Chapter 15

Cytoskeletal
Systems

Lectures by
Kathleen Fitzpatrick
Simon Fraser University

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Cytoskeletal Systems
• The interior of a cell is highly structured

• The cytoskeleton is a network of interconnected

filaments and tubules extending through the
cytosol

• It plays roles in cell movement and division

• It is dynamic and changeable

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Major Structural Elements of the
Cytoskeleton
• The major structural elements of the
cytoskeleton are

–Microtubules

–Microfilaments

–Intermediate filaments

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Microtubules
• Microtubules are the largest of the
cytoskeletal components of a cell

• There are two types of microtubules

• They are involved in a variety of functions in

the cell

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Two Types of Microtubules Are
Responsible for Many Functions in
the Cell
• Cytoplasmic microtubules pervade the
cytosol and are responsible for a variety of
functions

- Maintaining axons
- Formation of mitotic and meiotic spindles
- Maintaining or altering cell shape
- Placement and movement of vesicles

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Two types of microtubules (MTs)
• Axonemal microtubules include the
organized and stable microtubules found in
structures such as

- Cilia
- Flagella
- Basal bodies to which cilia and flagella attach

• The axoneme, the central shaft of a cilium or

flagellum, is a highly ordered bundle of MTs

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Tubulin Heterodimers Are the Protein
Building Blocks of Microtubules
• MTs are straight, hollow cylinders of different
length that consist of (usually 13) longitudinal
arrays of polymers called protofilaments

• The basic subunit of a protofilament is a

heterodimer of tubulin, one -tubulin and one -
tubulin

• These bind noncovalently to form an -

heterodimer, which does not normally dissociate
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Figure 15-2

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Subunit structure
•  and subunits have very similar 3-D structure,
but only 40% amino acid identity

• Each has an N-terminal GTP binding domain, a

central domain to which colchicine can bind, and
a C-terminal domain that interacts with MAPs
(microtubule-associated proteins)

• All the dimers in the MT are oriented the same

way

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MT polarity and isoforms
• Because of dimer orientation, protofilaments
have an inherent polarity

• The two ends differ both chemically and

structurally

• Most organisms have several closely related

genes for slight variants of - and -tubulin,
referred to as isoforms

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Microtubules Can Form as Singlets,
Doublets, or Triplets
• Cytoplasmic MTs are simple tubes, or singlet
MTs, with 13 protofilaments

• Some axonemal MTs form doublet or triplet MTs

• Doublets and triplets contain one 13-

protofilament tubule (the A tubule) and one or two
additional incomplete rings (B and C tubules) of
10 or 11 protofilaments
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Microtubules Form by the Addition
of Tubulin Dimers at Their Ends
• MTs form by the reversible polymerization of
tubulin dimers in the presence of GTP and Mg 2+

• Dimers aggregate into oligomers, which serve as

“nuclei” from which new MTs grow

• This process is called nucleation; the addition

of more subunits at either end is called
elongation

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Microtubule assembly
• MT formation is slow at first, the lag phase,
due to the slow process of nucleation

• The elongation phase is much faster

• When the mass of MTs reaches a point

where the amount of free tubulin is
diminished, the assembly is balanced by
disassembly; the plateau phase

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Figure 15-3

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Critical concentration
• Microtubule assembly in vitro depends on
concentration of tubulin dimers

• The tubulin concentration at which MT assembly

is exactly balanced by disassembly is called the
critical concentration

• MTs grow when the tubulin concentration

exceeds the critical concentration and vice versa

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Addition of Tubulin Dimers Occurs
More Quickly at the Plus Ends of
Microtubules
• The two ends of an MT differ chemically, and one
can grow or shrink much faster than the other
• This can be seen by mixing basal bodies
(structures found at the base of cilia) with tubulin
heterodimers
• The rapidly growing MT end is the plus end and
the other is the minus end

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Microtubule treadmilling
• The plus and minus ends of microtubules have
different critical concentrations

• If the [tubulin subunits] is above the critical

concentration for the plus end but below that of
the minus end, treadmilling will occur

• Treadmilling: addition of subunits at the plus end,

and removal from the minus end

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Figure 15-5

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Drugs Can Affect the Assembly of
Microtubules
• Colchicine binds to tubulin monomers,
inhibiting their assembly into MTs and promoting
MT disassembly

• Vinblastin, vincristine are related compounds

• Nocodazole inhibits MT assembly, and its

effects are more easily reversed than those of
colchicine
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Antimitotic drugs
• These drugs are called antimitotic drugs
because they interfere with spindle assembly
and thus inhibit cell division

• They are useful for cancer treatment

(vinblastine, vincristine) because cancer cells
are rapidly dividing and very sensitive to drugs
that inhibit mitosis

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Taxol
• Taxol binds tightly to microtubules and
stabilizes them, causing a depletion of free
tubulin subunits

• It causes dividing cells to arrest during mitosis

• It is also used in cancer treatment, especially for

breast cancer

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GTP Hydrolysis Contributes to the
Dynamic Instability of Microtubules
• Each tubulin heterodimer binds two GTP molecules, -
tubulin binds one and -tubulin binds a second

• The GTP bound to the -subunit is hydrolyzed to GDP

after the heterodimer is added to the MT

• GTP is needed to promote heterodimer interactions

and addition to MTs, but its hydrolysis is not required
for MT assembly

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Dynamic instability
• Dynamic instability model: one population of
MTs grows by polymerization at the plus ends
whereas another population shrinks by
depolymerization

• Growing MTs have GTP at the plus ends, and

shrinking MTs have GDP

• The GTP cap at the plus end prevents subunit

removal

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Figure 15-6A

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GTP-tubulin and dynamic instability
• If [GTP-tubulin] is high, it is added to an MT,
quickly creating a large GTP-tubulin cap

• If the concentration falls, the rate of tubulin

addition decreases

• At a low [GTP-tubulin], the rate of GTP

hydrolysis exceeds the rate of subunit addition
and the cap shrinks

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Catastrophe and rescue

• Individual MTs can go through periods of growth

and shrinkage; a switch from growth to
shrinkage is called microtubule catastrophe

• A sudden switch back to growth phase is called

microtubule rescue

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Figure 15-6B

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Microtubules Originate from
Microtubule- Organizing Centers
Within the Cell
• MTs originate from a microtubule-organizing
center (MTOC)
• Many cells have an MTOC called a centrosome
near the nucleus
• In animal cells the centrosome is associated with
two centrioles, surrounded by pericentriolar
material

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Centriole structure
• Centriole walls are formed by 9 pairs of triplet
microtubules

• They are oriented at right angles to each other

• They are involved in basal body formation for cilia

and flagella

• Cells without centrioles have poorly organized

mitotic spindles

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Figure 15-8A,B

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-tubulin

• Centrosomes have large ring-shaped protein

complexes in them; these contain -tubulin
(along with gamma tubulin ring proteins: GRiPs)

• -tubulin ring complexes (-TuRCs) nucleate

the assembly of new MTs away from the
centrosome

• Loss of -TuRCs prevents a cell from nucleating

MTs

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Figure 15-9A

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MTOCs Organize and Polarize the
Micotubules Within Cells
• MTOCs nucleate and anchor MTs

• MTs grow outward from the MTOC with a fixed

polarity—the minus ends are anchored in the
MTOC

• Because of this, dynamic growth and shrinkage

of MTs occurs at the plus ends, near the cell
periphery
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Figure 15-10A-C

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Figure 15-10D

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Microtubule Stability Is Tightly Regulated
in Cells by a Variety of Microtubule-
Binding Proteins

• Some MT-binding proteins use ATP to drive

vesicle or organelle transport or to generate
sliding forces between MTs

• Others regulate MT structure

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Microtubule-Stabilizing/Bundling
Proteins
• MAPs, microtubule-associated proteins, bind at
regular intervals along a microtubule wall, allowing
for interaction with other cellular structures and
filaments

– A MAP called Tau causes MTs to form tight

bundles in axons

– MAP2 promotes the formation of looser bundles

in dendrites
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MAPs that promote bundling
• MAPs such as Tau and MAP2 have two regions

• One region binds to the MT wall and another

part of the protein extends at right angles to the
MT to allow for interaction with other proteins

• The length of the extended “arm” controls the

spacing of MTs in the bundle

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Figure 15-11A

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+–TIP Proteins
• MTs can be stabilized by proteins that
“capture” and protect the growing plus ends

• These are +–TIP proteins (+-end tubulin

interacting proteins)

• These decrease the likelihood that MTs will

undergo catastrophic subunit loss

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Figure 15-11B

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Microtubule-Destabilizing/Severing
Proteins
• Some proteins promote depolarization of MTs

- Stathmin/Op18 binds to tubulin heterodimers and

prevents their polymerization

- Catastrophins act at the ends of MTs and promote

the peeling of subunits from the ends

- Proteins such as katanins sever MTs

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Figure 15-11C

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Microfilaments
• Microfilaments are the smallest of the
cytoskeletal filaments

• They are best known for their role in muscle

contraction

• They play a role in cell migration, amoeboid

movement, and cytoplasmic streaming

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Additional roles of microfilaments
• Development and maintenance of cell shape
(via microfilaments just beneath the plasma
membrane at the cell cortex)

• Structural core of microvilli

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Actin Is the Protein Building Block
of Microfilaments
• Actin is a very abundant protein in all eukaryotic
cells

• Once synthesized, it folds into a globular-shaped

molecule that can bind ATP or ADP
(G-actin; globular actin)

• G-actin molecules polymerize to form

microfilaments, F-actin
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Figure 15-12A

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Different Types of Actin Are Found
in Cells
• Actin is highly conserved, but there are some
variants

• Actins can be divided into muscle-specific actins

(-actins) and nonmuscle actins (- and
-actins)

• - and -actin localize to different regions of a cell

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G-Actin Monomers Polymerize into
F-Actin Microfilaments
• G-actin monomers can polymerize reversibly into
filaments with a lag phase, and elongation
phase, similar to tubulin assembly

• F-actin filaments are composed of two linear

strands of polymerized G-actin, wound into a
helix

• All the actin monomers in the filament have the

same orientation
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Demonstration of microfilament polarity

• Myosin subfragment 1 (S1) can be incubated with

microfilaments (MFs)

• S1 fragments bind and decorate the actin MFs in

a distinctive arrowhead pattern

• The plus end of an MF is called the barbed end

and the minus end is called the pointed end,
because of this pattern

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Figure 15-13A,B

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Figure 15-13C

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Polarity of microfilaments
• The polarity of MFs is reflected in more rapid
addition or loss of G-actin at the plus end than
the minus end

• After the G-actin monomers assemble onto a

microfilament, the ATP bound to them is slowly
hydrolysed

• So, the growing MF ends have ATP-actin,

whereas most of the MF is composed of ADP-
actin

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Specific Drugs Affect
Polymerization of Microfilaments
• Cytochalasins are fungal metabolites that
prevent the addition of new monomers to
existing MFs

• Latrunculin A is a toxin that sequesters actin

monomers and prevents their addition to MFs

• Phalloidin stabilizes MFs and prevents their

depolymerization

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Actin-Binding Proteins Regulate the
Polymerization, Length, and
Organization of Actin
• Cells can control where actin assembles and the
structure of the resulting network

• They use a variety of actin-binding proteins to do

so

• Control occurs at the nucleation, elongation, and

severing of MFs, and the association of MFs into
networks
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Figure 15-16

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Proteins That Regulate Polymerization
• If the concentration of ATP-bound G-actin is high,
microfilaments will assemble until the G-actin is
limiting

• In the cell, a large amount of free G-actin is not

available because it is bound by thymosin 4

• Profilin competes with thymosin 4 for G-actin

binding

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Proteins That Cap Actin Filaments
• Whether MFs can grow depends on whether
their filament ends are capped

• Capping proteins bind the ends of a filament

to prevent further loss or addition of subunits

• CapZ binds to plus ends to prevent addition

of subunits there; tropomodulins bind to
minus ends, preventing loss of subunits there

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Proteins That Crosslink Actin Filaments
• Often, actin networks form as loose networks of
crosslinked filaments

• One of the proteins important in the formation of

these networks is filamin

• Filamin act as splices, joining two MFs together

where they intersect

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Proteins That Sever Actin Filaments
• MFs are broken up by proteins that sever and/or
cap them

• Gelsolin breaks actin MFs and caps the newly

exposed plus ends, preventing further
polymerization

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Proteins That Bundle Actin Filaments
• Some actin-containing structures can be highly
ordered

• Actin may be bundled into tightly organized

arrays, called focal contacts or focal adhesions

• -actinin is a protein that is prominent in such

structures

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Microvilli
• Actin bundles in microvilli are the best-studied
examples of ordered actin structures

• Microvilli are prominent features of intestinal

mucosal cells; they increase the surface area of
the cells

• The core of a microvillus consists of a tight

bundle of microfilaments with the ends pointed
toward the tip

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Crosslinks
• The MFs are connected to the plasma
membrane by crosslinks made of myosin I and
calmodulin

• The MFs in the bundle are tightly bound together

by crosslinking proteins fimbrin and villin

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Figure 15-17

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Proteins That Link Actin to Membranes
• MFs are connected to the plasma membrane
and exert force on it during cell movement or
cytokinesis

• This (indirect) connection to the membrane

requires one or more linking proteins

• One group of such proteins is the band 4.1,

ezrin, radixin, and moesin family; another is
spectrin and ankyrin

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Figure 15-19A

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Proteins That Promote Actin Branching
and Growth
• Besides loose networks and bundles, actin can
form a dendritic (treelike) network

• A complex of actin-related proteins, the Arp2/3

complex, nucleates new branches on the sides of
filaments

• Arp2/3 branching is activated by a family of

proteins that includes WASP (Wiskott-Aldrich
syndrome protein) and WAVE/Scar
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Figure 15-20B

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Long actin filaments
• For some cell functions long actin filaments are
needed

• In this case, actin polymerization is regulated

independently of the Arp2/3 complex, through
proteins called formins

• Formins move along the end of the growing

filament as they promote polymerization

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Figure 15-20C

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Cell Signaling Regulates Where and
When Actin-Based Structures Assemble

• Both plasma membrane lipids and several

small G proteins related to Ras regulate the
formation, stability, and breakdown of MFs

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Inositol Phospholipids
• Phosphatidylinositol-4,5-bisphosphate (PIP2)
can bind to profilin, CapZ, and proteins such a
ezrin

• PIP2 recruits these proteins to the membrane

and regulates their interactions with actin

• CapZ binds tightly to PIP2 resulting in its

removal from the end of a MF, promoting
disassembly

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Rho Family GTPases
• The cytoskeleton of cells exposed to certain
growth factors can undergo a dramatic change

• Many signals that result in these changes act via

a family of monomeric G proteins called Rho
GTPases

• Three key family members are Rho, Rac, and

Cdc42

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Rho family GTPases, effect on
cytoskeleton
• Activation of the Rho pathway results in
formation of stress fibers

• Rac activation results in extension of

lamellipodia

• Cdc42 activation results in the formation of

filopodia

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Figure 15-21A-D

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Figure 15-21E

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Regulation of Rho GTPases
• Rho GTPases are stimulated by guanine-
nucleotide exchange factors (GEFs) through the
exchange of bound GDP for GTP

• GTPase activating proteins (GAPs) inactivate

Rho GTPases by causing them to hydrolyze
their bound GTPs to GDP

• Guanine-nucleotide dissociation inhibitors

(GDIs) sequester inactive Rho GTPases in the
cytosol

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Intermediate Filaments
• Intermediate filaments are the most stable
and least soluble cytoskeletal components
and are not polarized

• An abundant intermediate filament (IF) is

keratin, an important component of structures
that grow from skin in animals

• IFs may support the entire cytoskeleton

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Intermediate Filament Proteins Are
Tissue Specific
• IFs differ greatly in amino acid composition from
tissue to tissue

• They are grouped into six classes

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Classes of intermediate filament proteins
• Class I: acidic keratins

• Class II: basic or neutral keratins

• Proteins of classes I and II make up the

tonofilaments found in epithelial surfaces
covering the body and lining its cavities

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Classes of intermediate filament proteins
(continued)
• Class III: includes vimentin (connective tissue),
desmin (muscle cells), and glial fibrillary acidic
(GFA) protein (glial cells)

• Class IV: These are the neurofilament (NF)

proteins found in neurofilaments of nerve cells

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Classes of intermediate filament proteins
(continued)
• Class V: includes the nuclear lamins A, B, and C
that form a network along the inner surface of the
nuclear membrane

• Class VI: Neurofilaments in the nerve cells of

embryos are made of nestin

• Animal cells can be distinguished based on the

types of IF proteins they contain—intermediate
filament typing
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Intermediate Filaments Assemble
from Fibrous Subunits
• IF proteins are fibrous rather than globular

• All have a homologous central rodlike domain

conserved in size, secondary structure, and to
some extent, in sequence

• Flanking the central helical domain are N- and

C-terminal domains that differ greatly among IF
proteins

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Intermediate filament assembly
• The basic structural unit consists of two IF
polypeptides intertwined into a coiled-coil

• The two polypeptides are aligned in parallel

• Two such dimers align laterally to form a

tetrameric protofilament

• Protofilaments overlap to build up a filamentous

structure about 8 protofilaments thick
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Figure 15-23

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