The Cytoskeleton: Components

and Structural Functions

The cytosol is a major site of cellular metabolism and contains a large number of different enzymes. Proteins
constitute
about 2030 percent of the cytosol by weight, and from a
quarter to half of the total protein within cells is in the cytosol.
Estimates of the protein concentration in the cytosol
range from 200 to 400 mg/ml. Because of the high concentration
of cytosolic proteins, complexes of proteins can form
even if the energy that stabilizes them is weak. Many investigators believe that the cytosol is highly organized, with
most soluble proteins either bound to filaments or otherwise
localized in specific regions. In an electron micrograph of a
typical animal cell, soluble proteins packing the cell interior
conceal much of the internal structure. If a cell is pretreated
with a nonionic detergent (e.g., Triton X-100), which permeabilizes
the membrane, soluble cytosolic proteins diffuse
away. In micrographs of detergent-extracted animal cells,
two types of structures stand outmembrane-limited organelles
and the filaments of the cytoskeleton, which fill the
cytosol (Figure 5-28).
5.4 The Cytoskeleton: Components and Structural Functions 173
Membranemicrofilament
linkages
Core actin
filaments
Actin filaments
(rootlets)
Spectrin
connecting
fibers
Keratin intermediate
filaments
FIGURE 5-28 Electron micrograph of the apical part of a
detergent-extracted intestinal epithelial cell. Microvilli,
fingerlike projections of the plasma membrane, cover the apical
surface of an intestinal epithelial cell. A bundle of microfilaments
in the core of each microvillus stabilizes the structure. The
plasma membrane surrounding a microvillus is attached to the
sides of the bundle by evenly spaced membranemicrofilament
linkages (yellow). The bundle continues into the cell as a short
rootlet. The rootlets of multiple microvilli are cross-braced by
connecting fibers (red) composed of an intestinal isoform of
spectrin. This fibrous actin-binding protein is found in a narrow
band just below the plasma membrane in many animal cells. The
bases of the rootlets are attached to keratin intermediate
filaments. These numerous connections anchor the rootlets in a
meshwork of filaments and thereby support the upright
orientation of the microvilli. [Courtesy of N. Hirokawa.]

In this section, we introduce the protein filaments that

compose the cytoskeleton and then describe how they support
the plasma and nuclear membranes and organize the
contents of the cell. Later chapters will deal with the dynamic
properties of the cytoskeletonits assembly and disassembly
and its role in cellular movements.
Three Types of Filaments Compose
the Cytoskeleton
The cytosol of a eukaryotic cell contains three types of filaments
that can be distinguished on the bases of their diameter,
type of subunit, and subunit arrangment (Figure 5-29). Actin
filaments, also called microfilaments, are 89 nm in diameter
and have a twisted two-stranded structure. Microtubules
are hollow tubelike structures, 24 nm in diameter, whose
walls are formed by adjacent protofilaments. Intermediate
filaments (IFs) have the structure of a 10-nm-diameter rope.
Each type of cytoskeletal filament is a polymer of protein
subunits (Table 5-4). Monomeric actin subunits assemble
into microfilaments; dimeric subunits composed of
_- and _-tubulin polymerize into microtubules. Unlike microfilaments
and microtubules, which are assembled from
one or two proteins, intermediate filaments are assembled
from a large diverse family of proteins. The most common
intermediate filaments, found in the nucleus, are composed
of lamins. Intermediate filaments constructed from other
proteins are expressed preferentially in certain tissues: for example,
keratin-containing filaments in epithelial cells,
desmin-containing filaments in muscle cells, and vimentincontaining
filaments in mesenchymal cells.

_ FIGURE 5-29 Comparison of the three types of

filaments that form the cytoskeleton. (a) Diagram of the basic
structures of an actin filament (AF), intermediate filament (IF),
and microtubule (MT). The beadlike structure of an actin filament
shows the packing of actin subunits. Intermediate filament
subunits pack to form ropes in which the individual subunits are
difficult to distinguish. The walls of microtubules are formed from
protofilaments of tubulin subunits. (b) Micrograph of a mixture of
actin filaments, microtubules, and vimentin intermediate
filaments showing the differences in their shape, size, and
flexibility. Purified preparations of actin, tubulin, and vimentin
subunits were separately polymerized in a test tube to form the
corresponding filaments. A mixture of the filaments was applied
to a carbon film on a microscope grid and then rinsed with a
dilute solution of uranyl acetate (UC), which surrounds but does
not penetrate the protein (c). Because uranyl acetate is a heavy
metal that easily scatters electrons, areas of the microscope grid
occupied by protein produce a negative image in metal film
when projected onto a photographic plate, as seen in part (b).
[Part (b) courtesy of G. Waller and P. Matsudaira.]

Most eukaryotic cells contain all three types of cytoskeletal

filaments, often concentrated in distinct locations.
For example, in the absorptive epithelial cells that
line the lumen of the intestine, actin microfilaments are
abundant in the apical region, where they are associated
with cellcell junctions and support a dense carpet of microvilli
(Figure 5-30a). Actin filaments are also present in
a narrow zone adjacent to the plasma membrane in the lateral
regions of these cells. Keratin intermediate filaments,

Protein Subunits in Cytoskeletal Filaments

Protein Subunits MW Expression Function
MICROFILAMENTS
Actin 42,000 Fungi, plant, animal Structural support, motility
MreB 36,000 Rod-shaped bacteria Width control
MICROTUBULES
Tubulin (_ and _) 58,000 Fungi, plant, animal Structural support, motility, cell polarity
FtsZ 58,000 Bacteria Cell division
INTERMEDIATE FILAMENTS
Lamins Various Plant, animal Support for nuclear membrane
Desmin, keratin, vimentin, others Various Animal Cell adhesion
OTHER
MSP 50,000 Nematode sperm Motility

forming a meshwork, connect microvilli and are tethered to

junctions between cells. Lamin intermediate filaments support
the inner nuclear membrane. Finally, microtubules,
aligned with the long axis of the cell, are in close proximity
to major cell organelles such as the endoplasmic reticulum,
Golgi complex, and vesicles.
The cytoskeleton has been highly conserved in evolution.
A comparison of gene sequences shows only a small
percentage of differences in sequence between yeast actin
and tubulin and human actin and tubulin. This structural

conservation is explained by the variety of critical functions

that depend on the cytoskeleton. A mutation in a cytoskeleton
protein subunit could disrupt the assembly of filaments
and their binding to other proteins. Analyses of
gene sequences and protein structures have identified bacterial
homologs of actin and tubulin. The absence of IF-like
proteins in bacteria and unicellular eukaryotes is evidence
that intermediate filaments appeared later in the evolution
of the cytoskeletal system. The first IF protein to arise was
most likely a nuclear lamin from which cytosolic IF proteins
later evolved.
The simple bacterial cytoskeleton controls cell length,
width, and the site of cell division. The FtsZ protein, a bacterial
homolog of tubulin, is localized around the neck of dividing
bacterial cells, suggesting that FtsZ participates in cell
division (Figure 5-30b). The results of biochemical experiments
with purified FtsZ demonstrate that it can polymerize
into protofilaments, but these protofilaments do not
assemble into intact microtubules. Another bacterial protein,
MreB, has been found to be similar to actin in atomic structure
and filament structurestrong evidence that actin
evolved from MreB. Clues to the function of MreB include
its localization in a filament that girdles rod-shaped bacterial
cells, its absence from spherical bacteria, and the finding that
mutant cells lacking MreB become wider but not longer.
These observations suggest MreB controls the width of rodshaped
bacteria.
5.4 The Cytoskeleton: Components and Structural Functions 175
AF IF MT
(a)
(b)
(c)
AF IF
MT
Carbon film
Actin
MreB FtsZ MreB
(a) MTs IFs
(b)
FIGURE 5-30 Schematic depiction of the distribution of
cytoskeletal filaments in eukaryotic cells and bacterial cells.
(a) In absorptive epithelial cells, actin filaments (red) are
concentrated in the apical region and in a narrow band in the
basolateral region. Microtubules (blue) are oriented with the long
axis of the cell, and intermediate filaments (green) are
concentrated along the cell periphery especially at specialized
junctions with neighboring cells and lining the nuclear
membrane. (b) In a rod-shaped bacterial cell, filaments of MreB,
the bacterial actin homolog, ring the cell and constrict its width.
The bacterial tubulin homolog, FtsZ, forms filaments at the site
of cell division.

Cytoskeletal Filaments Are Organized

into Bundles and Networks
On first looking at micrographs of a cell, one is struck by
the dense, seemingly disorganized mat of filaments present in
the cytosol. However, a keen eye will start to pick out
areasgenerally where the membrane protrudes from the
cell surface or where a cell adheres to the surface or another
cellin which the filaments are concentrated into bundles.
From these bundles, the filaments continue into the cell interior,
where they fan out and become part of a network of
filaments. These two structures, bundles and networks, are
the most common arrangements of cytoskeletal filaments in
a cell.
Structurally, bundles differ from networks mainly in the
organization of the filaments. In bundles, the filaments are
closely packed in parallel arrays. In a network, the filaments
crisscross, often at right angles, and are loosely
packed. Networks can be further subdivided. One type, associated
with the nuclear and plasma membranes, is planar
(two-dimensional), like a net or a web; the other type,
present within the cell, is three-dimensional, giving the
cytosol gel-like properties. In all bundles and networks, the
filaments are held together by various cross-linking proteins.

We will consider various cytoskeletal cross-linking proteins

and their functions in Chapters 19 and 20.
Microfilaments and Membrane-Binding
Proteins Form a Skeleton Underlying
the Plasma Membrane
The distinctive shape of a cell depends on the organization of
actin filaments and proteins that connect microfilaments to the
membrane. These proteins, called membranemicrofilament
binding proteins, act as spot welds that tack the actin
cytoskeleton framework to the overlying membrane. When
attached to a bundle of filaments, the membrane acquires the
fingerlike shape of a microvillus or similar projection (see Figure
5-28). When attached to a planar network of filaments, the
membrane is held flat like the red blood cell membrane. The
simplest membranecytoskeleton connections entail the binding
of integral membrane proteins directly to actin filaments.
More common are complex linkages that connect actin filaments
to integral membrane proteins through peripheral membrane
proteins that function as adapter proteins. Such linkages
between the cytoskeleton and certain plasma-membrane proteins
are considered in Chapter 6.

IGURE 5-31 Cortical cytoskeleton supporting the

plasma membrane in human erythrocytes. (a) Electron
micrograph of the erythrocyte membrane showing the spokeand-
hub organization of the cytoskeleton. The long spokes are
composed mainly of spectrin and can be seen to intersect at the
hubs, or membrane-attachment sites. The darker spots along the
spokes are ankyrin molecules, which cross-link spectrin to

integral membrane proteins. (b) Diagram of the erythrocyte

cytoskeleton showing the various components. See text for
discussion. [Part (a) from T. J. Byers and D. Branton, 1985, Proc. Natl.
Acad. Sci. USA 82:6153. Courtesy of D. Branton. Part (b) adapted from
S. E. Lux, 1979, Nature

The richest area of actin filaments in many cells lies in the

cortex, a narrow zone just beneath the plasma membrane.
In this region, most actin filaments are arranged in a network
that excludes most organelles from the cortical cytoplasm.
Perhaps the simplest cytoskeleton is the two-dimensional
network of actin filaments adjacent to the erythrocyte
plasma membrane. In more complicated cortical cytoskeletons,
such as those in platelets, epithelial cells, and muscle,
actin filaments are part of a three-dimensional network that
fills the cytosol and anchors the cell to the substratum.
A red blood cell must squeeze through narrow blood capillaries
without rupturing its membrane. The strength and
flexibility of the erythrocyte plasma membrane depend on a
dense cytoskeletal network that underlies the entire membrane
and is attached to it at many points. The primary component
of the erythrocyte cytoskeleton is spectrin, a
200-nm-long fibrous protein. The entire cytoskeleton is
arranged in a spoke-and-hub network (Figure 5-31a). Each
spoke is composed of a single spectrin molecule, which extends
from two hubs and cross-links them. Each hub comprises
a short (14-subunit) actin filament plus adducin,
tropomyosin, and tropomodulin (Figure 5-31b, inset). The
last two proteins strengthen the network by preventing the
actin filament from depolymerizing. Six or seven spokes radiate
from each hub, suggesting that six or seven spectrin
molecules are bound to the same actin filament.
To ensure that the erythrocyte retains its characteristic
shape, the spectrin-actin cytoskeleton is firmly attached to
the overlying erythrocyte plasma membrane by two peripheral
membrane proteins, each of which binds to a specific
integral membrane protein and to membrane phospholipids.
Ankyrin connects the center of spectrin to band 3 protein, an
anion-transport protein in the membrane. Band 4.1 protein,
a component of the hub, binds to the integral membrane protein
glycophorin, whose structure was discussed previously
(see Figure 5-12). Both ankyrin and band 4.1 protein also
contain lipid-binding motifs, which help bind them to the
membrane (see Table 5-3). The dual binding by ankyrin and
band 4.1 ensures that the membrane is connected to both the
spokes and the hubs of the spectrin-actin cytoskeleton (see
Figure 5-31b).
Intermediate Filaments Support the Nuclear
Membrane and Help Connect Cells into Tissues
Intermediate filaments typically crisscross the cytosol, forming
an internal framework that stretches from the nuclear envelope
to the plasma membrane (Figure 5-32). A network of
intermediate filaments is located adjacent to some cellular
membranes, where it provides mechanical support. For example,
lamin A and lamin C filaments form an orthogonal
lattice that is associated with lamin B. The entire supporting
structure, called the nuclear lamina, is anchored to the inner
nuclear membrane by prenyl anchors on lamin B.
At the plasma membrane, intermediate filaments are attached
by adapter proteins to specialized cell junctions called
desmosomes and hemidesmosomes, which mediate cellcell
adhesion and cellmatrix adhesion, respectively, particularly
in epithelial tissues. In this way, intermediate filaments in one
cell are indirectly connected to intermediate filaments in a
neighboring cell or to the extracellular matrix. Because of the
important role of cell junctions in cell adhesion and the stability
of tissues, we consider their structure and relation to
cytoskeletal filaments in detail in Chapter 6.
Microtubules Radiate from Centrosomes
and Organize Certain Subcellular Structures
Like microfilaments and intermediate filaments, microtubules
are not randomly distributed in cells. Rather, microtubules
radiate from the centrosome, which is the primary
microtubule-organizing center (MTOC) in animal cells (Figure
5-33). As detailed in Chapter 20, the two ends of a microtubule
differ in their dynamic properties and are
commonly designated as the (_) and (_) ends. For this reason,
microtubles can have two distinct orientations relative
to one another and to other cell structures. In many nondividing
animal cells, the MTOC is located at the center of the
cell near the nucleus, and the radiating microtubules are all
oriented with their (_) ends directed toward the cell periphery.
Although most interphase animal cells contain a single
perinuclear MTOC, epithelial cells and plant cells contain
hundreds of MTOCs. Both of these cell types exhibit distinct

functional or structural properties or both in different regions

of the cell. The functional and structural polarity of
these cells is linked to the orientation of microtubules within
them.
Findings from studies discussed in Chapter 20 show that
the association of microtubules with the endoplasmic reticulum
and other membrane-bounded organelles may be critical
to the location and organization of these organelles
within the cell. For instance, if microtubules are destroyed by
drugs such as nocodazole or colcemid, the ER loses its networklike
organization. Microtubules are also critical to the
formation of the mitotic apparatusthe elaborate, transient
structure that captures and subsequently separates replicated
chromosomes in cell division.

FIGURE 5-33 Fluorescence micrograph of a Chinese

hamster ovary cell stained to reveal microtubles and the
MTOC. The microtubules (green), detected with an antibody to
tubulin, are seen to radiate from a central point, the microtubuleorganizing
center (MTOC), near the nucleus. The MTOC (yellow)
is detected with an antibody to a protein localized to the
centrosome. [Courtesy of R. Kuriyame.]

Microfilaments are assembled from monomeric actin

subunits; microtubules, from _,_-tubulin subunits; and intermediate
filaments, from lamin subunits and other tissuespecific
proteins.
In all animal and plant cells, the cytoskeleton provides
structural stability for the cell and contributes to cell movement.
Some bacteria have a primitive cytoskeleton.
Actin bundles form the core of microvilli and other fingerlike
projections of the plasma membrane.
Cortical spectrin-actin networks are attached to the cell
membrane by bivalent membranemicrofilament binding
proteins such as ankyrin and band 4.1 (see Figure 5-31).
Intermediate filaments are assembled into networks and
bundles by various intermediate filamentbinding proteins,
which also cross-link intermediate filaments to the plasma
and nuclear membranes, microtubules, and microfilaments.
In some animal cells, microtubules radiate out from a
single microtubule-organizing center lying at the cell center
(see Figure 5-33). Intact microtubules appear to be necessary
for endoplasmic reticulum and Golgi membranes to
form into organized structures.