CH16 Cytoskeleton.pdf - finedrafts
CH16 Cytoskeleton.pdf - finedrafts
CH16 Cytoskeleton.pdf - finedrafts
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THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS 973<br />
c<br />
o<br />
E<br />
o<br />
c<br />
c<br />
l<br />
! f<br />
c<br />
o<br />
(A)<br />
n ucleation<br />
(lag phase)<br />
elongation<br />
(growth phase)<br />
steady state<br />
(equilibrium phase)<br />
time after salt addition +<br />
Nucleation ls the Rate-Limiting Step in the Formation of a<br />
Cytoskeletal Polymer<br />
c<br />
o<br />
E<br />
o<br />
c<br />
c<br />
f<br />
.o f<br />
c<br />
o<br />
0<br />
elongation<br />
(growth phase)<br />
There is an important additional consequence of the multiple-protofilament<br />
organization of cltoskeletal polymers. Short oligomers composed of a few subunits<br />
can assemble spontaneously, but they are unstable and disassemble readily<br />
because each monomer is bonded only to a few other monomers. For a new<br />
large filament to form, subunits must assemble into an initial aggregate, or<br />
nucleus, that is stabilized by many subunit-subunit contacts and can then elongate<br />
rapidly by addition of more subunits. The initial process of nucleus assembly<br />
is called filament nucleation, and it can take quite a long time, depending on<br />
how many subunits must come together to form the nucleus.<br />
The instability of smaller aggregates creates a kinetic barrier to nucleation,<br />
which is easily observed in a solution of pure actin or tubulin-the subunits of<br />
actin filaments and microtubules, respectively. When polymerization is initiated<br />
in a test tube containing a solution of pure individual subunits (by raising the<br />
temperature or raising the salt concentration), there is an initial lag phase, during<br />
which no filaments are observed. During this lag phase, however, a few of the<br />
small unstable aggregates succeed in making the transition to the more stable<br />
filament form, so that the lag phase is followed by a phase of rapid filament elongation,<br />
during which subunits add quickly onto the ends of the nucleated filaments<br />
(Figure f 6-f 0A). Finally, the system approaches a steady state at which<br />
the rate of addition of new subunits to the filament ends exactly balances the<br />
rate of subunit dissociation from the ends. The concentration of free subunits<br />
left in solution at this point is called the critical concentration, C.. As explained<br />
in Panel f6-2 (pp. 978-979), the value ofthe critical concentration is equal to the<br />
rate constant for subunit loss divided by the rate constant for subunit additionthat<br />
is, Cc = koff I kon.<br />
The lag phase in filament growth is eliminated if preexisting seeds (such as<br />
filament fragments that have been chemically cross-linked) are added to the<br />
solution at the beginning of the polymerizalion reaction (Figure l6-108). The<br />
cell takes great advantage of this nucleation requirement: it uses special proteins<br />
to catalyze filament nucleation at specific sites, thereby determining the location<br />
at which new cytoskeletal filaments are assembled. Indeed, the regulation<br />
of filament nucleation is a primary way for cells to control their shape and their<br />
movement.<br />
The Tubulin and Actin Subunits Assemble Head-to-Tail to Create<br />
Polar Filaments<br />
Microtubules are formed from protein subunits of tubulin. The tubulin subunit<br />
is itself a heterodimer formed from two closely related globular proteins called<br />
ct-tubulin and B-tubulin, tigll'tly bound together by noncovalent bonds (Figure<br />
(B)<br />
steady state<br />
(equilibrium phase)<br />
preformed filament seeds added here<br />
coming on and off<br />
time after salt addition +<br />
Figure 16-1 0 The time course of actin<br />
polymerization in a test tube.<br />
(A) Polymerization is begun by raising the<br />
salt concentration in a solution of pure<br />
actin subunits. (B) Polymerization is<br />
begun in the same way, but with<br />
preformed fragments of actin filaments<br />
present to act as nuclei for filament<br />
growth. As indicated, the 0/o free subunits<br />
reflects the critical concentration (Cc), the<br />
point at which there is no net change in<br />
porymer.
974 Chapter 1 6: The <strong>Cytoskeleton</strong><br />
(A)<br />
P-tubulin<br />
d-tubulin<br />
R<br />
f,,uo tubulin heterodimer<br />
(= microtubule subunit)<br />
protofilament<br />
prus<br />
eno<br />
mtnus<br />
eno<br />
1<br />
5U nm<br />
I<br />
f 6-l f ). These two tubulin proteins are found only in this heterodimer. Each a or<br />
B monomer has a binding site for one molecule of GTP The GTP that is bound<br />
to the cr-tubulin monomer is physically trapped at the dimer interface and is<br />
never hydrolyzed or exchanged; it can therefore be considered to be an integral<br />
part of the tubulin heterodimer structure. The nucleotide on the p-tubulin, in<br />
contrast, may be in either the GTP or the GDP form, and it is exchangeable. As<br />
we shall see, the hydrolysis of GTP at this site to produce GDP has an important<br />
effect on microtubule dynamics.<br />
A microtubule is a hollow cylindrical structure built from l3 parallel protofilaments,<br />
each composed of alternating s-tubulin and B-tubulin molecules.<br />
\Mhen the tubulin heterodimers assemble to form the hollow cylindrical microtubule,<br />
they generate two new types of protein-protein contacts. Along the longitudinal<br />
axis of the microtubule, the "top" of one B-tubulin molecule forms an<br />
interface with the "bottom" of the a-tubulin molecule in the adjacent heterodimer.<br />
This interface is very similar to the interface holding the a and B<br />
monomers together in the dimer subunit, and the binding energy is strong. perpendicular<br />
to these interactions, neighboring protofilaments form lateral contacts.<br />
In this dimension, the main lateral contacts are between monomers of the<br />
same type (cr-cx and 0-0). Togetnel the longitudinal and lateral contacts are<br />
repeated in the regular helical lattice of the microtubule. Because multiple contacts<br />
within the lattice hold most of the subunits in a microtubule in place, the<br />
addition and loss of subunits occurs almost exclusively at the microtubule ends<br />
(see Figure 16-8). These multiple contacts among subunits make microtubules<br />
stiff and difficult to bend. The stiffness of a filament can be characterized by its<br />
persistence length, a property of the filament describing how long it must be<br />
before random thermal fluctuations are likely to cause it to bend. The persistence<br />
length of a microtubule is several millimeters, making microtubules the<br />
stiffest and straightest structural elements found in most animal cells.<br />
The subunits in each protofilament in a microtubule all point in the same<br />
direction, and the protofilaments themselves are aligned in parallel (in Figure<br />
16-11, for example, the cr-tubulin is dornm and the B-tubulin up in each hetero-<br />
microtubule<br />
Figure 1 6-1 1 The structure of a<br />
microtubule and its subunit. (A) The<br />
subunit of each protofilament is a tubulin<br />
heterodimer, formed from a very tightly<br />
linked pair of cr- and P-tubulin monomers.<br />
The GTP molecule in the o-tubulin<br />
monomer is so tightly bound that it can<br />
be considered an integral part ofthe<br />
protein. The GTP molecule in the B-tubulin<br />
monomer, however, is less tightly bound<br />
and has an important role in filament<br />
dynamics. Both nucleotides are shown in<br />
red. (B) One tubulin subunit<br />
(cr-p heterodimer) and one protofilament<br />
are shown schematically. Each<br />
protofilament consists of many adjacent<br />
subunits with the same orientation.<br />
(C) The microtubule is a stiff hollow tube<br />
formed from 13 protofilaments aligned in<br />
parallel. (D) A short segment of a<br />
microtubule viewed in an electron<br />
microscope. (E) Electron micrograph of a<br />
cross section of a microtubule showing a<br />
ring of 13 distinct protofilaments.<br />
(D, courtesy of Richard Wade; E, courtesy<br />
of Richard Linck.)
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THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTs<br />
triphosphate. As we explained with our ant-trail analogy at the beginning of the<br />
chapter, the advantage to the cell seems to be the spatial and temporal flexibility<br />
that is inherent in a structural system with constant turnover. Individual subunits<br />
are small and can diffuse very rapidly; an actin or tubulin subunit can diffuse<br />
across the diameter of a typical eucaryotic cell in several seconds. As noted<br />
I<br />
.*<br />
(A) I ""<br />
rapid growth with GTP-capped end<br />
accidental loss of GTp cao<br />
I rapid shrinkage<br />
|<br />
I<br />
F<br />
CATASTROPHE<br />
l."ouin of GTP cap REscUE<br />
t<br />
rapid growth with GTP-capped end<br />
rl<br />
GTP-tubulin dimer<br />
--;---l-<br />
-<br />
straight protofilament<br />
I<br />
GTP HYDROLYSIS CHANGES SUBUNIT CONFORMATION<br />
AND WEAKENS BOND IN THE POLYMER<br />
curved protofilament<br />
i<br />
F<br />
GTP cap<br />
less stable<br />
region of<br />
microtubule<br />
containing<br />
GDP-tubulin<br />
dimers<br />
GROWING<br />
0'@<br />
s os<br />
o<br />
o<br />
ro<br />
SHRINKING<br />
0,.orn<br />
(B) (c)<br />
Figure I 6-16 Dynamic instability due to the structural differences between a growing and a shrinking microtubule end. (A) lf the free<br />
tubulin concentration in solution is between the critical values indicated in Figure 16-148, a single microtubule end may undergo transitions<br />
between a growing state and a shrinking state. A growing microtubule has GTP-containing subunits at its end, forming a GTP cap' lf nucleotide<br />
hydrolysis proceeds more rapidly than subunit addition, ih".up is lost and the microtubule begins to shrink, an event called a "catastrophel'<br />
But GTP-containing subunits may still add to the shrinking end, and if enough add to form a new cap, then microtubule growth resumes, an<br />
event called "rescue." (B) Model for the structural consequences of GTP hydrolysis in the microtubule lattice. The addition of GTP-containing<br />
tubulin subunits to the end of a protofilament causes the end to grow in a linear conformation that can readily pack into the cylindrical wall of<br />
the microtubule. Hydrolysis of Gip after assembly changes the conformation of the subunits and tends to force the protofilament into a curved<br />
shape that is less able to pack into the microtubule wall. (C) In an intact microtubule, protofilaments made from GDP-containing subunits are<br />
forced into a linear conformation by the many lateral bonds within the microtubule wall, given a stable cap of GTP-containing subunits. Loss of<br />
the GTP cap, however, allows the GDP-containing protofilaments to relax into their more curved conformation.This leads to a progressive<br />
disruption of the microtubule. Above the drawings of a growing and a shrinking microtubule, electron micrographs show actual microtubules<br />
in each of these two states, as observed in preparations in vitreous ice. Note particularly the curling, disintegrating GDP-containing<br />
protofilaments at the end of the shrinking microtubule. (C, courtesy of E.M. MandelkoW E. Mandelkow and R.A. Milligan, J. Cell Biol.<br />
114:977-991,1991. With permission from The Rockefeller University Press.)<br />
c<br />
981
982 Chapter 1 6: The <strong>Cytoskeleton</strong><br />
time 0 sec<br />
1 25 sec 307 sec<br />
previously, the rate-limiting step in the formation of a new filament is nucleation,<br />
so these rapidly diffusing subunits tend to assemble either on the ends of<br />
preexisting filaments or at particular sites where special proteins catalyze the<br />
nucleation step. The new filaments in either case are highly dynamic, and unless<br />
specifically stabilized, they have only a fleeting existence. By controlling where<br />
filaments are nucleated and selectively stabilized, a cell can control the location<br />
of its filament systems, and hence its structure. It seems that the cell is continually<br />
testing a wide variety of internal structures and only preserving those that<br />
are useful. \Arhen external conditions change, or when internal signals arise (as<br />
during the transitions in the cell cycle), the cell is poised to change its structure<br />
rapidly (see Figures 16-2 to 16-4).<br />
Actin and tubulin have independently evolved their nucleoside triphosphate<br />
hydrolysis to enable their filaments to depolymerize readily aftei they<br />
have polymerized. These two proteins are completely unrelated in amino acid<br />
sequence: actin is distantly related in structure to the glycolltic enzyme hexokinase,<br />
whereas tubulin is distantly related to a large family of GTpases that<br />
includes the heterotrimeric G proteins and monomeric GTpases such as Ras<br />
tion you are reading now is transferred into long-term memory, even a cell as<br />
stable as a neuron can grow new elongated processes to make new slmapses. To<br />
do this, a neuron requires the dynamic, exploratory activities of its cytoikeletal<br />
filaments.<br />
Tubulin and Actin Have Been Highly conserved During Eucaryotic<br />
Evolution<br />
tube. However, they can have distinct locations in a cell and perform subtly different<br />
functions. As a striking example, a specific form of 0ltubulin forms the<br />
microtubules in six specialized touch-sensitive neurons in the nematode<br />
Caenorhabditis elegans. Mutations that eliminate this protein result in the loss of<br />
touch-sensitivity, with no apparent defect in other functions.<br />
Figure 16-1 7 Direct observation of the<br />
dynamic instability of microtubules in a<br />
living cell. Microtubules in a<br />
newt lung epithelial cell were observed<br />
after the cell was injected with a small<br />
amount of rhodamine labeled tubulin, as<br />
in Figure 16-1 5. Notice the dynamic<br />
instability of microtubules at the edge of<br />
the cell. Four individual microtubules are<br />
highlighted for clarity; each of these<br />
shows alternating shrinkage and growth.<br />
(Courtesy of Wendy C. Salmon and Clare<br />
Waterman-Storer.)
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THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS<br />
the central rod domain, demonstrating the importance of this particular part of<br />
the protein for correct filament assembly.<br />
A second family of intermediate filaments, called neurofilaments, is found<br />
in high concentrations along the axons of vertebrate neurons (Figure 16-22).<br />
Three types of neurofilament proteins (NF-L, NF-M, NF-H) coassemble in uiuo,<br />
forming heteropolymers that contain NF-L plus one of the others. The NF-H and<br />
NF-M proteins have lengthy C-terminal tail domains that bind to neighboring<br />
filaments, generating aligned arrays with a uniform interfilament spacing. During<br />
axonal growth, new neurofilament subunits are incorporated all along the<br />
axon in a dgramic process that involves the addition of subunits along the filament<br />
length, as well as the addition of subunits at the filament ends. After an<br />
axon has gro\,&'n and connected with its target cell, the diameter of the axon may<br />
increase as much as fivefold. The level of neurofilament gene expression seems<br />
to directly control axonal diameter, which in turn influences how fast electrical<br />
signals travel dor,tm the axon.<br />
The neurodegenerative disease amyotrophic lateral sclerosis (ALS, or Lou<br />
Gehrig's Disease) is associated with an accumulation and abnormal assembly of<br />
neurofilaments in motor neuron cell bodies and in the axon, which may interfere<br />
with normal axonal transport. The degeneration of the axons leads to muscle<br />
weakness and atrophy, which is usually fatal. The over-expression of human<br />
NF-L or NF-H in mice results in mice that have an ALS-like disease.<br />
The vimentin-like filaments are a third family of intermediate filaments.<br />
Desmin, a member of this family, is expressed in skeletal, cardiac, and smooth<br />
muscle. Mice lacking desmin shownormal initial muscle development, but adults<br />
have various muscle cell abnormalities, including misaligned muscle fibers.<br />
Drugs Can Alter Filament Polymerization<br />
Because the survival of eucaryotic cells depends on a balanced assembly and<br />
disassembly of the highly conserved cytoskeletal filaments formed from actin<br />
and tubulin, the two types of filaments are frequent targets for natural toxins.<br />
These toxins are produced in self-defense by plants, fungi, or sponges that do<br />
not wish to be eaten but cannot run away from predators, and they generally<br />
disrupt the filament polymerization reaction. The toxin binds tightly to either<br />
the filament form or the free subunit form of a polymer, driving the assembly<br />
reaction in the direction that favors the form to which the toxin binds. For example,<br />
the drug latrunculin, extracled from the sea sponge Latrunculia magnifica,<br />
binds to actin monomers and prevents their assembly into filaments; it thereby<br />
987<br />
Figure 16-22 Two types of intermediate<br />
filaments in cells of the nervous system.<br />
(A) Freeze-etch electron microscopic<br />
image of neurofilaments in a nerve cell<br />
axon, showing the extensive cross-linking<br />
through protein cross-bridges-an<br />
arrangement believed to give this long<br />
cell process great tensile strength. The<br />
cross-bridges are formed by the long,<br />
nonhelical extensions at the C-terminus<br />
of the largest neurofilament protein<br />
(NF-H). (B) Freeze-etch image of glial<br />
filaments in glial cells, showing that these<br />
intermediate filaments are smooth and<br />
have few cross-bridges. (C) Conventional<br />
electron micrograph of a cross section of<br />
an axon showing the regular side-to-side<br />
spacing of the neurofilaments, which<br />
greatly outnumber the microtubules.<br />
(A and B, courtesy of Nobutaka Hirokawa;<br />
C, courtesy of John Hopkins.)
988 Chapter 1 6: The <strong>Cytoskeleton</strong><br />
Table 16-2 Drugs That Affect Actin Filaments and Microtubules<br />
Phalloidin<br />
Cytochalasin<br />
Swinholide<br />
Latrunculin<br />
Taxol<br />
Colchicine, colcemid<br />
Vinblastine, vincristine<br />
Nocodazole<br />
binds and stabilizes filaments<br />
caps filament plus ends<br />
severs filaments<br />
binds subunits and prevents their polymerization<br />
binds and stabilizes microtubules<br />
binds subunits and prevents their polymerization<br />
binds subunits and prevents their polymerization<br />
binds subunits and prevents their polymerization<br />
stabilizes free tubulin, causing microtubule depolymerization. In contrast,<br />
taxol, extracted from the bark of a rare species of yew tree, binds to and stabilizes<br />
microtubules, causing a net increase in tubulin polyrnerization. These and some<br />
other natural products that are commonly used by cell biologists to manipulate<br />
the cyoskeleton are listed in Table 16-2.<br />
(A)<br />
o o<br />
C -NH-CH-CH-C -O<br />
15 pm<br />
o<br />
H3C-C -O<br />
H,C<br />
c-o<br />
o<br />
9oH<br />
CH<br />
o-c -<br />
ll<br />
o<br />
CH:<br />
Figure 1 6-23 Effect of the drug taxol on<br />
microtubule organization. (A) Molecular<br />
structure of taxol. Recently, organic<br />
chemists have succeeded in synthesizing<br />
this complex molecule, which is widely<br />
used for cancer treatment.<br />
(B) | mmunofluorescence micrograph<br />
showing the microtubule organization in<br />
a liver epithelial cell before the addition<br />
of taxol. (C) Microtubule organization in<br />
the same type of cell after taxol<br />
treatment. Note the thick circumferential<br />
bundles of microtubules around the<br />
periphery of the cell. (D) A Pacific yew<br />
tree, the natural source of taxol.<br />
(8, C from N.A. Gloushankova et al., Proc.<br />
Natl Acad. Sci. U.S.A. 91 :8597 -8601, 1 994.<br />
With permission from National Academy<br />
of Sciences; D courtesy of A.K. Mitchell<br />
2001. o Her Majesty the Queen in Right<br />
of Canada, Canadian Forest Service.)
THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS<br />
Figure 16-24The bacterial FtsZ protein, a tubulin homolog in<br />
procaryotes. (A) A band of FtsZ protein forms a ring in a dividing bacterial<br />
cell. This ring has been labeled by fusing the FtsZ protein to the green<br />
fluorescent protein (GFP), which allows it to be observed in living E coli<br />
cells with a fluorescence microscope. Iop, side view shows the ring as a bar<br />
in the middle of the dividing cell. Bottom, rotated view showing the ring<br />
structure. (B) FtsZ filaments and rings, formed in vitro, as visualized using<br />
electron microscopy. Compare this image with that of the microtubule<br />
shown on the right in Figure 16-16C. (A, from X. Ma, D.W. Ehrhardt and<br />
W. Margolin, Proc. Natl Acad. Sci. IJ.S.A.93:12998-13003, 1996; B, from<br />
:i,:5:Tffi'-.'#l;illir'ifl ,':?y.',i.'"::i1e-s23'lee6Arrw*h<br />
(A)<br />
Bacterial Cell Organization and Cell Division Depend on<br />
Homologs of the Eucaryotic <strong>Cytoskeleton</strong><br />
\Mhile eucaryotic cells are typically large and morphologically complex, bacterial<br />
cells are usually only a few micrometers long and assume simple, modest shapes<br />
such as spheres or rods. Bacteria also lack the elaborate networks of intracellular<br />
membrane-enclosed organelles such as the endoplasmic reticulum and<br />
Golgi apparatus. For many years, biologists assumed that the lack of a bacterial<br />
cltoskeleton was one reason for these striking differences between cell organization<br />
in the eucaryotic and bacterial kingdoms. This assumption was challenged<br />
with the discovery in the early 1990s that nearly all bacteria and many<br />
archaea contain a homolog of tubulin, FtsZ, that can polymerize into filaments<br />
and assemble into a ring (called the Z-ring) at the site where the septum forms<br />
during cell division (Figure 16-24).<br />
The three-dimensional folded protein structure of FtsZ is remarkably simi-<br />
Iar to the structure of o or B tubulin and, like tubulin, hydrolysis of GTP is triggered<br />
by polymerization and causes a conformational change in the filament<br />
structure. Although the Z-ring itself persists for many minutes, the individual filaments<br />
within it are highly dynamic, with an average half-life of about thirty seconds.<br />
As the bacterium divides, ttre Z-ring becomes smaller until it has completely<br />
disassembled, and it is thought that the shrinkage of the Z-ring may contribute<br />
to the membrane invagination necessary for the completion of cell division.<br />
The Z-ringmay also serve as a site for localization of specialized cell wall<br />
slnthesis enzymes required for building the septum between the two daughter<br />
cells. The disassembled FtsZ subunits later reassemble at the new sites of septum<br />
formation in the daughter cells (Figure f 6-25).<br />
More recently, it has been found that many bacteria also contain homologs<br />
of actin. TWo of these, MreB and Mbl, are found primarily in rod-shaped or spiral-shaped<br />
cells, and mutations disrupting their expression cause extreme<br />
abnormalities in cell shape and defects in chromosome segregation (Figure<br />
f6-26).MreBandMblfilamentsassemble inuiuotoformlarge-scalespiralsthat<br />
(A)<br />
time 0<br />
(min)<br />
35<br />
, trm<br />
Figure l6-25 Rapid rearrangements of FtsZ through the bacterial cell cycle. (A) After chromosome segregation is complete,<br />
thl ring formed by FtsZ at the middle of the cell becomes smaller as the cell pinches in two, much like the contractile ring<br />
formed by actin and myosin filaments in eucaryotic cells. The FtsZ filaments that have disassembled as the cells have separated<br />
then reassemble to form two new rings at the middle of the two daughter cells. (B) Dividing chloroplasts (red\ from a red alga<br />
also make use of a protein ring made from FtsZ (yeltow)for cleavage. (A, from Q. Sun and W. Margolin, J. Bacteriol.<br />
1 80:2050-2056, 1 998. With peimission from American Society for Microbiology; B, from S. Miyagishima et al., Plant Cell<br />
13:2257-2268,2001. With permission from American Society of Plant Biologists')<br />
37<br />
(B)<br />
(B)<br />
1 rm<br />
989<br />
il;
990 Chapter 1 6: The <strong>Cytoskeleton</strong><br />
L-----.1 (B)<br />
5um<br />
span the length of the cell and apparently contribute to cell shape determination<br />
by serving as a scaffold to direct the synthesis of the peptidoglycan cell wall,<br />
in much the same way that microtubules help organize the synthesis of the cellulose<br />
cell wall in higher plant cells (see Figure 19-82). As with Ftsz, the filaments<br />
within the MreB and Mbl spirals are highly dynamic, with half-lives of a<br />
few minutes; as for actin, ATP hydrolysis accompanies the polymerization pro-<br />
CCSS.<br />
Diverse relatives of MreB and Mbl have more specialized roles. A particularly<br />
intriguing bacterial actin homolog is parM, which is encoded on certain bacterial<br />
plasmids that also carry genes responsible for antibiotic resistance and frequently<br />
cause the spread of multi-drug resistance in epidemics. Bacterial plasmids<br />
typically encode all the gene products that are necessary for their owrsegregation,<br />
presumably as a strategy to ensure their faithful inheritance and propagation<br />
in their bacterial hosts. rn uiuo, parM assembles into a filamentous<br />
structure that associates at each end with a copy of the plasmid that encodes it,<br />
and growth of the ParM filament appears to push the replicated plasmid copies<br />
apart, rather like a mitotic spindle operating in reverse (Figure 16-2z).Altholgh<br />
ParM is a structural homolog of actin, its dynamic behavior differs significantiy.<br />
ParM filaments undergo dramatic dynamic instability in uitro, more closeiy<br />
resembling microtubules than actin filaments in the way that they grow and<br />
shrink. The spindle-like structure is apparently built by the selective stabilization<br />
of spontaneously nucleated filaments that bind to specialized proteins<br />
recruited to the origins of replication on the plasmids.<br />
The various bacterial actin homologs share similar molecular structures but<br />
their amino acid sequence similarity to each other is quite low (- l0-15% identical<br />
residues). They assemble into filaments with distinct helical packing patterns,<br />
which may also have very different dynamic behaviors. Rather than uiing<br />
the same well-conserved actin for many different purposes, as eucaryotic celli<br />
do, bacteria have apparently opted to proliferate and specialize their actin<br />
homologs for distinct purposes.<br />
It is now clear that the general principle of organizing cell structure by the<br />
self-association of nucleotide-binding proteins into dynamic helical filaments is<br />
used in all cells, and that the two major families of actin and tubulin are verv<br />
monomers replication f ilaments proteins<br />
(A)<br />
(B)<br />
(c)<br />
5[m<br />
r<br />
2ttm<br />
Figure 16-26 Actin homologs in<br />
bacteria determine cell shape. (A) The<br />
common soil bacterium Bacillus subtilis<br />
normally forms cells with a regular rodlike<br />
shape. (B) B. subtilis cells lacking the<br />
actin homolog Mbl grow into irregular<br />
twisted tubes and eventually die. (C) The<br />
Mbl protein forms long helices made of<br />
up many short filaments that run the<br />
length of the bacterial cell and help to<br />
direct the sites of cell wall synthesis.<br />
(From L.J. Jones, R. Carbadillo-Lopez and<br />
J. Errington, Cell 104: 913-922, 2001. With<br />
permission from Elsevier.)<br />
Figure 16-27 Role of the actin homolog<br />
ParM in plasmid segregation. (A) Some<br />
bacterial drug-resistance plasmids<br />
(yellow) encode an actin homolog, ParM,<br />
that will spontaneously nucleate to form<br />
small, dynamic filaments (green)<br />
throughout the bacterial cytoplasm. A<br />
second plasmid-encoded protein (b/ue)<br />
binds to specific DNA sequences in the<br />
plasmid, and also stabilizes the dynamic<br />
ends of the ParM filaments. When the<br />
plasmid has duplicated, so that the ParM<br />
filaments can be stabilized at both ends,<br />
the filaments grow and push the<br />
duplicated plasmids to opposite ends of<br />
the cell. (B) In these bacterial cells<br />
harboring a drug-resistance plasmid, the<br />
plasmids are labeled in red and the ParM<br />
protein in green.Left, a short ParM<br />
bundle connects the two daughter<br />
plasmids shortly after their duplication.<br />
Right, the fully assembled ParM filament<br />
has pushed the duplicated plasmids to<br />
the cell poles. (A, adapted from<br />
E.C. Garner, C.S. Campbell ano<br />
R.D. Mullins, Science 306:1 021 -1 025,<br />
2004. With permission from AAAS; B, from<br />
J. Moller-Jensen et al., Mol. Cell<br />
1 2:1 47 7 -1 487, 2003. With permission<br />
from Elsevier.)
THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS<br />
L__ t (B)<br />
zpm<br />
ancient, probably predating the split between the eucaryotic and bacterial kingdoms.<br />
However, the uses to which bacteria put their cltoskeletons appear somewhat<br />
different from their eucaryotic homologs. For example, in bacteria it is the<br />
tubulin (FtsZ) that is involved in cytokinesis (the pinching apart of a dividing cell<br />
into two daughters), while actin drives this process in eucaryotic cells. Conversely,<br />
eucaryotic microtubules are responsible for chromosome segregation,<br />
while bacterial actins (ParM and possibly MreB) help to segregate replicated<br />
DNA in bacteria.<br />
At least one bacterial species with an unusual crescent shape, Caulobacter<br />
crescentus, even appears to harbor a protein with significant structural similarity<br />
to the third major class of cytoskeletal filaments found in animal cells, the<br />
intermediate filaments. A protein called crescentin forms a filamentous structure<br />
that seems to influence the cell shape; when the gene encoding crescentin<br />
is deleted, the Caulobacter cells grow as straight rods (Figure f 6-28)'<br />
Since we now know that bacteria do in fact have sophisticated dynamic<br />
cltoskeletons, why then do they remain so small and morphologically simple?<br />
As yet there have been no motor proleins identified that walk along the bacterial<br />
filaments; perhaps the evolution of motor proteins was a critical step allowing<br />
morphological elaboration in the eucaryotes.<br />
Summary<br />
(A)<br />
I<br />
2pm<br />
The cytoplasm of eucaryotic cells is spatielly organized by a network of protein ftla'<br />
ments known as the cytoskeleton. This network contains three principal types of filaments:<br />
microtubules, actin filaments, and intermediate filaments. All three types of filaments<br />
form as helical assemblies of subunits that self-associate using a combination<br />
of end-to-end and side-to-side protein contacts. Dffirences in the structure of the subunits<br />
and the manner of their self-assembly giue the fitaments dffirent mechanical<br />
properties. Intermediate filaments are rope-like and easy to bend but hard to break.<br />
Microtubules are strong, rigid hollow tubes. Actin filaments are the thinnest of the<br />
three and are easy to break.<br />
In liuing cells, the assembly and disassembly of their subunits constantly remodels<br />
all three types of cytoskeletal filaments. Microtubules and actin filaments add and lose<br />
subunits only at their ends, with one end (the plus end) growing faster than the other.<br />
Tubulin and actin (the subunits of microtubules and actin filaments, respectiuely)<br />
birul and hydrolyze nucleoside triphosphates (tubulin binds GTP and actin binds<br />
ATP). Nucleotide hydrolysis underlies the characteristic dynamic behauior of these two<br />
fiIaments. Actin filaments in cells seem to predominantly undergo treadmilling, where<br />
a filament assembles at one end while simultaneously disassembling at the other end.<br />
Microtubules in cells predominantly display dynamic instability, where a microtubule<br />
end undergoes alternating bouts of growth and shrinkage.<br />
tnAlhereas tubulin and actin haue been strongly conserued in euolution, the family<br />
of intermediate filaments is uery diuerse. There are many tissue-speciftc forms found in<br />
the cytoplasm of animal cells, including keratin filaments in epithelial cells, neurofilaments<br />
in nerue cells, and desmin filaments in muscle cells. In all these cells, the primary<br />
job of intermediate ftlaments is to prouide mechanical strength.<br />
Bacterial cells also containhomologs of tubulin, actin and intermediate filaments<br />
that form dynamic filamentous structures inuolued in determining cell shape and in<br />
cell diuision.<br />
991<br />
Figure 1 6-28 Caulobacter and<br />
crescentin. The sickle-shaped bacterium<br />
Caulobacter crescenfus expresses a protein,<br />
crescentin, with a series of coiled-coil<br />
domains similar in size and organization to<br />
the domains of eucaryotic intermediate<br />
filaments. In cells, the crescentin protein<br />
forms a fiber that runs down the inner side<br />
of the curving bacterial cell wall. When the<br />
gene is disrupted, the bacteria are viable<br />
but grow in a straight rod-shaped form.<br />
(From N. Ausmees, J.R. Kuhn and<br />
C. Jacobs-Wagn er, Cell 115:705-713,2003'<br />
With oermission from Elsevier.)
992 Chapter 16:The <strong>Cytoskeleton</strong><br />
FILAM ENTS<br />
Microtubules, actin filaments, and intermediate filaments are much more<br />
dynamic in cells than they are in the test tube. The cell regulates the length and<br />
stability of its cytoskeletal filaments, as well as their number and geometry. It<br />
does so largely by regulating their attachments to one another and to other components<br />
of the cell, so that the filaments can form a wide variety of higher-order<br />
structures. Direct covalent modification of the filament subunits regulates some<br />
filament properties, but most of the regulation is performed by a large array of<br />
accessory proteins that bind to either the filaments or their free subunits. Some<br />
of the most important accessory proteins associated with microtubules and<br />
actin filaments are outlined in Panel 16-3 (pp. gg4-gg5). This section describes<br />
how these accessory proteins modify the dynamics and structure of cltoskeletal<br />
filaments. we begin with a discussion of the way that microtubules and actin filaments<br />
are nucleated in cells, because this plays a major part in determining the<br />
overall organization of the cell's interior.<br />
A Protein complex containing y-Tubulin Nucleates Microtubules<br />
\.{/hile cr- and B-tubulins are the regular building blocks of microtubules, another<br />
type of tubulin, carled y-tubulin, ]nas a more specialized role. present in much<br />
smaller amounts than cr- and B-tubulin, this protein is involved in the nucleation<br />
of microtubule growth in organisms ranging from yeasts to humans.<br />
Microtubules are generally nucleated from a specific intracellular location<br />
knor,rm as a microtubule-organizing center (MToc). Antibodies against y-tubulin<br />
stain the MToc in virtually all species and cell types thus far examined.<br />
Microtubules<br />
Emanate<br />
from the centrosome<br />
in Animal cells<br />
Most animal cells have a single, well-defined MToc called the centrosome,<br />
located near the nucleus. From this focal point, the cytoplasmic microtubules<br />
emanate in a star-like, "astral" conformation. Microtubules are nucleated at the<br />
centrosome at their minus ends, so the plus ends point outward and grow<br />
toward the cell periphery. Microtubules nucleated at ihe centrosome continuously<br />
grow and shrink by dFramic instabiliry probing the entire three-dimensional<br />
volume of the cell. A centrosome is composed of a fibrous centrosome<br />
y-tu bul in<br />
accessory proteins in<br />
y-tubulin ring complex<br />
Figure 16-29 Polymerization of tubulin<br />
nucleated by ytubulin ring complexes.<br />
(A) Structure of the y-tubulin ring<br />
complex, reconstructed from averaging<br />
electron micrographs of individual<br />
purified complexes. (B) Model for the<br />
nucleation of microtubule growth by the<br />
lTuRC. The red outline indicates a pair of<br />
proteins bound to two molecules of<br />
y-tubulin; this group can be isolated as a<br />
separate subcomplex of the larger ring.<br />
Note the longitudinal discontinuity<br />
between two protofilaments.<br />
Microtubules generally have one such<br />
"seam" breaking the otherwise uniform<br />
helical packing of the protofilaments.<br />
(C) Electron micrograph of a single<br />
microtubule nucleated from the purified<br />
y-tubulin ring complex. (A and C, from<br />
M. Moritz et al., Ndf. Cell Biol.2:365-370,<br />
2000. With permission from Macmillan<br />
Publishers Ltd.)
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ACTIN FILAMENTS<br />
./ a<br />
{<br />
A<br />
cofilin<br />
formin<br />
nucleates assembly and remains<br />
associated with the growing<br />
plus end<br />
thymosin<br />
binds subunits,<br />
prevents assembly<br />
binds ADP-actin f ilaments,<br />
accelerates disassembly<br />
fimbrin<br />
a-actinin<br />
ar<br />
rro<br />
a't C<br />
./<br />
gelsolin<br />
severs filaments and<br />
binds to plus end<br />
F<br />
oa o -V<br />
O2or<br />
oo.<br />
actin subunits<br />
actin filament<br />
ARP complex<br />
nucleates assembly<br />
to form a web and remains<br />
associated with the minus end<br />
'r t<br />
-\<br />
o\t.<br />
a<br />
capping protein<br />
prevents assembly and<br />
disassembly at plus end<br />
filament bundling, cross-linking, and attachment to membranes<br />
f ilamin<br />
profilin<br />
binds subunits,<br />
speeds elongation<br />
Some of the major accessory proteins of the actin cytoskeleton. Except for the myosrn motor proteins,<br />
a later,section,<br />
to be discussed<br />
an<br />
in<br />
example oi each. major type is shown. Each of theie is discussed in the text. HoweveL<br />
more than<br />
most cells<br />
a hundred<br />
contain<br />
different actin-bindin! proteins, and it is rit"rvihaiih*e are important<br />
proteins<br />
types of actin-associated<br />
that are not yet recognized.
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HOW CELLS REGULATE THEIR CYTOSKELETAL FILAMENTS<br />
by two different tlpes of regulated factors, the ARP complex and the formins<br />
(discussed below). The first of these is a complex of proteins that includes two<br />
actin-related proteins, or ARPs, each of which is about 45% identical to actin.<br />
Analogous to the function of the y-TuRC, the ARP complex (also known as the<br />
Arp 2/3 complex) nucleates actin filament growth from the minus end, allowing<br />
rapid elongation at the plus end (Figure 16-34A and B). The complex can also<br />
(A)<br />
T..-l<br />
".,in ll ]<br />
activating<br />
factor<br />
ARP complex active ARP complex<br />
(B)<br />
t.\<br />
\!/<br />
nrpz [ ]<br />
mrnus<br />
eno<br />
nucleated actin f ilament<br />
(D)<br />
arpr [ ]<br />
Figure 16-34 Nucleation and actin web formation by the ARP comple;<br />
actin. Afthough the face of the molecule equivalent to the plus end (toP.<br />
differences on the sides and minus end (bottom) prevent these actin-reli<br />
into filaments with actin. (B) A model for actin filament nucleation by th<br />
are held by their accessory proteins in an orientation that prevents then<br />
indicated by the blue triangle binds the complex, Arp2 and Arp3 are bro<br />
an actin filament. Actin subunits can then assemble onto this structure,<br />
1 6-10). (C) The ARP complex nucleates filaments most efficiently when i<br />
filament branch that grows at a 70. angle relative to the original filament. Repeated rounds of branching nucleation result in a treelike web of<br />
actin filaments. (D) Top, electron micrographs of branched u'.tin filur"ntt formed by mixing purified actin subunits with purified ARP<br />
complexes. Bottom, reconstructed image of a branch where the crystal structures oi actin and the ARP complex have been fitted to the<br />
electron density. The mother filament runs from top to bottom, and the daughter filament branches off to the right where the ARP complex<br />
binds to three actin subunits in the mother filament (D, from R.D. Mullins et al., Proc. Natl Acad. Sci. IJ.S.A.95:6181-61 86, 1 998' With permission<br />
from National Academy of Sciences, and from N. Volkmann et al., Science 293:2456-2459, 2001. With permission from Macmillan Publishers Ltd')<br />
100 nm<br />
10 nm<br />
997
998 Cha pter 1 6: The <strong>Cytoskeleton</strong><br />
rR\<br />
20 um 100 pm<br />
attach to the side of another actin filament while remaining bound to the minus<br />
end of the filament that it has nucleated, thereby building individual filaments<br />
into a treelike web (Figure l6-34C and D).<br />
near the plasma membrane in yeast, where it is required to form cortical actin<br />
patches (see Figure 16-6), and in plant cells, where it directs the formation of<br />
actin bundles at the surface that are required for the growth of complex cell<br />
shapes in a variety ofdifferent tissues (Figure f6-3b).<br />
The Mechanism of Nucleation Infruences Large-scale Filament<br />
Organization<br />
or microtubule and prevent both subunit addition and subunit loss at this end.<br />
Figure 1 6-35 Function of the ARP<br />
complex in plant cells. (A) Cells in the<br />
maize leaf epidermis form small, actinrich<br />
lobes that lock neighboring cells<br />
together like pieces of a jigsaw puzzle.<br />
(B) The regular pattern of interlocking<br />
cells covers the leaf surface. (C) Epidermal<br />
cells in a mutant plant lacking the ARp<br />
complex do not form the interlocking<br />
lobes. The brick-shaped cells are normal<br />
in size and spacing, but form leaves that<br />
appear too shiny to the naked eye. (From<br />
M.J. Frank, H.N. Cartwright and<br />
L.G. Smith, Development 1 30:7 53-7 62,<br />
2003. With permission from the Company<br />
of Biologists.)
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666
1000 Chapter 16;The <strong>Cytoskeleton</strong><br />
Figure 16-38 Profilin and formins. Some members of the formin protein<br />
family have unstructured domains or"whiskers"that contain several binding<br />
sites for profilin or the profilin-actin complex. These flexible domains serve as<br />
a staging area for addition of actin to the growing prus end of the actin<br />
filament when formin is bound. Under some conditions, this can enhance the<br />
rate of actin filament elongation so that filament growth is faster than that<br />
expected for a diffusion-controlled reaction, and faster in the presence of<br />
formin and profilin than the rate for pure actin alone (see also Figure 3-g0C).<br />
motile structures such as filopodia and lamellipodia (see below). Besides binding<br />
to actin and phospholipids, profilin also binds to various other intracellular<br />
proteins that have domains rich in proline; these proteins can also help to localize<br />
profilin to sites that require rapid actin asrembly.<br />
As it does with actin monomers, the cell sequesters unpolymerized tubulin<br />
subunits to maintain the subunit pool at a level substantially higher than the<br />
critical concentration. one molecule of the small protein smnmtibinds to two<br />
tubulin heterodimers and prevents their additlon onto the ends of microtubules.<br />
Stathmin thus decreases the effective concentration of tubulin subunits<br />
that are available for polymerization (an action analogous to that of the drug<br />
colchicine). Furthermore, stathmin enhances the rk;hhood that a growing<br />
microtubule will undergo the catastrophic transition to the shrinkin"g statel<br />
Phosphorylation of stathmin inhibits iis binding ro tubulin, and signils that<br />
c.ause stathmin phosphorylation can increase the rate of microtubui=e elongation<br />
andsuppress dynamic instability. Cancer cells frequently overexpress stuihmin,<br />
and the increased rate of microtubule turnover that results is thought to<br />
contribute to the characteristic change in cell shape associated with mahlnant<br />
transformation.<br />
severing Proteins Regulate the Length and Kinetic Behavior of<br />
Actin Filaments and Microtubules<br />
In some situations, a cell may break an existing long filament into many smaller<br />
filaments. This generates a large number of new filament ends: one iong filament<br />
with just one plus end and one minus end might be broken into dozens of<br />
short filaments, each with its own minus end and plus end. under some intracellular<br />
conditions, these newly formed ends nucleite filament elongation, and<br />
in-this case severing accelerates the assembly of new filament structures. under<br />
other conditions, severing promotes the depolymerization of old filaments,<br />
speeding up the depolymerization rate by tenfbldor more. In addition, severing<br />
filaments changes the physical and mechanical properties of the cytoplasmi<br />
stiff, large bundles and gels become more fluid when the filaments are severed.<br />
To sever a microtubule, thirteen longitudinal bonds must be broken, one for<br />
each protofilament. The protein katanin, named after the Japanese word for<br />
"sword," accomplishes-this demanding task (Figure 16-39). Kaianin is made up<br />
of two subunits, a smaller subunit thai hydrolyies Arp and performs the actual<br />
severing, and a larger one that directs katanin to the centrosome. Katanin<br />
releases microtubules from their attachment to a microtubule organizing center,<br />
and it is thought to have an important role in the rapid microtub"ule dep"oll,rnerization<br />
observed at the poles of spindles during mei,osis and mitosis. It may also<br />
be involved in microtubule release and depolyrnerization in proliferating cells in<br />
interphase and in postmitotic cells such ai nerr.ons.<br />
In contrast to microtubule severing by katanin, which requires ArB the severing<br />
of actin filaments does not requiie an extra energy input. Most actin-severing<br />
proteins are members of the gekotin superfamity,'whbse severing activity<br />
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in the protofilament. According to one model for gelsolin severing, gelsolin<br />
binds on the side of an actin filament and waits until a thermal fluctuation hanpens<br />
to create a small gap between neighboring subunits in the protofilament;<br />
gelsolin then insinuates its subdomain into the gap, breaking the filament.<br />
't<br />
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I nreear wrrH<br />
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HOW CELLS REGULATE THEIR CYTOSKELETAL FILAMENTS<br />
Some of the +TIPs, such as the kinesin-related catastrophe factors and<br />
XMAP215 mentioned above, modulate the growth and shrinkage of the microtubule<br />
end to which they are attached. Others control microtubule positioning<br />
by helping to capture and stabilize the growing microtubule end at the location<br />
of specific target proteins in the cell cortex. EBl, a +TIP present in both yeasts<br />
and humans, for example, is essential for yeast mitotic spindle positioning,<br />
directing the growing plus ends of yeast spindle microtubules to a specific docking<br />
region in the yeast bud and then helping to anchor them there.<br />
Filaments Are Organized into Higher-Order Structures in Cells<br />
So far, we have described how cells use accessory proteins to regulate the location<br />
and dynamic behavior of cltoskeletal filaments. These proteins can nucleate<br />
filament assembly, bind to the ends or sides of the filaments, or bind to the<br />
free subunits of filaments. But in order for the cytoskeletal filaments to form a<br />
useful intracellular scaffold that gives the cell mechanical integrity and determines<br />
its shape, the individual filaments must be organized and attached to one<br />
another in larger-scale structures. The centrosome is one example of such a<br />
cytoskeletal organizer; in addition to nucleating the growth of microtubules, it<br />
holds them together in a defined geometry, with all of the minus ends buried in<br />
the centrosome and the plus ends pointing outward. In this way, the centrosome<br />
creates the astral array of microtubules that is able to find the center of each cell<br />
(see Figure 16-32).<br />
Another mechanism that cells use to organize filaments into large structures<br />
is filament cross-linking. As described earlier, some MAPs can bundle microtubules<br />
together: they have two domains-one that binds along the microtubule<br />
side (and thereby stabilizes the filament) and another that projects outward to<br />
contact other MAP-coated microtubules. In the actin cytoskeleton, the stabilizing<br />
and cross-linking functions are separated. Tropomyosin binds along the<br />
sides of actin filaments, but it does not have an outward projecting domain. As<br />
we shall see shortly, filament cross-linking is instead mediated by a second<br />
group of actin-binding proteins that have only this function. Intermediate filaments<br />
are different yet again; they are organized both by a lateral self-association<br />
of the filaments themselves and by the cross-linking activity of accessory<br />
proteins, as we describe next.<br />
lntermediate Filaments Are Cross-Linked and Bundled Into<br />
Strong Arrays<br />
Each individual intermediate filament forms as a long bundle of tetrameric subunits<br />
(see Figure 16-19). Many intermediate filaments further bundle themselves<br />
by self-association; for example, the neurofilament proteins NF-M and<br />
NF-H (see Table 16-I, p. 985) contain a C-terminal domain that extends outward<br />
from the surface of the assembled intermediate filament and binds to a neighboring<br />
filament. Thus groups of neurofilaments form robust parallel arrays that<br />
are held together by multiple lateral contacts, giving strength and stability to the<br />
long cell processes ofneurons (see Figure 16-22).<br />
Other tlpes of intermediate filament bundles are held together by accessory<br />
proteins, such as filaggrin, which bundles keratin filaments in differentiating<br />
cells of the epidermis to give the outermost layers of the skin their special toughness.<br />
Plectinis a particularly interesting cross-linking protein. Besides bundling<br />
intermediate filaments, it also links the intermediate filaments to microtubules,<br />
actin filament bundles, and filaments of the motor protein myosin II (discussed<br />
below), as well as helping to attach intermediate filament bundles to adhesive<br />
structures at the plasma membrane (Figure 16-46).<br />
Mutations in the gene for plectin cause a devastating human disease that<br />
combines epidermolysis bullosa (caused by disruption of skin keratin filaments),<br />
muscular dystrophy (caused by disruption of desmin filaments), and<br />
neurodegeneration (caused by disruption of neurofilaments). Mice lacking a<br />
1 005
1006 Chapter 16:The <strong>Cytoskeleton</strong><br />
u.5 um<br />
functional plectin gene die within a few days of birth, with blistered skin and<br />
abnormal skeletal and heart muscles. Thus, although plectin may not be necessary<br />
for the initial formation and assembly of intermediate filaments, its crosslinking<br />
action is required to provide cells with the strength they need to withstand<br />
the mechanical stresses<br />
inherent to vertebrate life.<br />
cross-linking Proteins with Distinct properties organize Different<br />
Assemblies of Actin Filaments<br />
Actin filaments in animal cells are organized into two types of arrays: bundles<br />
and weblike (gel-like) networks (Figure lHz). As described earlier, these different<br />
structures are initiated by the action of distinct nucleating proteins: the<br />
long straight filaments produced by formins make bundles and the ARp complex<br />
makes webs. The actin filament cross-linking proteins that help to stabilize<br />
and maintain these distinct structures are divided into tvvo classes: bundling<br />
proteins and gel-forming proteins. Bundling proteins cross-link actin filaments<br />
into a parallel array, while gel-forming proteins hold two actin filaments<br />
together at a large angle to each other, thereby creating a looser meshwork. Both<br />
Each type of bundling protein also determines which other molecules can<br />
interact with an actin filament. Myosin II (discussed later) is the motor protein<br />
in stress fibers and other contractile arrays that enables them to contract. The<br />
contractile bundle<br />
gel-like network<br />
100 nm<br />
Figure 16-46 Plectin cross-linking of<br />
diverse cytoskeletal elements. Plectin<br />
(green) is seen here making cross-links<br />
from intermediate filaments (blue) to<br />
microtubules ( red). ln this electron<br />
micrograph, the dots (yellow) are gold<br />
particles linked to anti-plectin antibodies.<br />
The entire actin filament network was<br />
removed to reveal these proteins. (From<br />
T.M. Svitkina and G.G.Borisy, J. Cell Biol.<br />
1 35:991-1 007, 1 996. With permission<br />
from The Rockefeller University Press.)<br />
Figure 16-47 Actin arrays in a cell.<br />
A fibroblast crawling in a tissue culture dish<br />
is shown with three areas enlarged to show<br />
the arrangement of actin filaments. The<br />
actin fifaments are shown in red, with<br />
arrowheads pointing toward the minus<br />
end. Stress fibers are contractile and exert<br />
tension. Filopodia are spike-like projections<br />
of the plasma membrane that allow a cell<br />
to explore its environment. The cortex<br />
underlies the plasma membrane.
HOW CELLS REGULATE THEIRCYTOSKELETAL FI LAMENTS<br />
spectrin (tetramer)<br />
5U nm<br />
very close packing of actin filaments caused by the small monomeric bundling<br />
protein fimbrin apparently excludes myosin, and thus the parallel actin filaments<br />
held together by fimbrin are not contractile; on the other hand, the looser<br />
packing caused by the larger dimeric bundling protein a-actinin allows myosin<br />
molecules to enter, making stress fibers contractile (Figure f6-49). Because of<br />
the very different spacing between the actin filaments, bundling by fimbrin<br />
automatically discourages bundling by o-actinin, and vice-versa, so that the two<br />
types of bundling protein are themselves mutually exclusive.<br />
Villinis another bundling protein that, like fimbrin, has two actin-filamentbinding<br />
sites very close together in a single pollpeptide chain. Villin (together<br />
with fimbrin) helps cross-link the 20 to 30 tightly bundled actin filaments found<br />
in microvilli, the finger-like extensions of the plasma membrane on the surface<br />
of many epithelial cells (Figure l$-50). A single absorptive epithelial cell in the<br />
human small intestine, for example, has several thousand microvilli on its apical<br />
surface. Each is about 0.08 pm wide and I pm long, making the cell's absorptive<br />
surface area about 20 times greater than it would be without microvilli. \'Vhen<br />
villin is introduced into cultured fibroblasts, which do not normally contain<br />
villin and have only a few small microvilli, the existing microvilli become greatly<br />
elongated and stabilized, and new ones are induced. The actin filament core of<br />
the microvillus is attached to the plasma membrane along its sides by lateral<br />
sidearms made of myosin I (discussed later), which has a binding site for filamentous<br />
actin on one end and a domain that binds lipids on the other end'<br />
These two types of cross-linkers, one binding actin filaments to each other and<br />
the other binding these filaments to the membrane, seem to be sufficient to<br />
form microvilli on cells. Interestingly, when the gene for villin is disrupted in a<br />
mouse, the intestinal microvilli form with apparently normal morphology, indicating<br />
that other bundling proteins provide sufficient redundant function for<br />
this purpose. However, the remodeling of intestinal microvilli in response to certain<br />
kinds of stress or starvation is impaired.<br />
(A)<br />
fimbrin<br />
(monomer)<br />
actin filaments and<br />
d-actinin<br />
contractile bundle<br />
loose packing allows myosin-ll<br />
to enter bundle<br />
0-actinin<br />
(dimer)<br />
50 nm<br />
actin filaments and<br />
f imbrin<br />
parallel bundle<br />
tight packing prevents myosin-ll<br />
from entering bundle<br />
(B)<br />
1 007<br />
Figure l6-48 The modular structures of<br />
four actin-cross-linking proteins. Each of<br />
the proteins shown has two actinbinding<br />
sites (red) that are related in<br />
sequence. Fimbrin has two directly<br />
adjacent actin-binding sites, so that it<br />
holds its two actin filaments very close<br />
together (14 nm apart), aligned with the<br />
same polarity (see Figure 16-494). The<br />
two actin-binding sites in cr-actinin are<br />
separated by a spacer around 30 nm<br />
long, so that it forms more looselY<br />
packed actin bundles (see Figure<br />
16-49A). Filamin has two actin-binding<br />
sites with a V-shaped linkage between<br />
them, so that it cross-links actin filaments<br />
into a network with the filaments<br />
oriented almost at right angles to one<br />
another (see Figure 16-51). Spectrin is a<br />
tetramer of two o and two 0 subunits,<br />
and the tetramer has two actin-binding<br />
sites spaced about 200-nm apart (see<br />
Figure 10-41).<br />
Figure 16-49 The formation of two<br />
types of actin filament bundles.<br />
(A) cr-actinin, which is a homodimet<br />
cross-links actin filaments into loose<br />
bundles, which allow the motor protein<br />
myosin ll (not shown) to participate in<br />
the assembly. Fimbrin cross-links actin<br />
filaments into tight bundles, which<br />
exclude myosin. Fimbrin and s-actinin<br />
tend to exclude one another because of<br />
the very different spacing of the actin<br />
filament bundles that theY form.<br />
(B) Electron micrograph of purified<br />
c[-actinin molecules. (8, courtesy of<br />
John Heuser.)<br />
100 nm
1008 Chapter 1 6: The <strong>Cytoskeleton</strong><br />
(A)<br />
amorpnous,<br />
densely<br />
staining region<br />
plus end of<br />
actin filament<br />
lateral sidearm<br />
(myosin-1,<br />
calmodulin)<br />
(B) (c) | |<br />
1rt<br />
microvillus<br />
acItn<br />
f ilament<br />
bundle<br />
Figure 16-50 A microvillus. (A) A bundle of parallel actin filaments cross-linked by the actin-bundling proteins villin and<br />
fimbrin forms the core of a microvillus. Lateral sidearms (composed of myosin I and the Ca2+-binding protein calmodulin)<br />
connect the sides of the actin filament bundle to the overlying plasma membrane. All the plus ends of the actin filaments<br />
are at the tip of the microvillus, where they are embedded in an amorphous, densely staining substance of unKnown<br />
composition. (B) Freeze-fracture electron micrograph of the apical surface of an intestinal epithelial cell, showing microvilli.<br />
Actin bundles from the microvilli extend down into the cell and are rooted in the terminal web, where they are linked<br />
together by a complex set of proteins that includes spectrin and myosin ll. Below the terminal web is a layer of<br />
intermediate filaments. (C) Thin section electron micrograph of microvilli. (8, courtesy of John Heuser; C, from<br />
P.T. Matsudaira and D.R. Burgess, Cold Spring Harb. Symp. Quont. Biol. 46:845-854,1 985. With permission from Cold Spring<br />
Harbor Laboratory Press.)<br />
Filamin and Spectrin Form Actin Filament Webs<br />
The various bundling proteins that we have discussed so far have straight, stiff<br />
connections between their two actin-filament-binding domains, and they tend<br />
to align filaments in parallel bundles. In contrast, those actin cross-linking proteins<br />
that have either a flexible or a stiff, bent connection between their two<br />
binding domains form actin filament webs or gels, rather than actin bundles.<br />
projections called lamellipodiathathelp them to crawl across solid surfaces. Fil-<br />
A very different well-studied web-forming protein i s spectrin,which was first<br />
identified in red blood cells. spectrin is a long, flexible protein made out of four<br />
elongated pollpeptide chains (two cr subunits and two B subunits), arranged so<br />
that the two actin-filament-binding sites are about 200 nm apart (compared
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Surprts lueIJUJa pue plder eqt roJ pazllelJads r(tq8q are ri.aql ,tla,rpcadsar 'speeq<br />
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te aqru lse] e uI sepqnloJJllu e^o{u uec suleu,{p lPluauoxB :}sa}sPJ eql Suolue<br />
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sr peaq uraulp aql leql eloN'speaq oMl<br />
seq sleurrue uorl urau,(p ^lerlr) :speaq<br />
aarql seq pue ueozoloro e LUoJJ sr uMoqs<br />
urau{p {rer1o aql'aln)alour papeaq<br />
-oml e sr urau{p rrr-use;doy(r'ursaury pue<br />
p; urso{tu a111 'urau,{p (leuouoxe) Iletltl<br />
Jo aln)aloLu e pue urau{p rrr.use;do1,{r<br />
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q)la-azearl'sulau{q 69-9 1 arn6r1<br />
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luaraJ,rp {ren aql'r e1 r Lu rs (len {l I er n}rnr}s<br />
arc (uo11at( ul papeqs) ursautl pue<br />
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ol srueas alrs srsflorpdq apl]oapnu aqt puu alrs Supurq-raurr(1od aql uaamleq<br />
sa8ueqc prntJnrts ;o .,{.e1ar aqJ 'ef,eJJetur Surpurq-raru.4.1od aql o} pa,{e1ar<br />
aq o1 alcitc srs,{lorpdq dJV eql Lq pasnec suoursuEJl lBrntJnrts aq} 3uu,ro11u<br />
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asoqt qll,lr ,{Janrsualxa s}cerelur dool qcllvrs e 'ursol.ur pue ulseurl ul<br />
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aql yo ped ]uaJaJJrp e Jo uot1elor<br />
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ueql JaqlEJ dJV puP 'suralord rolo(u oe\l eql JoJ JeJJIp luarue^oru eql Jo slrEiep<br />
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'aroJ<br />
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uorleJrJrporu pue uorleorldnp aua8 erl esoJe suorlcunJ pazrTerJads snorJel<br />
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a.re surso,{ru eq} pue sursauDl eq} qtoq leqt rq8noqt sI lI ,(larrpcadsar ,ursauDl<br />
pue ursodur eql ur salrs Surpurq-elnqntoJcrru pue Surpurq-uuJe aq] epnlour<br />
sdool asaql'{JeDJo ecroqJ aql JoJ alqrsuodsa; eJe pue azrs ureruop ur ecualaJJrp<br />
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IBuorlJeJrprun<br />
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asn qloq suralord Surpurq-41g pue suralord Jolotu plala>1so{c aqr q8noqrry<br />
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,i.q palerauaS sr urlce Suop luaruanotu aq];o dals qcea 'ursodru JoC 'elnJelotu<br />
utalord eq] Jo lred ur ]uarualoru a8rel e eJJoJ ol srsdlorp,,(q pue Surpurq<br />
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aql uo drr8 slr asealeJ ol peeq ursodru aql 8umo1e 'urlJe JoJ rtfpgJe Surpurq s1r<br />
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pcnuaqooueqcaru<br />
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epnoalonu'Supurq epuoelonuJo aycrtc<br />
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speaq o^ l aq1 ur salrdc srsdlorpdq eppoelcnu eqJ 'peeq ]sJU aqt JoJ etrs Surpurq<br />
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MOLECULAR MOTORS I 019<br />
(B)<br />
+<br />
attachment to cargo<br />
or another microtubule<br />
(c)<br />
AAA domains AAA domains<br />
ADP + Pr<br />
power stroKe,<br />
8nm<br />
c<br />
AAA domains<br />
in motorhead<br />
major ATPase<br />
C-terminal domain<br />
15 nm<br />
shaped linker region connects the hear,y-chain tail to the AAA domain that is<br />
most active as an ATPase. Between the fourth and fifth AAA domains is a heavy<br />
chain domain that forms a long anti-parallel coiled-coil stalk. This stalk extends<br />
from the top of the ring, with an ATP hydrolysis-regulated microtubule-binding<br />
site at its tip. Dlmein's "power stroke" is driven by the release of ADP and inorganic<br />
phosphate, and it causes the ring to rotate relative to the tail (Figure f 6-64).<br />
Although kinesin, myosin, and dynein all undergo analogous mechanochemical<br />
cycles, the exact nature of the coupling between the mechanical and<br />
chemical cycles differs in the three cases. For example, myosin without any<br />
nucleotide is tightly bound to its actin track, in a so-called "rigor" state, and it is<br />
released from this track by the association of ATP In contrast, kinesin forms a<br />
rigor-like tight association with a microtubule when ATP is bound to the kinesin,<br />
and it is hydrolysis of AIP that promotes release of the motor from its track. The<br />
mechanochemical cycle of dlmein is more similar to myosin than to kinesin, in<br />
that nucleotide-free dynein is tightly bound to the microtubule and it is released<br />
by binding AIP However, for dynein the inorganic phosphate and ADP appear<br />
to be released at the same time, causing the conformational change driving the<br />
power stroke, while for myosin the phosphate is released first and the power<br />
stroke does not occur until the ADP subsequently dissociates from the motor<br />
head.<br />
Thus, cytoskeletal motor proteins work in a manner highly analogous to<br />
GTP-binding proteins, except that in motor proteins the small protein conformational<br />
changes (a few tenths of a nanometer) associated with nucleotide<br />
hydrolysis are amplified by special protein domains-the lever arm in the case<br />
of myosin, the linker in the case of kinesin, and the ring and stalk in the case of<br />
dgrein-to generate large-scale (several nanometers) conformational changes<br />
that move the motor proteins stepwise along their filament tracks. The analogy<br />
Figure 1 6-64 The power stroke of dynein.<br />
(A) The organization of the domains in each<br />
dynein heavy chain. This is a huge<br />
molecule, containing nearly 5000 amino<br />
acids. The number of heavy chains in a<br />
dynein is equal to its number of motor<br />
heads. (B) Dynein c is a monomeric flagella<br />
dynein found in unicellular green alga<br />
Ch I omyd o m on a s rei n hardtii. rhe large<br />
dynein motor head is a planar ring<br />
containing a C-terminal domain (gray) and<br />
six AAA domains, four of which retain ATPbinding<br />
sequences, but only one of which<br />
(dark red) has the major ATPase activity.<br />
Extending from the head are a long, coiledcoil<br />
stalk with the microtubule binding site<br />
at the tip, and a tail with a cargoattachment<br />
site. In the ATP-bound state,<br />
the stalk is detached from the microtubule,<br />
but ATP hydrolysis causes stalk-microtubule<br />
attachment. Subsequent release of ADP<br />
and Pi then leads to a large conformational<br />
"power stroke" involving rotation of the<br />
head and stalk relative to the tail. Each<br />
cycle generates a step of about 8 nm along<br />
the microtubule towards its minus end.<br />
(C) Electron micrographs of purified<br />
dyneins in two different conformations<br />
representing different steps in the<br />
mechanochemical cycle. (B, from<br />
S.A. Burgess et al., Noture 421:715-718,<br />
2003. With permission from Macmillan<br />
Publishers Ltd.)
1020 Chapter 16:The <strong>Cytoskeleton</strong><br />
between the GTPases and the cytoskeletal motor proteins has recently been<br />
extended by the observation that one of the GTP-binding proteins-the bacterial<br />
elongation factor G-translates the chemical energy of GTP hydrolysis into<br />
directional movement of the mRNA molecule on the ribosome.<br />
Motor Protein Kinetics Are Adapted to Cell Functions<br />
The motor proteins in the myosin and kinesin superfamilies exhibit a remarkable<br />
diversity of motile properties, well beyond their choice of different polymer<br />
tracks. Most strikingly, a single dimer of kinesin-1 moves in a highly processiue<br />
fashion, traveling for hundreds of ATPase cycles along a microtubule without<br />
dissociating. Skeietal muscle myosin II, in contrast, cannot move processively<br />
and makes just one or a few steps along an actin filament before letting go. These<br />
differences are critical for the motors' various biological roles. A small number<br />
of kinesin- I molecules must be able to transport an organelle all the way down<br />
a nerve cell axon, and therefore require a high level of processivity. Skeletal muscle<br />
myosin, in contrast, never operates as a single molecule but rather as part of<br />
a huge array of myosin II molecules in a thick filament. Here processivity would<br />
actually inhibit biological function, since efficient muscle contraction requires<br />
that each myosin head perform its power stroke and then quickly get out of the<br />
way-in order to avoid interfering with the actions of the other heads attached<br />
to the same actin filament.<br />
There are two reasons for the high degree of processivity of kinesin-1 movement.<br />
The first is that the mechanochemical cycles of the two motor heads in a<br />
kinesin- I dimer are coordinated with each other, so that one kinesin head does<br />
not let go until the other is poised to bind. This coordination allows the motor<br />
protein to operate in a hand-over-hand fashion, never allowing the organelle<br />
cargo to diffuse away from the microtubule track. In contrast, there is no apparent<br />
coordination between the myosin heads in a myosin II dimer. The second<br />
reason for the high processivity of kinesin- I movement is that kinesin- 1 spends<br />
a relatively large fraction of its ATPase cycle tightly bound to the microtubule.<br />
For both kinesin- I and myosin II, the conformational change that produces the<br />
force-generating working stroke must occur while the motor protein is tightly<br />
bound to its polymer, and the recovery stroke in preparation for the next step<br />
must occur while the motor is unbound. But myosin II spends only about 5% of<br />
its AIPase cycle in the tightly bound state, and it is unbound the rest of the time.<br />
\.Vhat myosin loses in processivity it gains in speed; in an array in which<br />
many motor heads are interacting with the same actin filament, a set of linked<br />
myosins can move its filament a total distance equivalent to 20 steps during a<br />
single cycle time, while kinesins can move only two. Thus, myosin II can typically<br />
drive filament sliding much more rapidly than kinesin-1, even though the<br />
two different motor proteins hydrolyze NIP at comparable rates and take molecular<br />
steps of comparable length. This property is particularly important in the<br />
rapid contraction of skeletal muscle, as we will discuss later.<br />
Within each motor protein class, movement speeds vary widely, from about<br />
0.2 to 60 pm/sec for myosins, and from about 0.02 to 2 pm/sec for kinesins.<br />
These differences arise from a fine-tuning of the mechanochemical cycle. The<br />
number of steps that an individual motor molecule can take in a given time, and<br />
thereby the velocity, can be decreased by either decreasing the motor protein's<br />
intrinsic ATPase rate or by increasing the proportion of cycle time spent bound<br />
to the filament track. For example, myosin v (which acts as a processive vesicle<br />
motor) spends up to 907o of its nucleotide cycle tightly bound to the actin filament,<br />
in contrast to 5% for myosin IL Moreover, a motor protein can evolve to<br />
change the size of each step by either changing the length of the lever arm (for<br />
example, the lever arm of myosin V is about three times longer than the lever<br />
arm of myosin II) or the angle through which the helix swings (Figure f 6-65).<br />
Each of these parameters varies slightly among different members of the myosin<br />
and kinesin families, corresponding to slightly different protein sequences and<br />
structures.<br />
mrnuS<br />
end<br />
Myosin ll<br />
Myosin V<br />
5 to 10 nm swing<br />
of lever arm<br />
30 to 40 nm swing<br />
of lever arm<br />
neao ---<br />
pl us<br />
end<br />
Figure 1 6-65 The effect of lever arm<br />
length on the step size for a motor<br />
protein. The lever arm of myosin ll is<br />
much shorter than the lever arm of<br />
myosin V. The power stroke in the head<br />
swings their lever arms through the same<br />
angle, so myosin V is able to take a bigger<br />
step than myosin ll.
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uiri 0 !
THE CYTOSKELETON AND CELL BEHAVIOR<br />
myosin light chains<br />
INACTIVE STATE:<br />
(light chains not phosphorylated)<br />
(A)<br />
actin-binding site<br />
myosin tail released<br />
@<br />
ACTIVE STATE:<br />
(light chains phosphorylated)<br />
bipolar filament<br />
of 15-20 molecules<br />
of the myosin superfamily is not as well understood, but the control of these<br />
myosins is likewise thought to involve site-specific phosphorylations.<br />
Summary<br />
Motor proteins use the energy of ATP hydrolysis to moue along microtubules or actin<br />
filaments. They mediate the sliding of ftlaments relatiue to one another and the transport<br />
of cargo along filament tracks. All known motor proteins that moue on actin filaments<br />
are members of the myosin superfamily. The motor proteins that moue on<br />
microtubules are either members of the kinesin superfamily or the dynein family. The<br />
myosin and kinesin superfamilies are diuerse, with about 40 genes encoding each type of<br />
protein in humans. The only structural element shared among all members of each<br />
superfamily is the motor "head" domain. These heads are fused to a wide uariery of different<br />
"tails," which attach to different types of cargo and enable the uarious family members<br />
to perform dffirent functions in the cell. These functions include the transportation<br />
and localization of specific proteins, membrane-enclosed organelles, and mRNAs.<br />
Although myosin and kinesin walk along different tracks and use different mech'<br />
anisms to produce force and mouement by ATP hydrolysis, they share a common structural<br />
core, suggesting that they are deriued from a common ancestor. The dynein motor<br />
protein has independently euolued, and it has a distinct structure and mechanism of<br />
action.<br />
THE CYTOSKELETON AND CELL BEH IOR<br />
A central challenge in all areas of cell biology is to understand how the functions<br />
of many individual molecular components combine to produce complex cell<br />
behaviors. The cell behaviors that we describe in this final section all rely on a<br />
coordinated deployment of the components and processes that we have<br />
explored in the first three sections of the chapter: the dynamic assembly and disassembly<br />
of cltoskeletal polymers, the regulation and modification of their<br />
structure by polymer-associated proteins, and the actions of motor proteins<br />
moving along the polymers. How does the cell coordinate all these activities to<br />
define its shape, to enable it to crawl, or to divide it neatly into two at mitosis?<br />
These problems of cltoskeletal coordination will challenge scientists for many<br />
years to come.<br />
1025<br />
Figure 16-72 Light-chain phosphorylation and the regulation of the assembly of myosin ll into thick filaments. (A) The<br />
controlled phosphorylation by the enzyme myosin light-chain kinase (MLCK) of one of the two light chains (the so-called<br />
regulatorylightchain,shownin lightblue)onnonmusclemyosinll inatesttubehasatleasttwoeffects: itcausesachange<br />
in the conformation of the myosin head, exposing its actin-binding site, and it releases the myosin tail from a "sticky patch"<br />
on the myosin head, thereby allowing the myosin molecules to assemble into short, bipolar, thick filaments. (B) Electron<br />
micrograph of negatively stained short filaments of myosin ll that have been induced to assemble in a test tube by<br />
phosphorylation of their light chains. These myosin ll filaments are much smaller than those found in skeletal muscle cells<br />
(see Figure 1 6-55). (8, courtesy of John Kendrick-Jones.)
1026 Chapter 16:The <strong>Cytoskeleton</strong><br />
To provide a sense of our present understanding, we first discuss examples<br />
where specialized cells build stable arrays of filaments and use highly ordered<br />
arrays of motor proteins sliding them relative to each other to generate the<br />
large-scale movements of muscle, cilia, and eucaryotic flagella. Next, we consider<br />
two important instances where filament dynamics collude with motor<br />
protein activity to generate complex, self-organized dynamic structures: the<br />
microtubule-based mitotic spindle and the actin arrays involved in cell crawling.<br />
Finally, we consider the extraordinary organization and behavior of the<br />
neuronal c)'toskeleton.<br />
Sliding of Myosin ll and Actin Filaments Causes Muscles to<br />
Contract<br />
Muscle contraction is the most familiar and the best understood form of movement<br />
in animals. In vertebrates, running, walking, swimming, and flying all<br />
depend on the rapid contraction of skeletal muscle on its scaffolding of bone,<br />
while involuntary movements such as heart pumping and gut peristalsis depend<br />
on the contraction of cardiac muscle and smooth muscle, respectively. All these<br />
forms of muscle contraction depend on the ATP-driven sliding of highly organized<br />
arrays of actin filaments against arrays of myosin II filaments.<br />
Skeletal muscle was a relatively late evolutionary development, and muscle<br />
cells are highly specialized for rapid and efficient contracrion. The long thin<br />
muscle fibers of skeletal muscle are actually huge single cells that form during<br />
development by the fusion of many separate cells, as discussed in Chapter 22.<br />
The large muscle cell retains the many nuclei of the contributing cells. These<br />
nuclei lie just beneath the plasma membrane (Figure f6-23). The bulk of the<br />
cltoplasm inside is made up of myofibrils, which is the name given to the basic<br />
contractile elements of the muscle cell. A myofibril is a cylindrical structure l-2<br />
pm in diameter that is often as long as the giant muscle cell itself. It consists of a<br />
long repeated chain of tiny contractile units-called snrcomeres, each about 2.2<br />
pm long, which give the vertebrate myofibril its striated appearance (Figure<br />
ro-7$.<br />
Each sarcomere is formed from a miniature, precisely ordered array of parallel<br />
and partly overlapping thin and thick filaments. Tlne thin fiIaments are composed<br />
of actin and associated proteins, and they are attached at their plus ends<br />
to a Z disc at each end of the sarcomere. The capped minus ends of the actin filaments<br />
extend in toward the middle of the sarcomere, where they overlap with<br />
thick fiIamenfs, the bipolar assemblies formed from specific muscle isoforms of<br />
myosin II (see Figure 16-55). \Mhen this region of overlap is examined in cross<br />
section by electron microscopy, the myosin filaments are seen to be arranged in<br />
a regular hexagonal lattice, with the actin filaments evenly spaced between them<br />
(Figure 16-75). cardiac muscle and smooth muscle also contain sarcomeres,<br />
although the organization is not as regular as that in skeletal muscle.<br />
Sarcomere shortening is caused by the myosin filaments sliding past the<br />
actin thin filaments, with no change in the length of either type of filament (Figure<br />
16-74 c and D). Bipolar thick filaments walk toward the plus ends of two sets<br />
of thin filaments of opposite orientations, driven by dozens of independent<br />
myosin heads that are positioned to interact with each thin filament. Because<br />
there is no coordination among the movements of the myosin heads, it is critical<br />
myofibril<br />
Figure 16-73 Skeletal muscle cells (also<br />
called muscle fibers). (A) These huge<br />
multinucleated cells form by the fusion of<br />
many muscle cell precursors, called<br />
myoblasts. In an adult human, a muscle<br />
cell is typically 50 pm in diameter and can<br />
be up to several centimeters long.<br />
(B) Fluorescence micrograph of rat muscle,<br />
showing the peripherally located nuclei<br />
(blue) in these giant cells. Myofibrils are<br />
stained red; see also Figure 23-468.<br />
(B, courtesy of Nancy L. Kedersha.)<br />
50 pm
THE CYTOSKELETON AND CELL BEHAVIOR<br />
Figure 16-74 Skeletal muscle myofibrils, (A) Low-magnification electron<br />
micrograph of a longitudinal section through a skeletal muscle cell of a<br />
rabbit, showing the regular pattern of cross-striations. The cell contains<br />
many myofibrils aligned in parallel (see Figure 16-73). (B) Detail of the<br />
skeletal muscle shown in (A), showing portions of two adjacent myofibrils<br />
and the definition of a sarcomere (black arrow). (C) Schematic diagram of a<br />
single sarcomere, showing the origin of the dark and light bands seen in<br />
the electron micrographs. The Z discs, at each end of the sarcomere, are<br />
attachment sites for the plus ends of actin filaments (thin filaments); the<br />
M line, or midline, is the location of proteins that link adjacent myosin ll<br />
filaments (thick filaments) to one another. The dark bands, which mark the<br />
location of the thick filaments, are sometimes called A bands because they<br />
appear anisotropic in polarized light (that is, their refractive index changes<br />
with the plane of polarization). The light bands, which contain only thin<br />
filaments and therefore have a lower density of protein, are relatively<br />
isotropic in polarized light and are sometimes called I bands. (D) When the<br />
sarcomere contracts, the actin and myosin filaments slide past one another<br />
without shortening. (A and B, courtesy of Roger Craig.)<br />
that they operate with a low processivity, remaining tightly bound to the actin<br />
filament for only a small fraction of each AIPase cycle so that they do not hold<br />
one another back. Each myosin thick filament has about 300 heads (294 in frog<br />
muscle), and each head cycles about five times per second in the course of a<br />
1um<br />
e;' ' -.<br />
2pm<br />
(B)<br />
Z disc<br />
dark band light band<br />
one sarcomere<br />
thick filament (myosin)<br />
thin filament (actinl -<br />
light band dark band light band<br />
1027<br />
--l<br />
I<br />
I<br />
)<br />
_d<br />
Figure 16-75 Electron micrographs of an<br />
insect flight muscle viewed in cross<br />
section. The myosin and actin filaments<br />
are packed together with almost crystalline<br />
regularity. Unlike their vertebrate<br />
counterparts, these myosin filaments have<br />
a hollow center, as seen in the<br />
enlargement on the right. The geometry of<br />
the hexagonal lattice is slightly different in<br />
vertebrate muscle. (From J. Auber, J. de<br />
Microsc. 8:197 -232, 1 969. With permission<br />
from Societ6 frangaise de microscopie<br />
6lectronique.)<br />
E
1028 Chapter 16:The <strong>Cytoskeleton</strong><br />
4!g!'llLlD<br />
.ra!i!rrtl,:)<br />
Z disc<br />
Cap Z tropomodulin<br />
blus end<br />
of actin<br />
f ilament<br />
mtnuseno<br />
myosin (thick f ilament)<br />
actin (thin filament)<br />
rapid contraction-sliding the myosin and actin filaments past one another at<br />
rates of up to 15 pm/sec and enabling the sarcomere to shorten by l0% of its<br />
length in less than 1/50th ofa second. The rapid synchronized shortening ofthe<br />
thousands of sarcomeres lying end-to-end in each myofibril enables skeletal<br />
muscle to contract rapidly enough for running and flying, or for playing the<br />
piano.<br />
Accessory proteins produce the remarkable uniformity in filament organizalion,length,<br />
and spacing in the sarcomere (Figure f 6-76). The actin filament<br />
plus ends are anchored in the Z disc, which is built fromcapzand o-actinin; the<br />
Z disc caps the filaments (preventing depolymerization), while holding them<br />
together in a regularly spaced bundle. The precise length of each thin filament is<br />
determined by a template protein of enormous size, called nebulin, which consists<br />
almost entirely of a repeating 35-amino-acid actin-binding motif. Nebulin<br />
stretches from the Z disc to the minus end of each thin filament and acts as a<br />
"molecular ruler" to dictate the length of the filament. The minus ends of the<br />
thin filaments are capped and stabilized by tropomodulin. Although there is<br />
some slow exchange of actin subunits at both ends of the muscle thin filament,<br />
such that the components of the thin filament turn over with a half-life of several<br />
days, the actin filaments in sarcomeres are remarkably stable compared to<br />
the dynamic actin filaments characteristic of most other cell t!?es that turn over<br />
with half-lives of a few minutes or less.<br />
Opposing pairs of an even longer template protein, called titin, position the<br />
thick filaments midway between the Z discs. Titin acts as a molecular spring,<br />
with a long series of immunoglobulin-like domains that can unfold one by one<br />
as stress is applied to the protein. A springlike unfolding and refolding of these<br />
domains keeps the thick filaments poised in the middle of the sarcomere and<br />
allows the muscle fiber to recover after being overstretched. In c. elegans,whose<br />
sarcomeres are longer than those in vertebrates, titin is also longer, suggesting<br />
that it too serves as a molecular ruler, determining in this case the overall length<br />
of each sarcomere (see Figure 3-33).<br />
A Sudden Rise in Cytosolic Ca2+ Concentration Initiates Muscle<br />
Contraction <br />
The force-generating molecular interaction between myosin thick filaments and<br />
actin thin filaments takes place only when a signal passes to the skeletal muscle<br />
from its motor nerve. Immediately upon arrival of the signal, the muscle cell<br />
act in rapid succession on the same thin filament without interfering with one<br />
another. second, a specialized membrane system relays the incoming signal<br />
rapidly throughout the entire cell. The signal from the nerve triggers an action<br />
potential in the muscle cell plasma membrane (discussed in Chapter ll), and<br />
' .:t<br />
Figure I 6-76 Organization of accessory<br />
proteins in a sarcomere. Each<br />
giant titin molecule extends from the<br />
Z disc to the M line-a distance of over<br />
1 pm. Part of each titin molecule is<br />
closely associated with a myosin thick<br />
filament (which switches polarity at the<br />
M line); the rest of the titin molecule is<br />
elastic and changes length as the<br />
sarcomere contracts and relaxes. Each<br />
nebulin molecule is exactly the length of<br />
a thin filament. The actin filaments are<br />
also coated with tropomyosin and<br />
troponin (not shown; see Figure i6-78)<br />
and are capped at both ends.<br />
Tropomodulin caps the minus end of the<br />
actin filaments, and CapZ anchors the<br />
plus end at the Z disc, which also<br />
contains o(-actinin.
THE CYTOSKELETON AND CELL BEHAVIOR<br />
polarized<br />
T-tubule<br />
t<br />
membrane<br />
CYTOSOL<br />
05pm<br />
depolarized T-tubule membrane<br />
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sa rcoplasm ic<br />
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:iji: 1 35 nm<br />
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this electrical excitation spreads rapidly into a series of membraneous folds, the<br />
transverse tubules, or T tubules, that extend inward from the plasma membrane<br />
around each myofibril. The signal is then relayed across a small gap to tlre sarcoplasmic<br />
reticulum, an adjacentweb-like sheath of modified endoplasmic reticulum<br />
that surrounds each myofibril like a net stocking (Figure lg-77{and B).<br />
rWhen the incoming action potential activates a Ca2+ channel in the T-tubule<br />
membrane, a Ca2+ influx triggers the opening of Ca2+-release channels in the<br />
sarcoplasmic reticulum (Figure l6-77C). CaZ* flooding into the cytosol then initiates<br />
the contraction of each myofibril. Because the signal from the muscle-cell<br />
plasma membrane is passed within milliseconds (via the T tubules and sarcoplasmic<br />
reticulum) to every sarcomere in the cell, all of the myofibrils in the<br />
cell contract at once. The increase in Ca2* concentration is transient because the<br />
Ca2* is rapidly pumped back into the sarcoplasmic reticulum by an abundant,<br />
AlP-dependent Ca2+-pump (also called a Caz*-ATPase) in its membrane (see<br />
Figure 1l-I3). Typically, the cytoplasmic Ca2* concentration is restored to resting<br />
levels within 30 msec, allowing the myofibrils to relax. Thus, muscle contraction<br />
depends on two processes that consume enormous amounts ofATP: filament<br />
sliding, driven by the ATPase of the myosin motor domain, and Ca2*<br />
pumping, driven by the Caz*-pump.<br />
The Ca2* dependence of vertebrate skeletal muscle contraction, and hence<br />
its dependence on motor commands transmitted via nerves, is due entirely to a<br />
set of specialized accessory proteins that are closely associated with the actin<br />
thin filaments. One of these accessory proteins is a muscle form of tropomyosin,<br />
an elongated molecule that binds along the groove of the actin helix. The other<br />
is troponin, a complex of three polypeptides, troponins T I, and C (named for<br />
their tropomyosin-binding, inhibitory, and Ca2*-binding activities, respectively).<br />
Troponin I binds to actin as well as to troponin T. In a resting muscle, the<br />
troponin I-T complex pulls the tropomyosin out of its normal binding groove<br />
into a position along the actin filament that interferes with the binding of<br />
(A)<br />
1 029<br />
Figure 16-77 T tubules and the<br />
sarcoplasmic reticulum. (A) Drawing of<br />
the two membrane systems that relay the<br />
signal to contract from the muscle cell<br />
plasma membrane to all of the myofibrils<br />
in the cell. (B) Electron micrograph<br />
showing two T tubules. Note the position<br />
of the large Ca2+-release channels in the<br />
sarcoplasmic reticulum membrane; they<br />
look like square-shaped "feet" that<br />
connect to the adjacent T-tubule<br />
membrane. (C) Schematic diagram<br />
showing how a Ca2+-release channel in<br />
the sarcoolasmic reticulum membrane is<br />
thought to be opened by the activation<br />
of a voltage-gated Ca2+ channel.<br />
(8, courtesy of Clara Franzini-Armstrong.)
1030 Chapter 16:The <strong>Cytoskeleton</strong><br />
,".<br />
10<br />
"t<br />
tropon in<br />
comPlex tropomvosin<br />
rc T<br />
+ ca2*<br />
ca2*<br />
myosin-binding site exposed<br />
by Ca'*-mediated tropomyosin<br />
movement<br />
Figure 16-78 The control of skeletal muscle contraction by troponin. (A) A skeletal muscle cell thin filament, showing the<br />
positions of tropomyosin and troponin along the actin filament. Each tropomyosin molecule has seven evenly spaced<br />
regions with similar amino acid sequences, each of which is thought to bind to an actin subunit in the filament.<br />
(B) A thin filament shown end-on, illustrating how Ca2+ (binding to troponin) is thought to relieve the tropomyosin<br />
blockage of the interaction between actin and the myosin head. (A, adapted from G.N. Phillips, J.P. Fillers and C. Cohen,<br />
J. Mol. Biol. 192:111-131,1986. With permission from Academic Press.)<br />
myosin heads, thereby preventing any force-generating interaction. \Mhen the<br />
level of Ca2* is raised, troponin C-which binds up to four molecules of Caz+causes<br />
troponin I to release its hold on actin. This allows the tropomyosin<br />
molecules to slip back into their normal position so that the myosin heads can<br />
walk along the actin filaments (Figure f 6-78). Troponin C is closely related to<br />
the ubiquitous Caz*-binding protein calmodulin (see Figure lS=44); it can be<br />
thought of as a specialized form of calmodulin that has acquired binding sites<br />
for troponin I and troponin ! thereby ensuring that the myofibril responds<br />
extremely rapidly to an increase in Ca2+ concentration.<br />
In smooth muscle cells, so-called because they lack the regular striations of<br />
skeletal muscle, contraction is also triggered by an influx of calcium ions, but the<br />
regulatory mechanism is different. Smooth muscle forms the contractile portion<br />
of the stomach, intestine, and uterus, the walls of arteries, and many other structures<br />
requiring slow and sustained contractions. smooth muscle is composed of<br />
sheets of highly elongated spindle-shaped cells, each with a single nucleus.<br />
Smooth muscle cells do not express the troponins. Instead, Caz* influx into the<br />
cell regulates contraction by two mechanisms that depend on the ubiquitous<br />
calcium binding protein calmodulin.<br />
First, Ca2+-bound calmodulin binds to an actin-binding protein, caldesmon,<br />
which blocks the actin sites where the myosin motor heads would normally<br />
bind. This causes the caldesmon to fall off of the actin filaments, preparing the<br />
filaments for contraction. Second, smooth muscle myosin is phosphorylated on<br />
one of its two light chains by myosin light chain kinase (MLCK.), as described<br />
previously for regulation of nonmuscle myosin II (see Figure 16-72).lVhen the<br />
light chain is phosphorylated, the myosin head can interact with actin filaments<br />
and cause contraction; when it is dephosphorylated, the myosin head tends to<br />
dissociate from actin and becomes inactive (in contrast to nonmuscle myosin II,<br />
light chain dephosphorylation does not cause thick filament disassembly in<br />
smooth muscle cells). MLCK requires bound ca2*/calmodulin to be fully active.<br />
External signaling molecules such as adrenaline (epinephrine) can also regulate<br />
the contractile activity of smooth muscle. Adrenaline binding to its G-protein-coupled<br />
cell surface receptor causes an increase in the intracellular level of<br />
cyclic AMf; which in turn activates cyclic-AMP-dependent protein kinase (pKA)<br />
(see Figure l5-35). PKA phosphorylates and inactivates MLCK, thereby causing<br />
the smooth muscle cell to relax.<br />
The phosphorylation events that regulate contraction in smooth muscle<br />
cells occur realtively slowly, so that maximum contraction often requires nearly<br />
a second (compared with the few milliseconds required for contraction of a<br />
skeletal muscle cell). But rapid activation of contraction is not important in<br />
smooth muscle: its myosin II hydrolyzes ArP about l0 times more slowly than<br />
skeletal muscle myosin, producing a slow cycle of myosin conformational<br />
changes that results in slow contraction.
THE CYTOSKELETON AND CELL BEHAVIOR<br />
Heart Muscle ls a Precisely Engineered Machine <br />
The heart is the most heavilyworked muscle in the body, contracting about 3 billion<br />
(3 x 10s) times during the course of a human lifetime. This number is about<br />
the same as the average number of revolutions in the lifetime of an automobile's<br />
internal combustion engine. Heart cells express several specific isoforms of cardiac<br />
muscle myosin and cardiac muscle actin. Even subtle changes in these contractile<br />
proteins expressed in the heart-changes that would not cause any<br />
noticeable consequences in other tissues-can cause serious heart disease (Figure<br />
16-79).<br />
The normal cardiac contractile apparatus is such a highly tuned machine<br />
that a tiny abnormality anywhere in the works can be enough to gradually wear<br />
it down over years of repetitive motion. Familial hypertrophic cardiomyopathy is<br />
a frequent cause of sudden death in young athletes. It is a genetically dominant<br />
inherited condition that affects about two out of every thousand people, and it<br />
is associated with heart enlargement, abnormally small coronary vessels, and<br />
disturbances in heart rhythm (cardiac arrhythmias). The cause of this condition<br />
is either any one of over 40 subtle point mutations in the genes encoding cardiac<br />
B myosin heavy chain (almost all causing changes in or near the motor domain),<br />
or one of about a dozen mutations in other genes encoding contractile proteins-including<br />
myosin light chains, cardiac troponin, and tropomyosin. Minor<br />
missense mutations in the cardiac actin gene cause another type of heart condition,<br />
called dilated cardiomyopathy, that also frequently results in early heart<br />
failure.<br />
Cilia and Flagella Are Motile Structures Built from Microtubules<br />
and Dyneins<br />
Just as myofibrils are highly specialized and efficient motility machines built<br />
from actin and myosin filaments, cilia and flagella are highly specialized and<br />
efficient motility structures built from microtubules and dynein. Both cilia and<br />
flagella are hair-like cell appendages that have a bundle of microtubules at their<br />
core. Flagella are found on sperm and many protozoa. By their undulating<br />
motion, they enable the cells to which they are attached to swim through liquid<br />
media (Figure f 6-80A). Cilia tend to be shorter than flagella and are organized<br />
in a similar fashion, but they beat with a whip-like motion that resembles the<br />
breast stroke in swimming (Figure f6-808). The cycles of adjacent cilia are<br />
almost but not quite in synchrony, creating the wave-like patterns that can be<br />
seen in fields of beating cilia under the microscope. Ciliary beating can either<br />
propel single cells through a fluid (as in the swimming of the protozoan Paramecium)<br />
or can move fluid over the surface of a group of cells in a tissue. In the<br />
human body, huge numbers of cilia (10s/cmz or more) line our respiratory tract,<br />
sweeping layers of mucus, trapped particles of dust, and bacteria up to the<br />
mouth where they are swallowed and ultimately eliminated. Likewise, cilia along<br />
the oviduct help to sweep eggs toward the uterus.<br />
The movement of a cilium or a flagellum is produced by the bending of its<br />
core, which is called the axoneme. The axoneme is composed of microtubules<br />
and their associated proteins, arranged in a distinctive and regular pattern. Nine<br />
special doublet microtubules (comprising one complete and one partial microtubule<br />
fused together so that they share a common tubule wall) are arranged in<br />
Figure 16-80 The contrasting motions of flagella and cilia. (A) The wave-like<br />
motion of the flagellum of a sperm cell from a tunicate. The cell was<br />
photographed with stroboscopic illumination at 400 flashes per second. Note that<br />
waves of constant amplitude move continuously from the base to the tip of the<br />
flagellum. (B) The beat of a cilium, which resembles the breast stroke in<br />
swimming. A fast power stroke (red arrows), in which fluid is driven over the<br />
surface of the cell, is followed by a slow recovery stroke. Each cycle typically<br />
requires 0.1 -0.2 sec and generates a force perpendicular to the axis of the<br />
axoneme (the ciliary core). (A, courtesy of C.J. Brokaw.)<br />
1 031<br />
Figure 16-79 Effect on the heart of a<br />
subtle mutation in cardiac myosin. left,<br />
normal heart from a 6-day old mouse<br />
pup. Right, heart from a pup with a point<br />
mutation in both copies of its cardiac<br />
myosin gene, changing Arg 403 to Gln.<br />
The arrows indicate the atria. In the heart<br />
from the pup with the cardiac myosin<br />
mutation, both atria are greatly enlarged<br />
(hypertrophic), and the mice die within a<br />
few weeks of birth. (From D. Fatkin et al.,<br />
J. CIin. lnvest. '103:147<br />
, 1999. With<br />
permission from The Rockefeller<br />
University Press.)
032 Chapter 1 6: The <strong>Cytoskeleton</strong><br />
100 nm<br />
radial spoke<br />
inner sheath<br />
central singlet<br />
microtubule<br />
plasma membrane<br />
a ring around a pair of single microtubules (Figure r6-sr). Almost all forms of<br />
eucaryotic flagella and cilia (from protozoans to humans) have this characteristic<br />
arrangement. The microtubules extend continuously for the length of the<br />
axoneme, which can be f 0-200 pm. At regular positions along the length of the<br />
microtubules, accessory proteins cross-link the microtubules together.<br />
Molecules of ciliary dynein form bridges between the neighboring doublet<br />
microtubules around the circumference of the axoneme (Figure f 6-S2). \fhen<br />
the motor domain of this dynein is activated, the dynein molecules attached to<br />
one microtubule doublet (see Figure 16-64) attempt to walk along the adjacent<br />
microtubule doublet, tending to force the adjacent doublets to slide relative to<br />
one another, much as actin thin filaments slide during muscle contraction.<br />
However, the presence of other links between the microtubule doublets prevents<br />
this sliding, and the dynein force is instead converted into a bending<br />
motion (Figure 16-83).<br />
The length of flagella is carefully regulared. If one of the two flagella on a<br />
chlamydomonas cell is amputated, the remaining one will transiently shrink as<br />
the stump regrows until they reach the same length, and then the two shortened<br />
flagella will continue to elongate until both are as long as they were on the<br />
unperturbed cell. New flagellar components including tubulin and dynein are<br />
incorporated into the growing flagella at the distal tips. Thus, even in these<br />
(A)<br />
(B)<br />
A microtubule B microtubule<br />
outer doublet microtubule<br />
outer dynein arm<br />
inner dynein arm<br />
Figure 16-81 The arrangement of<br />
microtubules in a flagellum or cilium.<br />
(A) Electron micrograph of the flagellum<br />
of a green-alga cell (Chlamydomonas)<br />
shown in cross section, illustrating the<br />
distinctive "9 + 2" arrangement of<br />
microtubules. (B) Diagram of the parts of<br />
a flagellum or cilium. The various<br />
projections from the microtubules link<br />
the microtubules together and occur at<br />
regular intervals along the length of the<br />
axoneme. (A, courtesy of Lewis Tilney.)<br />
50 nm 100 nm<br />
Figure 16-82 Ciliary dynein. Ciliary (axonemal) dynein is a large protein assembly (nearly 2 million daltons) composed of<br />
9-12 polypeptide chains, the largest of which is the heavy chain of more than 500,000 daltons. (A) The heavy chains form<br />
the major portion of the globular head and stem domains, and many of the smaller chains are clustered around the base of<br />
the stem. There are two heads in the outer dynein in metazoans, but three heads in protozoa, each formed from their own<br />
heavy chain (see Figure 1 6-598 for a view of an isolated molecule). The tail of the molecule binds tightly to an A<br />
microtubule in an ATP-|ndependent manner, while the large globular heads have an ATP-dependent binding site for a B<br />
microtubule (see Figure 16-81). When the heads hydrolyze their bound ATP, they move toward the minus end of the B<br />
microtubule, thereby producing a sliding force between the adjacent microtubule doublets in a cilium or flagellum. For<br />
details, see Figure 16-64. (B) Freeze-etch electron micrograph of a cilium showing the dynein arms projecting at regutar<br />
intervals from the doublet microtubules. (8, courtesy of John Heuser.)
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THE CYTOSKELETON AND CELL BEHAVIOR<br />
capprng 0<br />
proTern<br />
lr<br />
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net filament<br />
assembly at<br />
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The pushing force created by the polymerization of a branched web of actin<br />
filaments plays an important role in many cell processes. The polymerization at<br />
the plus end can push the plasma membrane outward, as in the example just<br />
discussed (see Figure 16-90), or it can propel vesicles or particles through the<br />
cell cltoplasm, as in the example of the bacterium Listeria monocytogenes discussed<br />
in Chapter 24 (see Figure 24-37 ). Moreover, when anchored in a<br />
more complex way to the membrane, the same t)?e of force drives plasma<br />
membrane invaginations, as it does during the endocytotic and phagocl'totic<br />
processes discussed in Chapter 13.<br />
It is interesting to compare the organization of the actin-rich lamellipodium<br />
to the organization of the microtubule-rich mitotic spindle. In both cases, the<br />
cell harnesses and amplifies the intrinsic dynamic behavior of the cltoskeletal<br />
filament systems to generate large-scale structures that determine the behavior<br />
of the whole cell. Both structures feature rapid turnover of their constituent<br />
cytoskeletal filaments, even though the structures themselves may remain intact<br />
at steady state for long periods of time. The leading edge plasma membrane in<br />
the lamellipodium fulfills an organizational role analogous to the condensed<br />
chromosomes in organizing and stimulating the dynamics of the mitotic spindle.<br />
In both cases, molecular motor proteins help to enhance cltoskeletal filament<br />
flux and turnover in the large-scale arrays.<br />
Figure 16-91 Contribution of myosin ll to polarized cell motility'<br />
(A) Myosin ll bipolar filaments bind to actin filaments in the dendritic<br />
lamellipodial meshwork and cause network contraction. The myosin-driven<br />
reorientation of the actin filaments in the dendritic meshwork forms an<br />
actin bundle that recruits more myosin ll and contributes to generating the<br />
contractile forces required for retraction of the trailing edge of the moving<br />
cell. (B) A fragment of the large lamellipodium of a keratocyte can be<br />
separated from the main cell body either by surgery with a micropipette or<br />
by treating the cell with certain drugs. Many of these fragments continue<br />
to move rapidly, with the same overall cytoskeletal organization as the<br />
intact keratocytes. Actin (btue) forms a protrusive meshwork at the front of<br />
the fragment. Myosin ll (pink) is gathered into a band at the rear' (From<br />
A. Verkovsky et al., Curr. Biol. 9:11-20,1 999. With permission from Elsevier,)<br />
o<br />
rd<br />
1039<br />
Figure 16-90 A model for protrusion of<br />
the actin meshwork at the leading edge,<br />
Two time points during advance of the<br />
lamellipodium are illustrated, with newly<br />
assembled structures at the later time<br />
point shown in a lighter color. Nucleation<br />
is mediated by the ARP complex at the<br />
front. Newly nucleated actin filaments are<br />
attached to the sides of preexisting<br />
filaments, primarily at a 70'angle.<br />
Filaments elongate, pushing the plasma<br />
membrane forward because of some sort<br />
of anchorage of the array behind. At a<br />
steady rate, actin filament plus ends<br />
become capped. After newly polymerized<br />
actin subunits hydrolyze their bound ATP<br />
in the filament lattice, the filaments<br />
become susceptible to depolymerization<br />
by cofilin. This cycle causes a spatial<br />
separation between net filament<br />
assembly at the front and net filament<br />
disassembly at the reat so that the actin<br />
filament network as a whole can move<br />
forward, even though the individual<br />
filaments within it remain stationary with<br />
resoect to the substratum.
1040 Chapter 16:The <strong>Cytoskeleton</strong><br />
leading edge of cell<br />
Cell Adhesion and Traction Allow Cells to PullThemselves<br />
Forward<br />
(c)<br />
20 pm<br />
Lamellipodia of all cells seem to share a basic, simple type of dynamic organization<br />
where actin filament assembly occurs preferentially at the leading edge and<br />
actin filament disassembly occurs preferentially at the rear. However, the interactions<br />
between the cell and its normal physical environment usually make the<br />
situation considerably more complex than for fish keratocFes crawling on a culture<br />
dish. Particularly important in locomotion is the intimate crosstalk between<br />
tightly to move forward. In these lamellipodia, the same cycle of localized nucleation<br />
of new actin filaments, depolymerization of old filaments, and myosindependent<br />
contraction continues to operate. But because the leading edge is<br />
prevented physically from moving forward, the entire actin mesh moves backward<br />
toward the cell body instead, pulled by myosins (Figure 16-92). The adhesion<br />
of most cells lies somewhere between these two extremes, and most lamellipodia<br />
exhibit some combination of forward actin filament protrusion (like keratocJ,tes)<br />
and rearward actin flux (like Ihe Aplysia neurons).<br />
As a lamellipodium, filopodium, or pseudopodium extends forward over a<br />
substratum, it can form new attachment sites at the cell front that remain stationary<br />
as the cell moves forward over them, persisting until the rear of the cell<br />
catches up with them. \.A/hen an individual lamellipodium fails to adhere to the<br />
(D)<br />
Hrc-cH'-cg,<br />
/- \<br />
HrC-CH<br />
CHOH<br />
Hzc<br />
HO<br />
Hzc<br />
HC<br />
au<br />
cytochalasin B<br />
CH<br />
il<br />
CH<br />
Figure 16-92 Rearward movement of<br />
the actin network in a growth-cone<br />
lamellipodium. (A) A growth cone from a<br />
neuron of the sea slug Aplysiais cultured<br />
on a highly adhesive substratum and<br />
viewed by differential-interferencecontrast<br />
microscopy. Microtubules and<br />
membrane-enclosed organelles are<br />
confined to the bright, rear area ofthe<br />
growth cone (to the /eft), while a<br />
meshwork of actin filaments fills the<br />
lamellipodium (onthe right). (B) After<br />
brief treatment with the drug<br />
cytochalasin, which caps the plus ends of<br />
actin filaments (see Table 16-2, p. 988),<br />
the actin meshwork has detached from<br />
the front edge of the lamellipodium and<br />
has been pulled backward. (C) At the<br />
time point shown in B, the cell was fixed<br />
and labeled with fluorescent phalloidin<br />
to show the distribution of the actin<br />
filaments. Some actin filaments persist at<br />
the leading edge, but the region behind<br />
the leading edge is devoid of filaments.<br />
Note the sharp boundary of the<br />
rearward-moving actin meshwork.<br />
(D) The complex cyclic structure of<br />
cytochalasin B. (A-C, courtesy of Paul<br />
Forscher.)<br />
Figure 16-93 Lamellipodia and ruffles at<br />
the leading edge of a human fibroblast<br />
migrating in culture. The arow in this<br />
scanning electron micrograph shows the<br />
direction of cell movement. As the cell<br />
moves forward, lamellipodia that fail to<br />
attach to the substratum are sweot<br />
backward over the dorsal surface of the<br />
cell, a movement known as rufflinq.<br />
(Courtesy of Julian Heath.)
THE CYTOSKELETON AND CELL BEHAVIOR<br />
substratum, it is usually lifted up onto the dorsal surface of the cell and rapidly<br />
carried backward as a "ruffle" (Figure 16-93).<br />
The attachment sites established at the leading edge serve as anchorage<br />
points, which allow the cell to generate traction on the substratum and pull its<br />
body forward. Myosin motor proteins, especially myosin II, seem to generate<br />
traction forces. In many locomoting cells, myosin II is highly concentrated at the<br />
posterior of the cell where it may help to push the cell body forward like toothpaste<br />
being squeezed out of a tube from the rear (Figure 16-94; see also Figure<br />
16-91). Dictyostelium amoebae that are deficient in myosin II are able to protrude<br />
pseudopodia at normal speeds, but the translocation of their cell body is<br />
much slower than that of wild-type amoebae, indicating the importance of<br />
myosin II contraction in this part of the cell locomotion cycle. In addition to<br />
helping to push the cell body forward, contraction of the actin-rich cortex at the<br />
rear of the cell may selectively weaken the older adhesive interactions that tend<br />
to hold the cell back. Myosin II may also transport cell body components forward<br />
over a polarized array of actin filaments.<br />
The traction forces generated by locomoting cells exert a significant pull on<br />
the substratum (Figure 16-95). In a living animal, most crawling cells move<br />
across a semiflexible substratum made of extracellular matrix, which can be<br />
deformed and rearranged by these cell forces. In culture, movement of fibroblasts<br />
through a gel of collagen fibrils aligns the collagen, generating an organized<br />
extracellular matrix that in turn affects the shape and direction of locomotion of<br />
the fibroblasts within it (Figure 16-96). Conversely, mechanical tension or<br />
stretching applied externally to a cell will cause it to assemble stress fibers and<br />
focal adhesions, and become more contractile. Although poorly understood,<br />
this two-way mechanical interaction between cells and their physical environment<br />
is thought to be a primary way that vertebrate tissues organize themselves.<br />
Members of the Rho Protein Family Cause Major Rearrangements<br />
of the Actin <strong>Cytoskeleton</strong><br />
Cell migration is one example of a process that requires long-distance communication<br />
and coordination between one end of a cell and the other. During<br />
directed migration, it is important that the front end of the cell remain structurally<br />
and functionally distinct from the back end. In addition to driving local<br />
mechanical processes such as protrusion at the front and retraction at the rear,<br />
the cltoskeleton is responsible for coordinating cell shape, organization, and<br />
mechanical properties from one end of the cell to the other, a distance which is<br />
typically several tens of micrometers for animal cells. In many cases, including<br />
but not limited to cell migration, large-scale cyoskeletal coordination takes the<br />
form of the establishment of cell polarity, where a cell builds different structures<br />
with distinct molecular components at the front vs. the back, or at the top vs. the<br />
100 $m<br />
1 041<br />
5[m<br />
Figure 16-94 The localization of myosin<br />
land myosin ll in a normal crawling<br />
Dictyostelium amoeba. This cell was<br />
crawling toward the upper right at the<br />
time that it was fixed and labeled with<br />
antibodies specific for two myosin<br />
isoforms. Myosin | (green) is mainly<br />
restricted to the leading edge of<br />
pseudopodia at the front of the cell.<br />
Myosin ll (red) is highest in the posterior,<br />
actin-rich cortex. Contraction of the<br />
cortex at the posterior of the cell by<br />
myosin ll may help to push the cell body<br />
forward. (Courtesy of Yoshio Fukui.)<br />
Figure 16-95 Adhesive cells exert<br />
traction forces on the substratum. These<br />
fibroblasts have been cultured on a very<br />
thin sheet of silicon rubber. Attachment of<br />
the cells, followed by contraction of their<br />
cytoskeleton, has caused the rubber<br />
substratum to wrinkle. (From A.K' Harris,<br />
P. Wild and D. Stopak, Science 208:177-179'<br />
1980. With permission from AAAS.)
1042 Chapter 16:The<strong>Cytoskeleton</strong><br />
Figure 16-96 Shaping of the extracellular matrix by cell pulling. This<br />
micrograph shows a region between two pieces of embryonic chick heart<br />
(tissue explants rich in fibroblasts and heart muscle cells) that were grown<br />
in culture on a collagen gel for 4 days. A dense tract of aligned collagen<br />
fibers has formed between the two explants, apparently as a result of<br />
fibroblasts tugging on the collagen. (From D. Stopak and A.K. Harris, Dey.<br />
Blol. 90:383-398, 1982. With permission from Academic press.)<br />
bottom. Cell locomotion requires an initial polarization of the cell to set it off in<br />
a particular direction. Carefully controlled cell polarization processes are also<br />
required for oriented cell divisions in tissues and for formation of a coherent,<br />
organized multicellular structure. Genetic studies in yeast, flies, and worms have<br />
provided most of our current understanding of the molecular basis of cell polarity.<br />
The mechanisms that generate cell polarity in vertebrates are only beginning<br />
to be explored. In all known cases, however, the c],toskeleton has a central role,<br />
and many of the molecular components have been evolutionarily conserved.<br />
For the actin cytoskeleton, diverse cell-surface receptors trigger global<br />
structural rearrangements in response to external signals. But all of these signals<br />
seem to converge inside the cell on a group of closely related monomeric<br />
GTPases that are members of the Rho protein family-cdc42, Rac, andRho. The<br />
same Rho family proteins are also involved in the establishment of many kinds<br />
of cell polarity.<br />
Like other members of the Ras superfamily, these Rho proteins act as molecular<br />
switches to control cell processes by cycling between an active, GTp-bound<br />
state and an inactive, GDP-bound state (see Figure 3-71). Activation of cdc42 on<br />
the plasma membrane triggers actin polymerization and bundling to form<br />
either filopodia or shorter cell protrusions called microspikes. Activation of Rac<br />
promotes actin polyrnerization at the cell periphery leading to the formation of<br />
sheet-like lamellipodial extensions and membrane ruffles, which are actin-rich<br />
protrusions on the cell's dorsal surface (see Figure l6-93). Activation of Rho promotes<br />
both the bundling of actin filaments with myosin II filaments into stress<br />
fibers and the clustering of integrins and associated proteins to form focal contacts<br />
(Figure 16-97). These dramatic and complex structural changes occur<br />
because each of these three molecular switches has numerous downsueam rarget<br />
proteins that affect actin organization and dynamics.<br />
actin staining actin staining<br />
(A) QUIE5CENT CELLS (B) Rho ACTIVATION<br />
(C) Rac ACTIVATION (D) Cdc42 ACTIVATION<br />
20 pm<br />
Figure 16-97 The dramatic effects of<br />
Rac, Rho, and Cdc42 on actin<br />
organization in fibroblasts. In each case,<br />
the actin filaments have been labeled<br />
with fluorescent phalloidin.<br />
(A) Serum-starved fibroblasts have actin<br />
filaments primarily in the cortex, and<br />
relatively few stress fibers.<br />
(B) Microinjection of a constitutively<br />
activated form of Rho causes the raoid<br />
assembly of many prominent stress<br />
fibers. (C) Microinjection of a<br />
constitutively activated form of Rac, a<br />
closely related monomeric GTPase,<br />
causes the formation of an enormous<br />
lamellipodium that extends from the<br />
entire circumference of the cell.<br />
(D) Microinjection of a constitutively<br />
activated form of Cdc42, another Rho<br />
family member, causes the protrusion of<br />
many long filopodia at the cell periphery.<br />
The distinct global effects of these three<br />
GTPases on the organization of the actin<br />
cytoskeleton are mediated by the actions<br />
of dozens of other protein molecules that<br />
are regulated by the GTPases. These<br />
target proteins include some of the<br />
various actin-associated proteins that we<br />
have discussed in this chapter. (From<br />
A. Hall, Science 279:509-514, 1998. With<br />
permission from AAAS.)
THE CYTOSKELETON AND CELL BEHAVIOR<br />
Some key targets of activated Cdc42 are members of the WASp protein family.<br />
Human patients deficient in WASp suffer fromWiskott-Aldrich Syndrome, a<br />
severe form of immunodeficiency where immune system cells have abnormal<br />
actin-based motility and platelets do not form normally. AlthoughWASp itself is<br />
expressed only in blood cells and immune system cells, other family members<br />
are expressed ubiquitously that enable activated Cdc42 to enhance actin polymerization.<br />
WASp proteins can exist in an inactive folded conformation and an<br />
activated open conformation. Association with Cdc42-GTP stabilizes the open<br />
form of WASp, enabling it to bind to the ARP complex and strongly enhancing<br />
this complex's actin-nucleating activity (see Figure 16-34). In this way, activation<br />
of Cdc42 increases actin nucleation.<br />
Rac-GTP also activates WASp family members, as well as activating the<br />
crosslinking activity of the gel-forming protein filamin, and inhibiting the contractile<br />
activity of the motor protein myosin II, stabilizing the lamellipodia and<br />
inhibiting the formation of contractile stress fibers (Figure l6-98A).<br />
Rho-GTP has a very different set of targets. Instead of activating the ARP<br />
complex to build actin networks, Rho-GTP turns on formin proteins to construct<br />
parallel actin bundles. At the same time, Rho-GTP activates a protein kinase that<br />
indirectly inhibits the activity of cofilin, leading to actin filament stabilization.<br />
The same protein kinase inhibits a phosphatase acting on myosin light chains<br />
(see Figure 16-72). The consequent increase in the net amount of myosin light<br />
chain phosphorylation increases the amount of contractile myosin motor protein<br />
activity in the cell, enhancing the formation of tension-dependent structures<br />
such as stress fibers (Figure 16-988).<br />
In some cell types, Rac-GTP activates Rho, usuallywith kinetics that are slow<br />
compared to Rac's activation of the ARP complex. This enables cells to use the<br />
Rac pathway to build a new actin structure while subsequently activating the<br />
Rho pathway to induce a contractility that builds up tension in this structure.<br />
This occurs, for example, during the formation and maturation of cell-cell contacts.<br />
As we will explore in more detail below the communication between the<br />
Rac and Rho pathways also facilitates maintenance of the large-scale differences<br />
between the cell front and the cell rear during migration.<br />
Extracellular Signals Can Activate the Three Rho Protein Family<br />
Members<br />
The activation of the monomeric GTPases Rho, Rac, and Cdc42 occurs through<br />
an exchange of GTP for a tightly bound GDP molecule, catalyzed by guanine<br />
nucleotide exchange factors (GEFs). Of the 85 GEFs that have been identified in<br />
_.:
1044 Chapter 16:The <strong>Cytoskeleton</strong><br />
the human genome, some are specific for an individual Rho family GTpase,<br />
whereas others seem to act on all three family members. The number of GEFs<br />
exceeds the number of Rho GTPases that they regulate because different GEFs<br />
are restricted to specific tissues and even specific subcellular locations, and they<br />
are sensitive to distinct kinds of regulatory inputs. Various cell-surface receptors<br />
activate GEFs. An example is the Eph receptor tyrosine kinase involved in neurite<br />
growth cone guidance, which is discussed in detail in chapter 15. Interestingly,<br />
several of the Rho family GEFs associate with the growing ends of microtubules<br />
by binding to one of the +TIPs. This provides a connection between the<br />
dlnamics of the microtubule cytoskeleton and the large-scale organization of<br />
the actin cltoskeleton, which is important for the overall integration of cell<br />
shape and movement.<br />
The Rho family GTPases are also primary determinants of cell polarity in<br />
budding yeast, where extensive genetic analyses have increased our understanding<br />
of the general mechanisms involved. on starvation, yeasts, like many<br />
other unicellular organisms, sporulate. But sporulation can occur only in diploid<br />
budding yeast cells, whereas budding yeasts mainly proliferate as haploid cells.<br />
A starving haploid individual must therefore locate a partner of the opposite<br />
mating type, woo it, and mate with it before sporulating. yeast cells are unable to<br />
swim and, instead, reach their mates by polarized growth. The haploid form of<br />
signal molecule, which under normal circumstances would direct it toward an<br />
amorous a cell located nearby.<br />
This polarized cell growth requires alignment of the actin cytoskeleton in<br />
response to the mating factor signal. \.A/hen the signal binds to its receptol the<br />
receptor activates cdc42, which in turn induces assembly of actin fllaments at<br />
the location closest to the source of the signal. Local activation of Cdc42 is further<br />
enhanced by a positive feedback loop, requiring actin-dependent transport of<br />
cdc42 itself as well as its GEF and other signaling components along the newly<br />
assembled actin structures toward the site of the signal. subsequently, actin<br />
cables are assembled pointing toward the site of cdc42 accumulation due to the<br />
upstream and dor,rmstream pathways have been identified through genetic<br />
Figure 16-99 Morphological polarization<br />
of yeast cells in response to mating<br />
factor. (A) Cells of Soccharomyces<br />
cerevisiae are usually spherical. (B) They<br />
become polarized when treated with<br />
mating factor from cells of the opposite<br />
mating type. The polarized cells are called<br />
"shmoos." (C) Al Capp's famous cartoon<br />
character, the original Shmoo. (A and B,<br />
courtesy of Michael Snyder; C, @ 1948<br />
Capp Enterprises, Inc. Used by permission.)
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THE CYTOSKELETON AND CELL EEHAVIOR<br />
(A)<br />
t<br />
localized<br />
signal<br />
Figure 1 6-1 03 The polarization of a cytotoxic T cell after target-cell<br />
recognition. (A) Changes in the cytoskeleton of a cytotoxic T cell after it<br />
has made contact with a target cell.The initial recognition event results in<br />
signals that cause actin polymerization in both cells at the site of contact.<br />
In theT cell, interactions between the actin-rich contact zone and<br />
microtubules emanating from the centrosome result in reorientation of the<br />
centrosome, so that the associated Golgi apparatus is directly apposed to<br />
the target cell. (B) lmmuno-fluorescence micrograph in which both the<br />
T cell (top)and its target cell (bottom) have been stained with an antibody<br />
against microtubules. The centrosome and the microtubules radiating from<br />
it in the T cell are oriented toward the point of cell-cell contact. In contrast,<br />
the microtubule array in the target cell is not polarized. (B' from B. Geiger,<br />
D. Rosen and G. Berke, J. Cett Biol.95137-143,1982. With permission from<br />
The Rockefeller University Press.)<br />
A similar cooperative feedback loop seems to operate in many other<br />
instances of cell polarization. A particularly interesting example is the killing of<br />
specific target cells byT lymphocytes. These cells are a critical component of the<br />
vertebrate's adaptive immune response to infection by viruses. T cells, like neutrophils,<br />
use actin-based motility to crawl through the body's tissue and find<br />
infected target cells. \Mhen a T cell comes into contact with a virus-infected cell<br />
and its receptors recognize foreign viral antigens on the surface of the target cell,<br />
the same polarization machinery is engaged in a very different way to facilitate<br />
kilting of the target cell. Rac is activated at the point of cell-cell contact and<br />
causes actin polymerization at this site, creating a specialized region of the cortex.<br />
This specialized site causes the centrosome to reorient, moving with its<br />
microtubules to the zone of T-cell-target contact (Figure f6-f03). The microtubules,<br />
in turn, position the Golgi apparatus right under the contact zone'<br />
focusing the killing machinery onto the target cell. The mechanism of killing is<br />
discussed in Chapter 25 (see Figure 25-47).<br />
The Complex Morphological Specialization of Neurons Depends<br />
on the <strong>Cytoskeleton</strong><br />
For our final case study of the ways that the intrinsic properties of the eucaryotic<br />
cytoskeleton enable specific and enormously complicated large-scale cell behaviors,<br />
we examine the neuron. Neurons begin life in the embryo as unremarkable<br />
cells, which use actin-based motility to migrate to specific locations. Once there,<br />
however, they send out a series of long specialized processes that will either<br />
receive electrical signals (dendrites) or transmit electrical signals (axons) to their<br />
target cells. The beautiful and elaborate branching morphology of axons<br />
and dendri les neurons to form tremendously complex signaling networks,<br />
interacting with many other cells simultaneously and making possible the<br />
complicated and often unpredictable behavior of the higher animals. Both axons<br />
and dendrites (collectively called. neurites) are filled with bundles of microtubules<br />
that are critical to both their structure and their function.<br />
In axons, all the microtubules are oriented in the same direction, with their<br />
minus end pointing back toward the cell body and their plus end pointing forward<br />
toward the axon terminals (Figure f 6-104). The microtubules do not reach<br />
(8)<br />
10 pm<br />
1047
1048 Chapter 16:The<strong>Cytoskeleton</strong><br />
o vesicle with bound dynein<br />
o vesicle with bound kinesin<br />
r microtubule<br />
(A) FIBRoBLAST (B) NEURoN<br />
from the cell body all the way to the axon terminals; each is typically only a few<br />
micrometers in length, but large numbers are staggered in an overlapping array.<br />
This set of aligned microtubule tracks acts as a highway to transport many specific<br />
proteins, protein-containing vesicles, and mRNAs to the axon terminals,<br />
where synapses must be constructed and maintained. The longest axon in the<br />
human body reaches from the base of the spinal cord to the foot, being up to a<br />
meter in length.<br />
Mitochondria, large numbers of specific proteins in transport vesicles, and<br />
synaptic vesicle precursors make the long journey in the forward (anterograde)<br />
direction. They are carried there by plus-end-directed kinesin-family moror proteins<br />
that can move them a meter in as little as two or three days, which is a great<br />
improvement over diffrrsion, which would take approximately several decades<br />
to move a mitochondrion this distance. Many members of the kinesin superfamily<br />
contribute to this anterograde axonal transport, most carrying sp"iific<br />
subsets of membrane-enclosed organelles along the microtubuler. ih" g."ut<br />
diversity of the kinesin family motor proteins used in axonal transport suggests<br />
that they are involved in targeting their cargo to specific structures near the terminus<br />
or along the way, as well as in cargo movement. old components from the<br />
axon terminals are carried back to the cell body for degradation and recycling by<br />
a retrograde axonal transport. This transport occurs along the same set of oriented<br />
microtubules, but it relies on cytoplasmic dynein, t-tri.tr is a minus-enddirected<br />
motor protein. Retrograde transport is also critical for communicating<br />
the presence of growth and survival signals received by the nerve terminus back<br />
to the nucleus, in order to influence gene expression.<br />
one form of human peripheral neuropathy, charcot-Marie-Tooth disease, is<br />
caused by a point mutation in a particular kinesin family member that transports<br />
slmaptic vesicle precursors dovrrn the axon. other kinds of neurodegenerative<br />
diseases such as Alzheimer's disease may also be caused in part by dlsruptions<br />
in neuronal trafficking; as pointed out previously, the amyloid pr".r.r^o,<br />
protein APP is part of a protein complex that serves as a receptor for kinesin-l<br />
binding to other axonal transport vesicles.<br />
Axonal structure depends on the axonal microtubules, as well as on the contributions<br />
of the other two major cytoskeletal systems-actin filaments and<br />
intermediate filaments. Actin filaments line the cortex of the axon, just beneath<br />
the plasma membrane, and actin-based motor proteins such as myosin v are<br />
also abundant in the rxon, presumably to help move materials. Neurofilaments,<br />
the specialized intermediate filaments of nerve cells, provide the most important<br />
structural support in the axon. A disruption in neurofilament structure, or<br />
in the cross-linking proteins that attach the neurofilaments to the microtubules<br />
and actin filaments distributed along the ixon, can result in axonal disorganization<br />
and eventually axonal degeneration.<br />
The construction ofthe elaborate branching architecture ofthe neuron during<br />
embryonic development requires actin-based motility. As mentioned earlier,<br />
the- tips of growing axons and dendrites extend by means of a growth cone, a specialized<br />
motile structure rich in actin (Figure 16-105). Mosi neuronal growth<br />
cones produce filopodia, and some make lamellipodia as well. The protrusion<br />
Figure l6-104 Microtubule organization<br />
in fibroblasts and neurons. (A) In a<br />
fibroblast, microtubules emanate<br />
outward from the centrosome in the<br />
middle of the cell. Vesicles with Dlus-enddirected<br />
kinesin attached move outward,<br />
and vesicles with minus-end-directed<br />
dynein attached move inward. (B) In a<br />
neuron, microtubule organization is more<br />
complex. In the axon, all microtubules<br />
share the same polarity, with the plus<br />
ends pointing outward toward the axon<br />
terminus. No one microtubule stretches<br />
the entire length ofthe axon; instead,<br />
short overlapping segments of parallel<br />
microtubules make the tracks for fast<br />
axonal transport. In dendrites, the<br />
microtubules are of mixed polarity, with<br />
some plus ends pointing outward and<br />
some pointing inward.
THE CYTOSKELETON AND CELL BEHAVIOR<br />
tt(B)<br />
10 pm 10 pm<br />
and stabilization of growth-cone filopodia are exquisitely sensitive to environmental<br />
cues. Some cells secrete soluble proteins such as netrin to attract or repel<br />
growth cones. These modulate the structure and motility of the growth cone<br />
cytoskeleton by altering the balance between Rac activity and Rho activity at the<br />
leading edge (see Figure 15-62).In addition, there are fixed guidance markers<br />
along the way, attached to the extracellular matrix or to the surfaces of cells.<br />
\Mhen a filopodium encounters such a "guidepost" in its exploration, it quickly<br />
forms adhesive contacts. It is thought that a myosin-dependent collapse of the<br />
actin meshwork in the unstabilized part of the growth cone then causes the<br />
developing ixon to turn toward the guidepost.<br />
Thus, a complex combination of positive and negative signals, both soluble<br />
and insoluble, accurately guide the growth cone to its final destination. Microtubules<br />
then reinforce the directional decisions made by the actin-rich protrusive<br />
structures at the leading edge of the growth cone. Microtubules from the<br />
axonal parallel array just behind the growth cone are constantly growing into the<br />
growth cone and shrinking back by dynamic instability. Adhesive guidance signals<br />
are somehow relayed to the dynamic microtubule ends, so that microtubules<br />
growing in the correct direction are stabilized against disassembly. In<br />
this way, a microtubule-rich axon is left behind, marking the path that the<br />
growth cone has traveled.<br />
Dendrites are generally much shorter projections than axons' and they<br />
receive synaptic inputs rather than being specialized for sending signals like<br />
axons. The microtubules in dendrites all lie parallel to one another but their<br />
polarities are mixed, with some pointing their plus ends toward the dendrite tip,<br />
while others point back toward the cell body. Nevertheless, dendrites also form<br />
as the result of growth-cone activity. Therefore, it is the growth cones at the tips<br />
of axons and dendrites that create the intricate, highly individual morphology of<br />
each mature neuronal cell (Figure f 6-f 06).<br />
cell body<br />
zlrm<br />
axon (less than 1 mm to<br />
more than 1 m in length)<br />
dendrites receive<br />
synaptic inputs<br />
terminal branches of<br />
axon make synapses on<br />
target cells<br />
104!l<br />
Figure 1 6-1 05 Neuronal growth cones.<br />
(A) Scanning electron micrograph<br />
of two growth cones at the end of a<br />
neurite, put out by a chick sympathetic<br />
neuron in culture. Here, a previously single<br />
growth cone has recently split into two'<br />
Note the many filopodia and the large<br />
lamellipodia. The taut appearance of the<br />
neurite is due to tension generated by the<br />
forward movement of the growth cones,<br />
which are often the only firm points of<br />
attachment of the axon to the substratum.<br />
(B) Scanning electron micrograph of the<br />
growth cone of a sensory neuron crawling<br />
over the inner surface of the epidermis of a<br />
Xenopus tadpole. (A, from D. Bray, in Cell<br />
Behaviour [R. Bellairs, A. Curtis and<br />
G. Dunn, eds.l. Cambridge, UK: Cambridge<br />
University Press, 1982; B, from A. Roberts,<br />
Brain Res. 1 1 8:526-530, 1 976. With<br />
permission from Elsevier.)<br />
Figure 1 6-106 The complex architecture<br />
of a vertebrate neuron. The neuron<br />
shown is from the retina of a monkey.<br />
The arrows indicate the direction of travel<br />
of the electrical signal along the axon.<br />
The longest and largest neurons in the<br />
human body extend for a distance of<br />
about 1 m (1 million Um), from the base<br />
of the spinal cord to the tip of the big<br />
toe, and have an axon diameter of 15 pm.<br />
(Adapted from B.B. Boycott, in Essays on<br />
the Nervous System [R. Bellairs and<br />
E.G. Gray, eds.l. Oxford, UK: Clarendon<br />
Press, 1 974.)
1050 Chapter 16:The <strong>Cytoskeleton</strong><br />
(B) 20 40<br />
time in mins<br />
60 | lolr# 80<br />
Although the neurons of the central nervous system are long-lived cells, they<br />
are by no means static. synapses are constantly being created, strengthened,<br />
weakened, and eliminated as the brain learns, evaluates, and forgets. uigh-r"rolution<br />
imaging of the structure of neurons in the brains of adult mice has<br />
revealed that neuronal morphology is undergoing constant rearrangement as<br />
synapses are forged and broken (Figure l6-10z). These actin-dependent rearrangements<br />
are rhought to be critical in learning and long-term memory. In this<br />
way, the cltoskeleton provides the engine for construction of the entire nervous<br />
system, as well as producing the supporting structures that strengthen, stabilize,<br />
and maintain its parts.<br />
Summary<br />
Two distinct types of specialized structures in eucaryotic cells are formed from highty<br />
ordered arrays of motor proteins that moue on stabilized filament tracks. The<br />
system function in the adult animal-is another prime example of such complex,<br />
coordinated cytoskeletal action. For a cell to crawl, it must generate and maintain an<br />
ouerall structural polarity, which is influenced by external cues. In addition, the cell<br />
must coordinate protrusion at the leading edge (by assembly of new actin filaments),<br />
adhesion of the newly protruded part of the cell to the substratum, forces generated by<br />
molecular motors to bring the cell body forward.<br />
Complex cells, such as neurons, require the coordinated assembly of microtubules,<br />
neurofilaments (neuronal intermediate filaments), and actin ftlaments, as well as the<br />
actions of dozens of highly specialized molecular motors that transport subcellular<br />
components to their appropriate destinations.<br />
PROBLEMS<br />
Which statements<br />
are true? Explain why or why not.<br />
16-1 The role of ATP hydrolysis in actin polymerization is<br />
similar to the role of GTP hydrolysis in tubulin polyrneriza_<br />
tion: both serve to weaken the bonds in the polymer and<br />
thereby promote depolymerization.<br />
Figure 16-107 Rapid changes in dendrite<br />
structure within a living mouse brain.<br />
(A) lmage of cortical neurons in a transgenic<br />
mouse that has been engineered to express<br />
green fluorescent protein in a small fraction<br />
of its brain cells. Changes in these brain<br />
neurons and their projections can be<br />
followed for months using highly sensitive<br />
fluorescence microscopy. To make this<br />
possible, the mouse is subjected to an<br />
operation that introduces a small<br />
transparent window through its skull, and it<br />
is anesthetized each time that an image is<br />
recorded. (B) A single dendrite, imaged over<br />
the period of 80 minutes, demonstrates that<br />
dendrites are constantly sending out and<br />
retracting tiny actin-dependent protrusions<br />
to create the dendritic spines that receive<br />
the vast majority of excitatory synapses<br />
from axons in the brain. Those spines that<br />
become stabilized and persist for months<br />
are thought to be important for brain<br />
function, and may be involved in long-term<br />
memory. (Courtesy of Karel Svoboda.)<br />
16-2 In most animal cells, minus end-directed micro_<br />
tubule motors deliver their cargo to the periphery of the cell,<br />
whereas plus end-directed microtubule motors deliver their<br />
cargo to the interior ofthe cell.<br />
16-3 Motor neurons trigger action potentials in muscle<br />
cell membranes that open voltage-sensitive Ca2* channels<br />
in T-tubules, allowing extracellular CaZ* to enter the cytosol,<br />
bind to troponin C, and initiate rapid muscle contraction.
END.OF-CHAPTER PROBLEMS<br />
Discuss the following problems.<br />
16-4 At 1.4 mg/ml pure tubulin, microtubules grow at a<br />
rate of about 2 pm/min. At this growth rate how many oBtubulin<br />
dimers (B nm in length) are added to the ends of a<br />
microtubule each second?<br />
16*5 A solution of pure aB-tubulin dimers is thought to<br />
nucleate microtubules by forming a linear protofilament<br />
about seven dimers in length. At that point, the probabilities<br />
that the next ap-dimer will bind laterally or to the end of the<br />
protofilament are about equal. The critical event for microtubule<br />
formation is thought to be the first lateral association<br />
(Figure Qf6-f). How does lateral association promote the<br />
subsequent rapid formation of a microtubule?<br />
o -,<br />
e<br />
od<br />
LINEAR GROWTH<br />
I<br />
I JaJ<br />
o<br />
od<br />
o.cd<br />
..ddd<br />
'lj,t'l<br />
oofo<br />
dddd<br />
dddd<br />
.t.s,lJ<br />
LATERAL ASSOCIATION<br />
e<br />
"tJ<br />
e<br />
.t<br />
,f<br />
Figure Q16-1 Model for microtubule nucleation by pure crB-tubulin<br />
dimers (Problem 16 5).<br />
16-6 How does a centrosome "know" when it has found<br />
the center of the cell?<br />
16-7 The concentration of actin in cells is 50-100 times<br />
greater than the critical concentration observed for pure<br />
actin in a test tube. How is this possible?'v\rhat prevents the<br />
actin subunits in cells from polymerizing into filaments?<br />
\A4ry is it advantageous to the cell to maintain such a large<br />
pool of actin subunits?<br />
16*8 The movements of single motor-protein molecules<br />
can be analyzed directly. Using polarized laser light, it is possible<br />
to create interference patterns that exert a centrally<br />
directed force, ranging from zero aI the center to a few<br />
piconewtons at the periphery (about 200 nm from the center).<br />
Individual molecules that enter the interference pattern<br />
are rapidly pushed to the center, allowing them to be<br />
captured and moved at the experimenter's discretion.<br />
Using such "optical tweezers," single kinesin molecules<br />
can be positioned on a microtubule that is fixed to a coverslip.<br />
Although a single kinesin molecule cannot be seen<br />
optically, it can be tagged with a silica bead and tracked indirectly<br />
by following the bead (Figure Qf 6-2,{). In the absence<br />
of ATB the kinesin molecule remains at the center of the<br />
interference pattern, but with AIP it moves toward the plus<br />
end of the microtubule. As kinesin moves along the microtubule,<br />
it encounters the force of the interference pattern,<br />
which simulates the load kinesin carries during its actual<br />
function in the cell. Moreover, the pressure against the silica<br />
bead counters the effects of Brownian (thermal) motion, so<br />
that the position of the bead more accurately reflects the<br />
position of the kinesin molecule on the microtubule.<br />
Traces of the movements of a kinesin molecule along a<br />
microtubule are sho'nr,.n in Figure Q16-28.<br />
(A) EXPERIMENTAL SETUP<br />
Euo<br />
a<br />
Fqo<br />
20<br />
(B) POSITION OF KINESIN<br />
1 051<br />
microtubule<br />
o z<br />
.,.1 o".onlu<br />
8<br />
Figure Q16-2 Movement of kinesin along a microtubule (Problent<br />
16 s). (A) Experimental setup with kinesin linked to a silica bead,<br />
moving along a microtubule. (B) Position of kinesin (as visualized by<br />
Dosition of silica bead) relative to center of interference pattern, as a<br />
function of time of movement alonq the microtubule' The jagged<br />
nature of the trace results from Brownian motion of the bead'<br />
A. As shown in Figure Ql6-2B, all movement of kinesin is<br />
in one direction (toward the plus end of the microtubule).<br />
\A4rat supplies the free energy needed to ensure a unidirectional<br />
movement along the microtubule?<br />
B. \Altrat is the average rate of movement of kinesin along<br />
the microtubule?<br />
c. \A/hat is the length of each step that a kinesin takes as it<br />
moves along a microtubule?<br />
D. From other studies it is knor,rm that kinesin has two<br />
globular domains that each can bind to B-tubulin, and that<br />
kinesin moves along a single protofilament in a microtubule.<br />
In each protofilament the B-tubulin subunit repeats<br />
at B-nm intervals. Given the step length and the interval<br />
between B-tubulin subunits, how do you suppose a kinesin<br />
molecule moves along a microtubule?<br />
E. Is there anything in the data in Figure Ql6-28 that tells<br />
you how many AIP molecules are hydrolyzed per step?<br />
16-9 How is the unidirectional motion of a lamellipodium<br />
maintained?<br />
1 6* 1 0 Detailed measurements of sarcomere length and tension<br />
during isometric contraction in striated muscle provided<br />
crucial early support for the sliding filament model of<br />
muscle contraction. Based on your understanding of the<br />
sliding filament model and the structure of a sarcomere,<br />
propose a molecular explanation for the relationship of tension<br />
to sarcomere length in the portions of Figure Qf 6-3<br />
marked L II, III, and IV (In this muscle, the length of the<br />
myosin filament is 1.6 pm and the lengths of the actin thin<br />
filaments that project from the Z discs are 1.0 pm.)<br />
E l<br />
E i 100<br />
o<br />
E<br />
b '-<br />
-50<br />
c<br />
az)<br />
q<br />
0<br />
23<br />
sarcomere length (Pm)<br />
Figure Ql6-3 Tension as a<br />
function of sarcomere length<br />
during isometric contraction<br />
(Problem 16-10).
1052 Chapter 16:The <strong>Cytoskeleton</strong><br />
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