13 Cytoskeletal Systems

The Cytoskeleton.
Fibroblast cells stained for
DNA (magenta), tubulin
(yellow), and F-actin
(blue) (fluorescence
microscopy).

T he cytosol of the eukaryotic cell was originally regarded as the generally uninteresting,
gel-like substance in which the nucleus and other organelles were suspended.
Advances in microscopy and other investigative techniques have revealed that the cytosol is
anything but uninteresting: The interior of a eukaryotic cell is highly structured and dynamic.
Part of this structure is provided by the cytoskeleton, a complex network of interconnected
filaments and tubules that extends throughout the cytosol, from the nucleus to the inner
surface of the plasma membrane.
The term cytoskeleton accurately expresses the role of this polymer network in providing
an architectural framework for cellular function. It confers a high level of internal organization
www.masteringbiology.com

on cells and enables them to assume and maintain complex shapes that would not
otherwise be possible. The electron micrograph in Figure 13-1 shows just how dense and
fibrous this network can be. The name does not, however, convey the dynamic, changeable
nature of the cytoskeleton and its critical involvement in a great variety of cellular processes.
The cytoskeleton plays important roles in cell movement and cell division, and in
eukaryotes it actively moves membrane-bounded organelles within the cytosol. It plays a
similar role for messenger RNA and other cellular components. The cytoskeleton is also
intimately related to other processes, such as cell signaling and cell-cell adhesion. The
cytoskeleton is altered by events at the cell surface and, at the same time, appears to
participate in and modulate these events. 375

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0.5 mm
Figure 13-1 The Cytoskeleton. In this colorized electron micro-
graph, the three major elements of the eukaryotic cytoskeleton can
be seen together. Yellow: actin microfilaments; blue: intermediate
filaments; pink: microtubules (deep-etch TEM).
13.1 Major Structural Elements of
the Cytoskeleton
A fundamental feature of the cytoskeleton in both eukaryotes
and prokaryotes is its modularity; that is, a small number of
cytoskeletal elements are deployed in different locations and
arranged in different ways to meet the needs of a particular
cellular structure. In this way the cytoskeleton is very much
like the skeleton of the human body, in which only a few ele-
ments (bone, cartilage, tendons, and ligaments) are used re-
peatedly to give the body its overall shape and structure. We
now turn to the basic elements of the cytoskeleton in eukary-
otes and prokaryotes.
Eukaryotes Have Three Basic Types
of Cytoskeletal Elements
The three major structural elements of the cytoskeleton in eu-
karyotes are microtubules, microfilaments, and intermediate fila-
ments. The existence of three distinct systems of filaments and
tubules was first revealed by electron microscopy. Biochemi-
cal and cytochemical studies then identified the distinctive
proteins of each system. The technique of immunofluorescence
(see Figure 1-A3 and the Appendix) was especially important
in localizing specific proteins to the cytoskeleton.
Each structural element of the cytoskeleton has a char-
acteristic size, structure, and intracellular distribution, and
each element is formed by the polymerization of a differ-
ent kind of protein subunit (Table 13-1). Microtubules are
composed of the protein tubulin and are about 25 nm in di-
ameter. Microfilaments, with a diameter of about 7 nm, are
polymers of the protein actin. Intermediate filaments have
diameters in the range of 8–12 nm. Intermediate filament sub-
units differ depending on the cell type. Each type of cytoskel-
etal filament has a number of other proteins associated with it.
These accessory proteins account for some of the remarkable
structural and functional diversity of cytoskeletal networks.
376
M13_HARD7694_09_GE_C13.indd 376
In addition to the three fundamental polymer systems
within eukaryotic cells, other polymer networks are located
within cells. One of these networks is composed of proteins
called septins. Originally identified in yeast, septins are some-
times called “the fourth cytoskeleton.” They are closely as-
sociated with the contractile ring, a structure involved in the
pinching off of daughter cells during cell division. You have
already encountered other proteins that act via polymeriza-
tion (see Chapter 12): many proteins that bend membranes do
so by forming large polymers. Each monomer imparts a slight
bend to the membrane, but many can collectively impart large
bends to the plasma membrane or internal membranes.
Bacteria Have Cytoskeletal Systems That Are
Structurally Similar to Those in Eukaryotes
Cytoskeletal proteins were once thought to be unique to
eukaryotes. However, it is now clear that bacteria and ar-
chaea have polymer systems that function similarly to mi-
crofilaments, microtubules, and intermediate filaments.
Figure 13-2 illustrates three examples. Based on the effects of
mutating prokaryotic cytoskeletal proteins, it is clear that they
play roles similar to those of their eukaryotic counterparts.
For example, the actin-like MreB protein helps establish cell
shape and is involved in DNA segregation during cell division;
the tubulin-like FtsZ protein is involved in determining where
bacterial cells will divide; and the intermediate filament-like
crescentin protein is an important regulator of cell shape.
Significantly, the FtsZ protein is produced by certain eu-
karyotic organelles, such as chloroplasts and mitochondria,
and localizes to sites where these organelles divide. This find-
ing provides further evidence for the endosymbiont theory
(discussed in Chapter 4). Although prokaryotic cytoskeletal
proteins are not very similar to their eukaryotic counterparts
at the level of amino acid sequence, X-ray crystallography
has shown that when they are assembled into polymers, their
overall structure is remarkably similar (Figure 13-2b). More-
over, the equivalent proteins bind the same phosphonucleo-
tides (for example, GTP, ATP) as their eukaryotic equivalents,
indicating striking similarities at the biochemical level.
The Cytoskeleton Is Dynamically Assembled
and Disassembled
The cytoskeleton is perhaps best known for its roles in cell mo-
tility. For example, microfilaments are essential components of
muscle fibrils, and microtubules are the structural elements of
cilia and flagella, appendages that enable certain cells to either
propel themselves through a fluid environment or move fluids
past the cell. These structures are large enough to be seen by
light microscopy and were therefore known and studied long
before it became clear that the same structural elements are
also integral parts of the cytoskeleton in most cells.
With the advent of sophisticated microscopy techniques,
it eventually became clear that most cells dynamically regu-
late where and when specific cytoskeletal structures are
assembled and disassembled. Recent progress in understand-
ing cytoskeletal structure relies heavily on a combination of
powerful techniques. Along with chemical disruption of the
cytoskeleton (which we discuss below), modern cell biologists
17/02/17 7:16 am
 Table 13-1 Properties of Microtubules, Microfilaments, and Intermediate Filaments

Microtubules Microfilaments Intermediate Filaments

Polymer Subunit

10 mm
25 nm
8–12 nm
7 nm

Polymer

Chapter 13
Protofilament

a b a b a b
Subunit

ab-tubulin heterodimers G-actin monomers IF dimer

|Cytoskeletal Systems
Structure Hollow tube with a wall consisting of typically Two intertwined chains Eight protofilaments joined end to end with
13 protofilaments of F-actin staggered overlaps
Diameter 25 nm 7 nm 8–12 nm

Monomers a-tubulin, b-tubulin G-actin Six classes of proteins; see Table 13-4, page 397
Polarity Plus, minus ends Plus, minus ends No known polarity
Nucleotide GTP ATP None
substrate
Functions Cytosolic MTs: Muscle contraction Structural support
Organization and maintenance of animal cell Cell locomotion Maintenance of animal cell shape
shape and polarity
Chromosome movements Cytoplasmic streaming Formation of nuclear lamina and scaffolding
Intracellular transport/trafficking, and movement Cytokinesis Strengthening of nerve cell axons
of organelles (neurofilament protein)
Axonemal MTs: Cell motility Maintenance of animal Keeping myofibrils in register (desmin)
cell shape
Intracellular transport/
trafficking

use fluorescence microscopy, digital video microscopy, and
various types of electron microscopy to study the cytoskeleton
(Table 13-2, page 379). (Each of these techniques is described
in more detail in the Appendix.) In parallel with increasing-
ly sophisticated biochemical studies, modern visualization
techniques have revealed the incredibly dynamic nature of
the cytoskeleton and the remarkably elaborate structures it
comprises.
In this chapter, we will focus on the structure of the cyto-
skeleton and how its components are dynamically assembled
and disassembled. In each case, we will consider the chemistry
of the subunit(s), the structure of the polymer and how it is
polymerized, the role of accessory proteins, and some of the
structural and functional roles each component plays within
the cell. In doing so, we will be discussing microtubules, mi-
crofilaments, and intermediate filaments as though they were
separate entities, each with its own independent functions. In
reality, the components of the cytoskeleton are linked together
both structurally and functionally, as we will see in the last
section of this chapter.
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 Rod Curved Two Types of Microtubules Are Responsible
(E. coli, B. subtilis) (C. crescentus)
for Many Functions in the Cell
Microtubules can be classified into two general groups, which
differ in both degree of organization and structural stability.
Spherical
(S. aureus) MreB The first group comprises an often loosely organized, dynamic
network of cytosolic microtubules. The existence of cytosolic
MTs was not recognized until the early 1960s, when better fixa-
FtsZ tion techniques permitted direct visualization of the network of
Crescentin
MTs now known to pervade the cytosol of most eukaryotic cells.
(a) Prokaryotic Since then, fluorescence microscopy has revealed the diversity
cytoskeletal and complexity of MT networks in different cell types.
proteins Cytosolic MTs are responsible for a variety of cellular
functions. In animals, for example, crosslinked bundles of
cytosolic MTs are required to maintain axons (specialized
extension of nerve cells). In plant cells, cytosolic MTs gov-
ern the orientation of cellulose microfibrils deposited during
GTP the growth of cell walls. Significantly, cytosolic MTs form the
GDP mitotic and meiotic spindles that are essential for the move-
ment of chromosomes during mitosis and meiosis. Cytosolic
microtubules also contribute to the spatial disposition and
directional movement of vesicles and organelles by providing
an organized system of fibers to guide their movement.
GTP The second group of microtubules, axonemal micro-
GDP
tubules, includes the highly organized, stable microtubules
found in subcellular structures specialized for cellular move-
ment, including cilia, flagella, and the basal bodies to which
these appendages are attached. The central shaft, or axoneme,
of a cilium or flagellum consists of a highly ordered bundle
GTP
of axonemal MTs and associated proteins. (You have already
GDP
encountered an example of such a structure; the axoneme of
the sperm tail shown in Figure 4-13 consists of MTs.) Given
FtsZ ab-tubulin their order and stability, it is not surprising that the axone-
mal MTs were the first of the two groups to be recognized and
(b) Structure comparison studied.
Figure 13-2 Cytoskeletal Proteins in Prokaryotes Are
Similar to Those in Eukaryotes. (a) The distribution of several
Tubulin Heterodimers Are the Protein Building
bacterial cytoskeletal proteins is shown. Blue: the microtubule- Blocks of Microtubules
like FtsZ protein; yellow: the actin-like MreB protein; red: the MTs are straight, hollow cylinders with an outer diame-
intermediate-filament-like protein crescentin. (b) Comparison of the ter of about 25 nm and an inner diameter of about 15 nm
structure of FtsZ homodimer (left) and an ab-tubulin heterodimer (Figure 13-3). Microtubules vary greatly in length. Some are
(based on X-ray crystallography). Note the similarity in monomer less than 200 nm long; others, such as axonemal MTs, can be
folding and filament structure.
many micrometers in length. The MT wall consists of longitu-
dinal arrays of linear polymers called protofilaments. There
are usually 13 protofilaments arranged side by side around
the hollow center, or lumen, of the MT. Although some MTs in
CONCEPT CHECK 13.1
some animals contain more or fewer protofilaments, 13 is by
Cytoskeletal polymers are modular, and their assembly is
far the most common number.
reversible. What advantages do you think there are to using
As shown in Figure 13-3, the basic subunit of a protofila-
repeating structural elements to construct the cytoskeleton?
ment is a heterodimer (from hetero, “different” and dimer, refer-
ring to two subunits) of the protein tubulin. Protofilaments
13.2 Microtubules are composed of one molecule of a-tubulin and one molecule
of b-tubulin. As soon as individual a- and b-tubulin mole-
Microtubules (MTs) are the largest structural elements of cules are synthesized, they bind noncovalently to each other
the eukaryotic cytoskeleton (see Table 13-1, page 377). They to produce an ab-heterodimer that does not dissociate un-
function in a wide array of cellular processes whose common der normal conditions.
theme is movement—either moving components within cells Individual a- and b-tubulin molecules have diameters
or moving fluid outside of cells. In this chapter, we will con- of about 4–5 nm and molecular weights of 55 kDa. Struc-
sider MTs structurally, and in Chapter 14, we will consider the tural studies show that a- and b-tubulins have nearly identi-
mechanisms by which MT-dependent movement is achieved. cal three-dimensional structures, even though they share only
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 Table 13-2 Techniques for Visualizing the Cytoskeleton

Technique Description Example

Fluorescence Fluorescent compounds directly A fibroblast stained with

microscopy on fixed bind to cytoskeletal proteins, or fluorescent antibodies directed
specimens* antibodies are used to indirectly against actin shows bundles of
label cytoskeletal proteins in actin filaments.
chemically preserved cells,
causing them to glow in the
fluorescence microscope.

Live-cell fluorescence Fluorescent versions of Fluorescent tubulin molecules

microscopy* cytoskeletal proteins are made
and introduced into living cells.
Fluorescence microscopy and
video or digital cameras are used
to view the proteins as they
function in cells.
were microinjected into living
fibroblast cells. Inside the cell,
the tubulin dimers become
incorporated into microtubules,
which can be seen easily with a
fluorescence microscope.
0 sec 145 sec

Computer-enhanced High-resolution images from a The two micrographs show

digital video video or digital camera attached several microtubules. The image

microscopy to a microscope are computer
processed to increase contrast
and remove background features
that obscure the image.
Chapter 13
on the right was processed
to make the details of the
microtubules more visible.

Electron microscopy Electron microscopy can resolve
individual filaments. Cells to be
visualized may be prepared by
thin section, quick-freeze deep-
etch, or direct-mount techniques.
|Cytoskeletal Systems
Unenhanced Enhanced
A fibroblast cell prepared by the
quick-freeze deep-etch method.
Bundles of actin microfilaments
are visible.

*Confocal, deconvolution, multiphoton, and total internal reflection fluorescence (TIRF) microscopy are often used to improve detection of fluorescent signals.
See the Appendix for more details.

40% amino acid sequence identity. Each has (1) a GTP-binding
domain at the N-terminus, (2) a domain in the middle to which
colchicine, an MT poison, can bind, and (3) a third domain
at the C-terminus that interacts with MT-associated proteins
(MAPs). (You will learn about MAPs later in this chapter.)
This uniform orientation of tubulin dimers means that
one end of the protofilament differs structurally from the
other. Because the orientation of tubulin dimers is the same
for all of the protofilaments in an MT, the MT itself is also a
polarized structure, with a plus (+) end and a minus ( - ) end,
whose properties we will examine shortly.
Most organisms have several closely related but nonidenti-
cal genes for each of the a- and b-tubulin subunits. These slightly
different forms of tubulin are called tubulin isoforms. In the mam-
malian brain, for example, there are five a- and five b-tubulin
isoforms. These isoforms differ mainly in the C-terminal domain,
which suggests that various tubulin isoforms may interact with
different proteins. In addition to different isoforms, tubulin can
be chemically modified. For example, acetylated tubulin tends to
form more stable MTs than nonacetylated tubulin does.
Microtubules Can Form as Singlets, Doublets,
or Triplets
Cytosolic MTs are simple tubes, or singlet MTs, built from 13
protofilaments. Some axonemal MTs are more complex, how-
ever: they can contain doublet or triplet MTs. Doublets and
triplets contain one complete 13-protofilament microtubule
(the A tubule) and one or two additional incomplete tubules
(called B and C tubules) consisting of 10 or 11 protofilaments
(see Figure 13-3c). Doublets are found in cilia and flagella;
triplets are found in basal bodies and centrioles. (You will learn
about cilia and flagella in much more detail in Chapter 14.)
Microtubules Form by the Addition of Tubulin
Dimers at Their Ends
Microtubules form by the reversible polymerization of tubulin
dimers. The polymerization process has been studied exten-
sively in vitro; a schematic representation of MT assembly in
vitro is shown in Figure 13-4. When a solution containing a
sufficient concentration of tubulin dimers, GTP, and Mg2+ is
379

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 25 nm
15 nm
+

a
8 nm
A A
b

a B B
Singlet
b C
a Doublet
-
Triplet

Protofilament

(a) Microtubule structure (b) Microtubules in an 100 nm (c) Different types of microtubules
axon

Figure 13-3 Microtubule Structure. (a) A schematic diagram showing a microtubule (MT) as a cylinder
enclosing a hollow lumen. The outside diameter is about 25 nm, the inside about 15 nm. The wall of the cylinder
consists of 13 protofilaments, one of them indicated by an arrow. A protofilament is a linear polymer of tubulin
dimers, each consisting of two polypeptides: a-tubulin and b-tubulin. All heterodimers in the protofilaments
have the same orientation; the plus (+) and minus ( - ) ends are indicated. (b) MTs in a longitudinal section of
an axon (TEM). (c) MTs can form as singlets (13 protofilaments around a hollow lumen), doublets, and triplets.
Doublets and triplets contain one complete 13-protofilament microtubule (the A tubule) and one or two ad-
ditional incomplete tubules (called B and C tubules) consisting of 10 protofilaments.

warmed from 0°C to 37°C, the polymerization reaction begins.
(MT formation in the solution can be readily measured as an
increase in light scattering with an instrument called a spec-
trophotometer.) A critical step in the formation of MTs is the
aggregation of tubulin dimers into clusters called oligomers.
These oligomers serve as “seeds” from which new microtubules
can grow; hence this process is referred to as nucleation. Once
an MT has been nucleated, it grows by addition of subunits at
either end, via a process called elongation.
Microtubule formation is initially slow, a period referred
to as the lag phase of MT assembly. This period reflects the rela-
tively slow process of MT nucleation. The elongation phase of
MT assembly—the addition of tubulin dimers—is relatively
fast compared with nucleation. Eventually, the mass of MTs

Lag phase Elongation Plateau

(nucleation) phase phase
100

Figure 13-4 The Kinetics of Microtubule

Assembly In Vitro. The kinetics of MT assem-
Percent tubulin in microtubules

Microtubule with bly can be monitored by observing the amount

subunits coming of light scattered by a solution containing
on and off GTP-tubulin after it is warmed from 0°C to 37°C.
- + (Microtubule assembly is inhibited by cold and
activated by warming.) Light-scattering mea-
surements reflect changes in the MT population
Growing as a whole, not the assembly of individual mi-
microtubule crotubules. MT assembly exhibits three phases:
lag, elongation, and plateau. The lag phase is
Individual Protofilaments the period of nucleation. During the elongation
dimers phase, MTs grow rapidly, causing the concen-
tration of tubulin subunits in the solution to
0 Oligomers decline. When this concentration is low enough
to limit further assembly, the plateau phase is
0 60
reached, during which subunits are added and
Time (min) removed from MTs at equal rates.
380

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 MT minus ends Basal body MT plus ends

0.5 mm

Figure 13-5 Polar Assembly of Microtubules In Vitro. The polarity of MT assembly can be demonstrated
by adding basal bodies to a solution of tubulin dimers. The dimers add to the plus and minus ends of the MTs
in the basal body. However, MTs that grow from the plus end are much longer than those growing from the
minus end.

Chapter 13
increases to a point where the concentration of free tubulin a given tubulin molecule incorporated at the plus end is dis-
becomes limiting. This leads to the plateau phase, where MT as- placed progressively along the MT and eventually lost by de-
sembly is balanced by disassembly. polymerization at the opposite end (Figure 13-6). By examin-

|
Microtubule growth in vitro depends on the concentra- ing fluorescently labeled MTs, treadmilling has been observed

tion of tubulin dimers. The tubulin heterodimer concentra-
tion at which MT assembly is exactly balanced with disassem-
bly is called the overall critical concentration. MTs tend to
grow when the tubulin concentration exceeds the critical con-
centration and depolymerize when the tubulin concentration
falls below the critical concentration.
Addition of Tubulin Dimers Occurs More
Quickly at the Plus Ends of Microtubules
Cytoskeletal Systems
in living cells, although it is uncertain how important it is to
overall MT dynamics.
Drugs Can Affect the Assembly and Stability
of Microtubules
A number of drugs affect microtubule assembly (Table 13-3,
page 382). One well-known drug of this sort is colchicine,
a naturally occurring compound from the autumn crocus,

The inherent structural polarity of microtubules also means

that the two ends differ chemically. An important difference - +
between the two ends of an MT is that one can grow or shrink
much faster than the other. This difference in assembly rate
can readily be visualized by mixing the MT-associated struc-
tures found at the base of cilia, known as basal bodies, with
tubulin heterodimers. Microtubule nucleation and assembly
occur at both ends of the basal body, but the MTs grow much
faster from one end than from the other (Figure 13-5). The
rapidly growing end of a microtubule is called the plus end,
and the other end is the minus end. As will be discussed be-
low, minus ends of MTs are often anchored at the centrosome;
in this case, MT dynamics are confined to plus ends.
The different growth rates of the plus and minus ends
of microtubules reflect the different critical concentrations
required for assembly at the two ends of an MT. The critical
concentration for the plus end is lower than that for the mi-
nus end. If the free tubulin concentration is higher than the
Figure 13-6 Treadmilling of Microtubules. Microtubule as-
critical concentration for the plus end but lower than the criti-
sembly occurs more readily at the plus end of an MT than at the
cal concentration for the minus end, assembly will occur at minus end. When the tubulin concentration is higher than the critical
the plus end while disassembly takes place at the minus end. concentration for the plus end but lower than the critical concentra-
This simultaneous assembly and disassembly produces the tion for the minus end, the MT can add tubulin heterodimers to its
phenomenon known as treadmilling. Treadmilling arises when plus end while losing them from its minus end.
381

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 Table 13-3 Chemical Agents Used to Perturb the Cytoskeleton
Agent Source
Agents Affecting Microtubules
Colchicine, colcemid Autumn crocus, Colchicum autumnale
Nocadazole Synthetic compound
Vinblastine, vincristine Periwinkle plant, Vinca rosea
Paclitaxel (taxol) Symbiotic fungi in bark of the Pacific yew
tree, Taxus brevifolia
Agents Affecting Microfilaments
Cytochalasin D Fungal metabolite
Latrunculin A Red sea sponge, Latrunculia magnifica
Phalloidin Death cap fungus, Amanita phalloides
Agents Affecting Intermediate Filaments
Acrylamide Synthetic compound
Colchicum autumnale. Colchicine binds to the b-subunit of tu-
bulin heterodimers. Colchicine binding strongly inhibits incor-
poration of tubulin heterodimers into microtubules. The result-
ing tubulin-colchicine complex can still add to the growing end
of an MT, but it then prevents any further addition of tubulin
molecules and destabilizes the structure, thereby promoting MT
disassembly. Vinblastine and vincristine are related compounds
from the periwinkle plant (Vinca rosea) that bind and seques-
ter tubulin dimers inside the cell. Nocodazole (a synthetic
agent) is another compound that inhibits MT assembly and is
frequently used in experiments instead of colchicine because its
effects are more readily reversible when the drug is removed.
These compounds are often called antimitotic drugs be-
cause they disrupt the mitotic spindle of dividing cells, block-
ing the further progress of mitosis. The sensitivity of the mi-
totic spindle to these drugs is understandable because spindle
fibers are composed of microtubules. Indeed, vinblastine and
vincristine find application in medical practice as anticancer
drugs because cancer cells divide in an uncontrolled fashion
and are therefore preferentially susceptible to drugs that inter-
fere with the mitotic spindle.
In contrast, paclitaxel, or taxol (originally isolated from
the bark of the Pacific yew tree, Taxus brevifolia) binds tightly
to microtubules and stabilizes them, causing much of the free
tubulin in the cell to be locked up within microtubules. Within
mitotic cells, paclitaxel blocks MT disassembly and arrests cell
division. Thus, both paclitaxel and colchicine block cells in mi-
tosis, but they do so by opposing effects on MTs and hence on
the fibers of the mitotic spindle. Paclitaxel is also used in the
treatment of some cancers, especially breast cancer.
GTP Hydrolysis Contributes to the Dynamic
Instability of Microtubules
In the previous section, you saw that tubulin can assemble
in vitro in the presence of Mg2+ and GTP. In fact, GTP is re-
quired for MT assembly. Each tubulin heterodimer binds two
382
M13_HARD7694_09_GE_C13.indd 382
Effect
Binds b-tubulin, inhibiting assembly
Binds b-tubulin, inhibiting polymerization
Aggregates tubulin heterodimers
Stabilizes microtubules
Prevents addition of new monomers to plus ends
Sequesters actin monomers
Binds and stabilizes assembled microfilaments
Causes loss of intermediate filament networks
GTP molecules. The a-tubulin binds one GTP; b-tubulin binds
the other. Although the GTP bound to the a subunit is not hy-
drolyzed, the GTP bound to the b subunit can be hydrolyzed to
GDP sometime after the heterodimer is added to an MT. Hydro-
lysis of GTP is not necessary for assembly, because MTs polym-
erize from tubulin heterodimers bound to a nonhydrolyzable
analogue of GTP. However, MT assembly apparently requires
that GTP be present in both the a- and b-subunits because the
association of GDP-bound tubulin heterodimers with each
other is thought to be too weak to support polymerization.
Studies of MT assembly in vitro using isolated centrosomes
(a structure we will discuss in detail below) as nucleation sites
show that some microtubules can grow by polymerization at
the same time that others shrink by depolymerization. As a re-
sult, some MTs effectively enlarge at the expense of others. To
explain how both polymerization and depolymerization might
occur simultaneously, Tim Mitchison and Marc Kirschner pro-
posed the dynamic instability model. This model presumes
two populations of microtubules, one growing in length by
continued polymerization at plus ends and the other shrink-
ing in length by plus-end depolymerization. The distinction
between the two populations is that growing MTs have GTP
bound to the b-tubulin at their plus ends, whereas shrinking
MTs have GDP instead.
Heterodimeric tubulin complexed with two GTPs, re-
ferred to as GTP-tubulin, is thought to protect MTs by prevent-
ing tubulin from peeling away from their plus ends. This GTP
cap provides a stable MT tip to which further dimers can be
added (Figure 13-7a). Hydrolysis of GTP by b-tubulin even-
tually results in an unstable tip, at which point depolymeriza-
tion may occur rapidly.
The concentration of GTP-tubulin is crucial to the dy-
namic instability model. When GTP-tubulin is readily avail-
able, it is added to the microtubule quickly, creating a large
GTP-tubulin cap. If the concentration of GTP-tubulin falls,
however, the rate of tubulin addition decreases. At a suf-
ficiently low concentration of GTP-tubulin, the rate of
17/02/17 7:16 am
 GTP cap which shows that dynamic instability is a key feature of
MTs in living cells. An individual MT can undergo alternat-
ing periods of growth and shrinkage (Figure 13-7b). When
an MT switches from growth to shrinkage, an event called
microtubule catastrophe, the MT can disappear completely, or
GROWTH
it can abruptly switch back to a growth phase, a phenomenon
known as microtubule rescue. The frequency of catastrophe
GDP-tubulin
GTP-tubulin added is inversely related to the free tubulin concentration. High
HIGH tubulin concentrations make catastrophe less likely, but it can
GTP-tubulin
concentration still occur. When catastrophe does occur, higher tubulin con-
- + centrations make the rescue of a shrinking MT more likely. At
any tubulin concentration, catastrophe is more likely at the
plus end of an MT—that is, dynamic instability is more pro-
LOW
GTP-tubulin GTP hydrolyzed; nounced at the plus end of the MT.
concentration GTP cap depleted

Microtubules Originate from Microtubule-

Organizing Centers Within the Cell
In previous sections, we primarily discussed properties that
tubulin and microtubules exhibit in vitro, providing a founda-
CATASTROPHE tion for understanding how MTs function in the cell. However,
MT formation in vivo is a more ordered and regulated process,
(a) Model for how the GTP cap functions one that produces sets of MTs in specific locations for specific

Chapter 13
cell functions.
1 GROWTH: 2 CATASTROPHE: 3 RESCUE: Microtubules commonly originate from a structure in the
GTP-tubulin added GTP hydrolyzed, MT Growth resumes cell called a microtubule-organizing center (MTOC). An
depolymerizes rapidly
MTOC serves as a site at which MT assembly is initiated and

|
acts as an anchor for one end of these MTs. During interphase

Cytoskeletal Systems
of the cell cycle, many cells have an MTOC called the centro-
Length change

some that is positioned near the nucleus. During cell division,

Plus end
Minus end
15 30 45
Minutes
(b) Evidence for dynamic instability
Figure 13-7 The GTP Cap and Its Role in the Dynamic
Dynamic Instability of Microtubules. (a) When the tubulin concentration is
Instability of high, GTP-tubulin is added to the microtubule tip faster than the in-
Microtubules
corporated GTP can be hydrolyzed. The resulting GTP cap stabilizes
the MT tip and promotes further growth. At lower tubulin concentra-
tions, the rate of growth decreases, thereby allowing GTP hydrolysis
to catch up. This creates an unstable tip (no GTP cap) that favors MT
depolymerization. (b) In an individual MT observed by light micros-
copy, ➊ growth and ➋ catastrophic shrinkage can occur. Plus and
minus ends grow and shrink independently; changes in length are
much more dynamic at the plus end. ➌ Rescue involves the switch
from shrinkage to growth.
hydrolysis of GTP on the b-tubulin subunits near the tip of
the MT exceeds the rate of addition of new GTP-tubulin. This
results in shrinkage of the GTP cap. When the GTP cap disap-
pears, the MT becomes unstable, and loss of subunits from its
tip is favored.
Direct evidence for dynamic instability comes from obser-
vation of individual microtubules in vitro via light microscopy,
centrosomes are duplicated, creating new MTOCs for each of
the daughter cells.
The centrosome in an animal cell is normally associ-
ated with two structures called centrioles surrounded by
a diffuse granular matrix known as pericentriolar material
(Figure 13-8a). In electron micrographs of the centrosome,
MTs can be seen to originate from the pericentriolar material
(Figure 13-8b). The symmetrical structure of centrioles is re-
markable: As Figure 13-8c shows, their walls are formed by
nine sets of triplet microtubules. In most cases, pairs of cen-
trioles are oriented at right angles to one another; the signifi-
cance of this arrangement is still unknown.
Centrioles are known to be involved in the formation of
basal bodies, which are important for the formation of cilia and
flagella (see Chapter 14). The role of centrioles in nonciliated
cells is less clear. In animal cells, centrioles may serve to recruit
pericentriolar material to the centrosome, which then nucle-
ates growth of microtubules. When centrioles are removed
from many animal cells, microtubule-nucleating material dis-
perses, and the MTOCs disappear. Cells lacking centrioles can
still divide, probably because chromosomes can recruit MTs to
some extent on their own. However, the resulting spindles are
poorly organized. In contrast to animal cells, the cells of higher
plants and most fungi lack centrioles; their absence indicates
that centrioles are not essential for the formation of MTOCs.
g-Tubulin, a form of tubulin found only in centrosomes,
has been implicated in MT nucleation. In conjunction with a
number of other proteins called GRiPs (gamma tubulin ring
proteins), g-tubulin forms large, ring-shaped structures called
g-tubulin ring complexes (g-TuRCs), which can be seen at
383

M13_HARD7694_09_GE_C13.indd 383 17/02/17 7:16 am

 Centrosome
+ + +
+
+
+ +

+
+

Centriole Centrosome +
+
pair
+ + g-tubulin
(a)
(a)

Microtubule

CENTRIOLE
Microtubule g-TuRC 100 nm
(b)

Figure 13-9 G-Tubulin Ring Complexes (G-TuRCs) Nucleate

Microtubules. (a) g-TuRCs are found at centrosomes, where the
minus ends (−) of MTs are anchored. The enlargement shows a
single g-TuRC. The plus end of the MT is oriented away from the g
-TuRC. (b) An MT in vitro, visualized by metal shadowing. Here a
GRiP component of the g-TuRC (Xgrip109) was labeled with antibod-
Centrioles ies to which small particles of gold were attached. These particles
appear as bright spheres (TEM).

Pericentriolar the base of MTs emerging from the centrosome (Figure 13-9).
material
The importance of g-TuRCs in MT nucleation has been demon-
strated by depleting cells of g-tubulin or other components of
Microtubule the g-TuRC. In the absence of these proteins, centrosomes can
no longer nucleate MTs. In addition to the centrosome, some
types of cells have other MTOCs. For example, the basal body at
the base of each cilium in ciliated cells also serves as an MTOC.
(b) TEM 0.5 mm
MTOCs Organize and Polarize Microtubules
Within Cells
MTOCs play important roles in controlling the organization of
microtubules in cells. Most important is probably the ability
of MTOCs to nucleate and anchor MTs. Because of this ability,
MTs extend outward from an MTOC toward the periphery of
the cell. Furthermore, they grow out from an MTOC with a
fixed polarity—their minus ends are always anchored in the
MTOC, and their plus ends always point outward toward the
cell membrane. It is for this reason that dynamic growth and
shrinkage of MTs tend to occur at the periphery of cells. The
relationship between MTOCs and the distribution and polarity
of MTs within nondividing cells are shown in Figure 13-10.
The MTOC also influences the number of microtubules
(c) Centriolar structure (TEM) 50 nm in a cell. Each MTOC has a limited number of nucleation and
anchorage sites at which MTs can form. However, the MT-
Figure 13-8 The Centrosome. (a) In animal cells, the centro-
nucleating activity of the MTOC can be modulated during
some contains two centrioles and associated pericentriolar mate-
rial. The walls of centrioles are composed of nine sets of triplet
certain processes such as mitosis, during which the organiza-
microtubules. (b) A centrosome showing the centrioles and the tion of MTs changes dramatically (Figure 13-11). For exam-
pericentriolar material. Notice that microtubules originate from the ple, centrosomes associated with spindle poles in mitotic cells
pericentriolar material. (c) A closeup of the centriole. Centrioles have the highest MT-nucleating activity during prophase and
form a characteristic “pinwheel” structure. metaphase, when the mitotic spindle is forming.
384

M13_HARD7694_09_GE_C13.indd 384 17/02/17 7:16 am

 + + + + Apical end
-
+
Dendrite
Basal bodies
(MTOCs) +
- - - - -
-
Centrosome - - - -
+
(MTOC) + -
- +
+ + + -
+ -+ +- +
-
Axon - Marginal bundle
of microtubules
Basal end (mixed polarity)
+
+
(a) Nerve cell (b) Ciliated epithelial cell (c) Red blood cell

Figure 13-10 Microtubule Polarity in Nondividing Animal Cells. In the cell, the distribution of most
microtubules is determined by microtubule-organizing centers (MTOCs). MT orientation in a cell (shown in
orange) may vary with that cell’s function. (a) Nerve cells contain two distinct sets of MTs, those of the axon
and those of the dendrite. Axonal MTs are attached at their minus ends to the centrosome, with their plus
ends at the tip of the axon. Dendritic MTs are not associated with the centrosome and are of mixed polarities.
(b) Ciliated epithelial cells have many MTOCs called basal bodies, one at the base of each cilium. Ciliary MTs
originate with their minus ends in the basal bodies and elongate with their plus ends toward the tips of the cilia.
(c) Mature human red blood cells have no nucleus or MTOC. MTs of mixed polarities persist as a circular band
at the periphery of the cell. This band helps maintain the cell’s disklike shape.

Chapter 13
Centrosome Centrosomes
+ + + +
+ + + (MTOCs)
-- + - +
+ -
- - -- - - -+ - -- + +
- + -- -
+ - - -- +

|
- -- -- -

Cytoskeletal Systems
+ +
- - - -
+ - - -- -
- +
+
+ + +
+ +

+ + + + + + Chromosomes

(a) Interphase (b) Early prophase (c) Metaphase

a-tubulin DNA g-tubulin

(d) Cell stained for spindle components 5 mm

Figure 13-11 Changes in Microtubule Orientation During Mitosis. MTs in a dividing cell are oriented
with their minus ends anchored in the centrosome and their plus ends pointing away from the centrosome.
(a) Cell division is preceded by the division of the centrosome. (b) The daughter centrosomes then separate, each
forming one pole of the mitotic spindle. (c) At metaphase, the two centrosomes are at opposite sides of the cell.
Each spindle pole anchors half of the spindle MTs. (d) A cell stained for spindle components. g-tubulin (yellow)
marks centrosomes, a-tubulin (red) marks spindle MTs, and DNA is stained blue (fluorescence micrograph).
385

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 KEY TECHNIQUE
Studying the Dynamic Cytoskeleton
PROBLEM: Cytoskeletal systems are dynamically involved
in many cellular functions (summarized in Table 13-1, page 377).
But to determine exactly how a particular cytoskeletal system is
required for a particular process, researchers need to be able
to follow where and when cytoskeletal polymers assemble in
living cells.
SOLUTION: Chemical agents that
disrupt specific cytoskeletal elements
can quickly help identify whether or not
a cytoskeletal system is required for a
particular process.
Key Tools: (1) Chemicals that
disrupt the cytoskeleton, (2)
fluorescently labeled cytoskeletal
monomers and/or cytoskeletal
binding proteins, and (3) a
fluorescence microscope and sensitive
camera.
Details: Different chemical agents Human keratinocyte (green, DNA;
are known to affect the assembly or yellow, microtubules; white, actin)
disassembly of specific cytoskeletal
elements at the biochemical level (see Table 13-3, page 382).
Some bind to monomers, taking them out of commission, or
block addition of new monomers to a polymer. These agents
are useful for studying whether new polymer assembly is nec-
essary for a particular cellular event. Conversely, others bind to
assembled polymers, preventing them from being disassem-
bled. Such agents are useful for determining whether a preex-
isting cytoskeletal element must be dismantled for a cellular
process to proceed.
The picture we have developed thus far is that MT networks
arising from MTOCs are highly organized. Equally important is
that they are dynamic systems. Techniques mentioned so far, such
as electron microscopy, however, are limited in that they cannot
be used to analyze the dynamic cytoskeleton. Fortunately, mod-
ern cell biologists have other tools to study the dynamic assembly
and disassembly of highly organized arrays of MTs. These include
the use of filament-perturbing chemical agents (see Table 13-3,
page 382), high-resolution fluorescence microcopy (see Table
13-2, page 379), and genetically engineered cells expressing fluo-
rescent proteins. In some cases, combinations of such tools can
be used to study MTs in action (see Key Technique, above).
Microtubule Stability Is Tightly Regulated
in Cells by a Variety of Microtubule-Binding
Proteins
You have seen that cellular microtubules exhibit dynamic
instability—they can grow out from the centrosome and
then disassemble. This process could account for randomly
386
M13_HARD7694_09_GE_C13.indd 386
Each kind of agent can be used in a “wash-out” experiment.
When the chemical is applied, a particular cytoskeletal system
is blocked from forming (or, conversely, is blocked from disas-
sembling). When the chemical is washed out of the medium
surrounding the cells, normal cytoskeletal dynamics will resume
over time.
Consider how researchers learned
that the centrosome is a microtubule-
organizing center (MTOC) in the
mid-1970s. When a microtubule-
depolymerizing drug called colcemid
was applied to cells, their cytosolic
microtubules disappeared, as veri-
fied by immunofluorescence micros-
copy using MT-specific antibodies
(Figure 13A-1a). After the colcemid
was washed out, MTs began to
reform, but always at the centrosome
(Figure 13A-1b). Such experiments
provided powerful evidence that the
plus ends of MTs are oriented away
from the centrosome in cells.
Antibody staining, however, has a
10 mm drawback: It requires killing and chemi-
cally preserving cells before they can
be analyzed. An even more powerful
approach is to observe formation and disassembly of cytoskel-
etal elements in living cells. In this method, fluorescently labeled
building blocks of a particular cytoskeletal element are intro-
duced into cells, either by injecting labeled monomers directly or
by genetically engineering cells to express monomers to which
a fluorescent protein such as GFP has been attached. As Figure
13A-2 shows, this approach was crucial for establishing that
dynamic instability of MTs occurs in living cells.
distributed and short-lived MTs, but not for organized and
stable arrays of MTs within cells. Indeed, cells regulate the as-
sembly and structure of MTs with great precision. To do so,
they use a variety of MT-binding proteins. Some MT-binding
proteins act as ATP-driven motors that transport vesicles and
organelles or generate sliding forces between MTs. These pro-
teins will be discussed in detail in Chapter 14. Here we will fo-
cus on proteins that regulate MT organization and structure.
Microtubule-Stabilizing/Bundling Proteins. Microtu-
bule-associated proteins (MAPs) account for 10–15%
of the mass of MTs isolated from cells. MAPs bind at regular
intervals along the walls of MTs, allowing interaction with
other filaments and cellular structures. Most MAPs have been
shown to promote MT stability by preventing the severing of
MTs and by keeping heterodimers locked into the MT. They
can also affect the density of bundles of MTs. MAP function
has been studied extensively in brain cells, as they are the
most abundant source of these proteins. Neurons have axons,
which carry electrical signals away from the cell body of the
17/02/17 7:16 am

(a) 0 minutes of recovery (b) 30 minutes of 10 mm
recovery
Figure 13A-1 A Wash-out Experiment Shows That Centro-
somes Polarize Microtubules in Cells. (a) Cells treated with
colcemid, then immunostained to visualize MTs. Only MTs associ-
ated with the centrosome (which are resistant to colcemid) persist
(arrowheads). (b) Another cell fixed 30 minutes after washing out
the colcemid. New MTs are growing away from the centrosome.
Labeling all polymers in a system can be effective for
studying cytoskeletal dynamics, but sometimes it is easier to
make sense of the fluorescent signal if a cytoskeletal element is
only partially labeled. One such technique, speckle microscopy,
involves introducing a limited amount of fluorescent monomer,
which results in speckles of fluorescence along the length of
a cytoskeletal element. This technique has been very useful
for analyzing the flow of cytoskeletal elements. If, for exam-
ple, treadmilling of MTs or MFs occurs, speckles of labeled
neuron, and dendrites, which receive signals from neighbor-
ing cells and carry them to the cell body. Within these project-
ing structures, or neurites, groups of microtubules are cross-
linked into bundles. The bundles are characteristically denser
in axons than they are in dendrites. A MAP called Tau causes
MTs to form tight bundles in axons. Another MAP, MAP2, is
present in dendrites and causes the formation of looser bun-
dles of MTs. One portion of MAPs such as Tau and MAP2
binds along the length of an MT; another portion protrudes
away from the microtubule, where it can interact with other
proteins (Figure 13-12a). The length of these “arms” con-
trols the spacing of MTs within bundles. MAP2 has a longer
arm than does Tau, and so MAP2 forms MT bundles that are
less densely packed than those formed with Tau.
The importance of MAPs can be demonstrated by forc-
ing non-neuronal cells to make Tau protein. When such cells
express large amounts of Tau, they extend single long pro-
cesses that look remarkably similar to the axons of neurons
(Figure 13-12b). Tau is important in human disease. Dense tan-
gles of neurites, known as neurofibrillary tangles, are a hallmark
M13_HARD7694_09_GE_C13.indd 387
0 sec
32 sec
B
Chapter 13
1 mm
Figure 13A-2 Dynamic Instability of Microtubules In Vivo.
MTs visualized by live-cell fluorescence microscopy exhibit dynamic
instability in vivo. Here, two individual MTs (A, B) have been labeled.
|
Over a 32-second time span, MT B grows whereas A shrinks.
Cytoskeletal Systems
monomer incorporated near the plus end of a cytoskeletal
filament will be displaced rearward as subunits are lost at the
minus end and additional monomers are added at the plus end.
QUESTION: How could you use paclitaxel, which makes
MTs less likely than normal to depolymerize, to study whether
the completion of mitosis requires disassembly of MTs?
of several diseases that result in dementia, such as Alzheimer
disease, Pick disease, and several types of palsy. In the case
of Alzheimer disease, these tangles contain large amounts of
hyperphosphorylated Tau protein, which forms paired helical
filaments. Mutations that result in defective Tau protein lead
to hereditary predisposition to form neurofibrillary tangles.
Such diseases are therefore sometimes called tauopathies.
+ −TIP Proteins. MTs are generally too unstable to remain in-
tact for long periods of time and will depolymerize unless they
are stabilized in some way. One way to stabilize MTs is to “cap-
ture” and protect their growing plus ends. To do so, +–TIP
proteins (+– end tubulin interacting proteins) associate with
MT plus ends. One important example of MT capture involves
kinetochores during mitosis. Other +–TIPs associate with
the cell cortex, an actin-based network underneath the plasma
membrane, and can stabilize MTs that extend there. Some +–
TIPs, including end-binding protein 1 (EB1), directly bind to
and stabilize plus ends, decreasing the likelihood that they will
undergo catastrophic subunit loss (Figure 13-12c).
387
17/02/17 7:16 am
 Tau (a MAP)

- +

40 mm
Normal Sf9 (insect) cells Sf9 cells expressing Tau
(a) MAPs
(b) Effects of Tau overexpression on insect cells
EB1 (a +–TIP)
- +

(c) +–TIPs

MCAK
(a catastrophin)
- +

(e) Catastrophins (d) EB1 localization in an insect 5 mm

cell

Figure 13-12 Microtubule-Binding Proteins Regulate Microtubule Function In Vivo. (a) Tau is a MAP
that binds along the length of an MT; another portion of Tau extends away from the MT, regulating MT spacing
in bundles. (b) The effects of Tau overexpression in a line of cultured non-neuronal cells called Sf9. (c) +–TIP
proteins, such as EB1, bind at or near the plus ends of MTs, stabilizing them. (d) An S2 cell stained for EB1
(green), MTs (red), and DNA (blue). (e) Catastrophins, such as MCAK (mitotic centromere-associated kinesin),
are kinesin family proteins that destabilize MTs.

EB1 associates with GTP-tubulin at MT plus ends, yield-

ing a characteristic “fireworks” pattern (Figure 13-12d). EB1
protein that is tagged with the green fluorescent protein (GFP)
is an incredibly useful tool for following the plus ends of MTs
dynamically (see Key Technique, page 386).
Microtubule-Destabilizing/Severing Proteins. Whereas
some MT-binding proteins stabilize microtubules, making
them less likely to depolymerize, others promote depolymer-
ization of MTs. For example, the protein stathmin/Op18 binds
to tubulin heterodimers, preventing them from polymerizing.
Other proteins act at the ends of MTs once they have poly-
merized, promoting the peeling of protofilaments and even-
tual loss of subunits from their ends. Several proteins of the
kinesin family, called catastrophins (Figure 13-12e), act in this
way. By tightly regulating where catastrophins act, a cell can
precisely control where and when MTs form and depolymer-
ize. A prime example of such regulation is the mitotic spindle.
Indeed, a catastrophin known as MCAK (mitotic centromere-
associated kinesin) plays just such a role. Still other proteins
sever MTs; one example of such proteins is katanin.
CONCEPT CHECK 13.2
The amount of dynamic instability exhibited by microtubules
varies depending on the kind of cell and whether it is divid-
ing. Under what circumstances might you expect dynamic
instability to be greatest, and why?
388
13.3 Microfilaments
With a diameter of about 7 nm, microfilaments (MFs) are
the smallest of the cytoskeletal filaments (see Table 13-1,
page 377). Microfilaments are best known for their role in the
contractile fibrils of muscle cells, where they interact with
thicker filaments of myosin to cause the contractions char-
acteristic of muscle (see Chapter 14). MFs are not confined
to muscle cells, however. They occur in almost all eukaryotic
cells and are involved in numerous other phenomena, includ-
ing a variety of locomotory and structural functions.
Examples of cell movements in which microfilaments play
a role include cell migration via lamellipodia and filopodia, amoe-
boid movement, and cytoplasmic streaming, a regular pattern of
flow in the cytosol of some plant and animal cells. (We will dis-
cuss all of these phenomena in detail in Chapter 14.) MFs also
produce the cleavage furrows that divide the cytosol of animal
cells during cytokinesis, and MFs are found at sites of attach-
ment of cells to one another and to the extracellular matrix.
In addition to mediating a variety of cell movements, MFs
are important in developing and maintaining cell shape. Most
animal cells, for example, have a dense network of microfila-
ments within the cell cortex just beneath the plasma membrane.
The cortex confers structural rigidity on the cell surface and facil-
itates shape changes and cell movement. Parallel bundles of MFs
also make up the structural core of microvilli, the fingerlike exten-
sions found on the surface of many animal cells (see Figure 4-4).

M13_HARD7694_09_GE_C13.indd 388 17/02/17 7:16 am