics and translocation by
motor proteins
• each half spindle contains a centrosome and 3 sets of MTs: astral (radiate outward to the
cortex), kinetochore (attached to kinetochores) and polar (interdigitate with opposing MTs
from the other centrosome).

Cilia and flagella (e.g., sperm tails) (Ross,p112-121):

• cilia are present on surface of epithelial cells (e.g., respiratory tract, reproductive tract)
• flagella are present in sperm
o the function of both structures is to generate movement
• flagella and cilia grow from a type of MTOC different from a centrosome that is called a basal
body
o basal bodies are closely related to centrioles
• basal bodies and centrioles are composed of nine triplets of MTs where each triplet consists of
three fused MTs. They are arranged in a (9*3 +0) array.
• basal body serves as anchoring point for the axoneme of the cilium
• minus ends of MTs in cilium are embedded in basal body; plus ends extend out to the tip of the
cilium.
• the axoneme forms the core of the cilium or flagellum. It is composed of stable MTs arranged in
a nine plus two (9*2+2) array, and includes other proteins associated with the MTs
o the nine outer doublets of MTs are composed of a complete MT fused with a partial MT
o the central pair is composed of two separate and complete MTs
o radial spokes composed of MAPs extend from the central pair to the doublets
o the motor protein called ciliary dynein is an ATPase that generates the sliding force that
moves MTs relative to one another
o accessory proteins bundle the ring of MT doublets together and convert the sliding into
bending and beating

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Clinical correlates:
Immotile cilia syndrome (Ross, p118, Folder 5.2) - a body-wide defect in axonemal structure that
results in obstructive lung disease, sterile males (due to lack of sperm motility), and increased
incidence of infertility in females (due to lack of ciliated epithelial cells in the lining of the oviduct.
These cells normally help move the ovum through the oviduct).
Kartagener’s syndrome - a combination of immotile cilia syndrome and situs inversus (reversal of
normal left-right asymmetry of internal organs). Situs inversus occurs because cilia normally
establish a leftward flow of fluid past the embryonal node during development that establishes the
left-right axis of the body. In the absence of ciliary movement, the axis is established randomly, so
approximately 50% of individuals with immotile cilia will also have situs inversus.
Polycystic kidney disease (PKD) (Ross, p114-116): kidney failure due to development of
numerous cysts from the epithelium of kidney tubules. Proteins known to be mutated in various
forms of PKD localize at least in part to cilia. Loss of functional cilia may lead to cyst formation.
Bardet-Beidl syndrome – a multisystem disorder that can include blindness due to loss of
photoreceptor function in the retina. Mutated proteins localize to cilia or basal bodies.
Spastic paraplegia – a spinal cord degenerative disease most commonly due to altered function of
spastin, a microtubule severing protein.
Cancer therapies (Ross, p65, Folder 2.2) – many cancer therapies use MT “poisons” to block
mitosis of rapidly dividing cells (such as most cancers) by over-stabilizing MTs (e.g., taxol),
depolymerizing MTs, or preventing their formation. However, these ‘poisons’ also have effects on
normal cells, resulting in undesirable side effects (e.g., neuropathies due to effects on axonal
microtubules, gastrointestinal malabsorption due to failure to replace intestinal absorptive cells, low
neutrophil counts due to the effect on the bone marrow).

III. ACTIN FILAMENTS (MICROFILAMENTS, F ACTIN) (Ross, p58-61)

Composition
• nonhollow polymers of the globular protein actin (G actin)
• helical in structure (two chains wrapping around each other)
• roughly 7 nm in diameter

Properties
• highly dynamic if not stabilized; more so than microtubules
• organized into many different configurations (e.g., bundles, meshworks) that are regulated by
accessory proteins
• Act as substrate for members of the myosin family of motor proteins to move cargo along the
filaments or generate force between filaments
• Unlike microtubules, microfilaments do not form at specific organizing centers such as the
centrosome; microfilaments can be nucleated almost anywhere in the cell

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Pointed end Barbed end

Ross, Fig. 2.47

Structure
• microfilaments are polarized, with a barbed end and a pointed end
o This terminology is derived from a method used to demonstrate the polarity of an actin
filament by coating it with isolated myosin heads
• the barbed end is analogous to the plus end of a microtubule because it is more dynamic; barbed
end is favored for assembly over the pointed end
• pointed end is analogous to the minus end (more stable)
• the myosins that move along microfilaments almost all move toward the barbed end

Functions of actin filaments

• microfilaments are concentrated in cell cortex beneath the plasma membrane. They are linked to
integral membrane proteins and thus attach the cytoskeleton to the plasma membrane
• the contractile ring of the cleavage furrow contains actin filaments and myosin; important in
cytokinesis at the end of cell division
• cell motility (amoeboid motion) is based on actin microfilaments
• short-range organelle transport (as opposed to moving an organelle from one end of the cell to the
other as a MT can do)
• contractility in muscle and non-muscle cells
• chromatin organization within the nucleus

Actin filaments
• are helical filaments
• actin is a conserved & abundant protein
• there are three major isoforms of actin (a, b, g) encoded by different genes
• the a-isoform is specific to muscle; the b- and g- isoforms are made by most cells
• have actin-associated proteins, just as MTs have microtubule-associated proteins
• actin-associated proteins differ according to the location and function of the particular actin
filament (e.g., tropomyosin is associated with the a-actin in muscle cells)

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Actin filament dynamics and nucleation (Ross, p59, Fig. 2.47)

• nucleation occurs when g- and b- subunits form a dimer or nucleus on which further polymerization
can occur
• polymerization is ATP-dependent (vs. the GTP-dependent polymerization of MTs); after
incorporation of ATP-actin monomers into the filament, the ATP is gradually hydrolyzed
• globular actin (G actin) monomers polymerize to form thin helical microfilaments (F- actin) that
associate with bound proteins
• nucleation of new microfilaments is slow, but their elongation is rapid
• nucleation is usually mediated by one of two major sets of proteins:
1. the Arp 2/3 complex, which adds actin sidechains to form mesh-works of branched
filaments as in lamellipodia (thin sheet-like projections found at the leading edge of motile
cells)
2. formins, which produce bundles of parallel unbranched filaments as in filopodia
(slender spike-like projections that extend from the lamellipodia)

Functions of actin-binding proteins:

• functions of different actin-binding proteins include: sequestering actin monomers from the
pool available for polymerization, capping actin filaments, cross-linking actin filaments to form
either a bundle of parallel filaments or a more angular meshwork, severing filaments, and
annealing (joining separate filaments together).
• actin-binding proteins are not homogeneously distributed within a cell. For example
1. stress fibers
o each stress fiber is a bundle of many actin filaments arranged in parallel (formed by
formin)
o are important for attachment of cell to substrate and generation of force on the substrate
via contraction of the stress fiber
o actin-binding proteins found in a stress fiber include myosins (allow the stress fiber to
contract) and a -actinin (stabilizes bundles)
2. at the leading edge of a moving cell
o actin filaments are arranged in a meshwork instead of a bundle, and are associated with
proteins such as Arp2/3
o the cell extends filopodia (microspikes) and lamellipodia containing dynamic
microfilaments

Myosins (Ross, p60)

• are force-generating mechanoenzymes that use energy from ATP hydrolysis to move along actin
filaments in the cell
• most myosins move toward the barbed end of the actin filament
• some myosins (e.g., myosin I) carry membrane bounded organelles along filaments (think short
distance movement); others (e.g., myosin II, the conventional myosin) move filaments relative
to one other
• in skeletal and cardiac muscle, myosin II is associated with actin to form sarcomeres
• myosin II polymerizes to form filaments (thick filaments) of its own. By interacting with actin
filaments and hydrolyzing ATP, myosin undergoes a conformational change that results in force
generation and movement of actin and myosin filaments relative to one another (i.e., muscle
contraction)

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• myosin II is also found in non-muscle cells where it is involved in establishing cell polarity and
in cell migration
• other myosins (unconventional myosins) do not form filaments or generate contractile forces

Cytokinesis
• actin filaments & myosin II are found in the contractile ring that forms during cell division
• myosin II gradually contracts the ring of actin filaments until cell separation (cytokinesis) occurs
• inhibiting myosin II would inhibit cytokinesis

Microvilli are actin-based structures (Ross, p109-113)

• found in diverse locations and have varying functions. For example in small intestine they
increase surface area for absorption of nutrients. In the inner ear (where they are called
stereocilia) they detect sound.
• have a bundle of parallel actin filaments as their core
• all barbed ends are oriented toward the tip of the microvillus
• myosin molecules are associated link the actin filaments to the plasma membrane
• between the actin filaments are proteins such as fimbrin and villin that cross-link the actin
filaments into a bundle; lateral arms that include myosin I attach the central bundle to the
membrane and allow for movement
• pointed lower ends of filaments end in a meshwork of actin filaments and intermediate filaments
called the terminal web. (Note that the Ross text includes only actin filaments as part of the
terminal web. We will go with the more inclusive definition of actin plus intermediate filaments
that are located just deep to the actin filaments.)
• stereocilia are defined as extremely long microvilli. Myosin XV is found at the tip of the
stereocilia, and regulates their length.

Cytoskeleton & cell attachment to the extracellular matrix (ECM)

• actin filaments are indirectly linked to the ECM
• cell membrane has transmembrane proteins (e.g., integrins) with an:
1. extracellular domain that binds to ECM components (e.g., integrins bind to fibronectin in
the ECM)
2. intracellular domain that is indirectly connected to actin filaments via a series of adaptor
proteins (talin, vinculin, a-actinin)
• because of these interconnections, contraction of the actin filaments inside the cell exerts force
on the extracellular matrix
• some transmembrane proteins bind to neighboring cells rather than to the ECM

Erythrocyte cytoskeleton
• is the classical model system for studying the cytoskeleton
• shape of the erythrocyte is determined by the cytoskeleton
• cytoskeleton forms a planar network just beneath the plasma membrane and is anchored to it
• provides increase resilience, flexibility and control of lateral mobility of membrane proteins
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• consists of a network of spectrin molecules joined together by protein complexes that include
short actin filaments
• other proteins bind the cytoskeleton to the plasma membrane; these proteins include band 3
dimer (a transmembrane protein), which binds to ankyrin, which binds to spectrin
• in muscle cells, dystrophin is the analog of spectrin

Clinical correlates:
Hereditary spherocytosis deforms red cells to fragile spherocytes.
Hereditary elliptocytosis deforms red cells to fragile elliptocytes.
In both these conditions the majority of cases are due to mutations in spectrin. Rarer forms are due
to mutations in spectrin &/or other associated cytoskeletal proteins.
Breast cancer - in some forms the actin-associated protein called tensin (which links integrin
receptors to the actin cytoskeleton) is defective, allowing migration of cancerous cells.
Deafness - Mutations in myosin VI, VII, and XV are associated with some forms of deafness.

IV. INTERMEDIATE FILAMENTS (Ross, p61-63

Structure
• are non-polarized filaments
• roughly 10 nm in diameter, hence intermediate between MTs (25 nm) and microfilaments (7 nm)
• are comparatively NON-dynamic structures; they form stable filaments with high tensile
strength
• are diverse filaments with many isoforms vs. 3 each for MTs and microfilaments

Functions
• have space filling functions, e.g. in neurons where diameter of axon depends on how many IFs it
contains, and diameter is important in determining conductivity
• provide tensile strength
• can have specialized functions, depending on cell type
• associated with the cytoplasmic face of certain cell junctions (desmosomes and
hemidesmosomes)

Important Points
• intermediate filament (IF) proteins are much less conserved across cell types (e.g., liver cell vs.
muscle cell vs. neuron) than MT or microfilament proteins; the IF proteins include many
different but homologous protein families
• different cell types have intermediate filaments composed of different IF proteins (can be used as
cell-specific “markers”)
• are classified as intermediate filaments (IFs) more on the basis of their size and filament structure
than on similarity in amino acid sequence
• play a more structural role as opposed to the more dynamic MTs and microfilaments
• found in most animal cells, particularly in cells that are subjected to mechanical stress
• cytoplasmic IFs usually form a network that surrounds the nucleus and extends to the cell
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periphery
o intercellular junctions called desmosomes anchor IFs to the plasma membrane to
transmit forces between adjacent cells
o Hemidesmosomes connect IFs across the plasma membrane to the extracellular matrix
(ECM)
• also forms the nuclear lamina to give support to the nuclear envelope and to aid in organizing
the chromosomal architecture in the interphase nucleus

Ross, p61, Fig. 2.50

General scheme of intermediate filament formation

• IF protein monomers have a globular amino-terminal head domain, a globular carboxyl-
terminal tail domain, and a central a-helical rod domain
• the rod domain structure is conserved among different types of IFs, while the head and tail
domains are variable and are responsible for the heterogeneity of IFs. (NOTE that this is the
opposite of what is stated in the Ross textbook at the bottom of page 60. The textbook is
incorrect.)
• two monomers form a coiled-coil dimer
• then two dimers associate in antiparallel arrangement to form tetramers
• antiparallel arrangement of dimers means there is no polarity to IFs
• tetramers associate in a staggered pattern like a brick wall
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• assembly does not require ATP or GTP hydrolysis

Types of intermediate filaments (Ross, p62, Table 2.3)

• there are five major types of IFs classified by their sequence homology
• most have a characteristic distribution in particular cell types
• they can be used as markers to determine the origin of cells such as tumor cells (i.e., did the
tumor cells arise from epithelial cells, mesenchymal cells, muscle cells, glia, or neurons).
• only lamin is widespread in many cell types (found in all nucleated cells)

TYPE SIZE DISTRIBUTION

(kDa)
Type I: acid keratins 40-70 Epithelial cells and epidermal derivatives (such as
Type II: neutral/basic keratins hair and nails), highly diverse
Type III:
Vimentin 53 Cells of mesenchymal origin (fibroblasts smooth
muscle cells)
Desmin 52 Muscle cells (smooth, skeletal, cardiac)
Glial fibrilary acidic protein 45 Glial cells
Type IV (neurofilament proteins): Neurons
NF-L
NF-M 70
NF-H 140
200

Type V: nuclear lamins A, B, C 65-75 Nuclear lamina of all nucleated cells

Clinical correlates
Epidermolysis bullosa simplex - mutation in keratin genes that causes disruption of the membrane
junctions that hold the epidermal cells together (desmosomes). This results in intraepidermal
blister formation and a skin that is very sensitive to mechanical injury.
Neuropathies – e.g., a pathological hallmark of Alzheimer’s disease is the formation of
neurofilament-containing ‘tangles’ in neurons.
Progeria – “fast aging disease”. One form (Hutchinson-Gilford progeria syndrome) is associated
with a single base substitution in the gene for one of the nuclear lamin proteins (lamin A).
Unknown exactly how this mutation results in the accelerated rate of aging that characterizes the
disease.

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Overall Note
I recommend you view an animation on YouTube: Inner Life of the Cell (Full Version - Narrated)
at https://www.youtube.com/watch?v=FzcTgrxMzZk It is 7.57 min long.

Take note of the animations of the actin filament and microtubule networks and the migration of a
vacuole attached to a kinesin motor protein as it walks along a MT trackway.

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