Cell Biology Study Guide – Chapter 17 – Cytoskeleton (copy)

The cytoskeleton is responsible for the determination of cell shape, the organization of cellular

compartments, intracellular transport of particles and organelles, cell movement and cell division.

The cytoskeleton is composed of three dynamic filament systems and their associated proteins (Figure

17-2). The intermediate filaments (IFs) provide mechanical support for both the nucleus and the

cytoplasm. The actin filaments provide flexible support for the cortex underlying the cell membrane and

for contractile movements. The microtubules (tubulin subunit) provide more rigid support for long-

range transport and motility and chromosome movements.

IFs are intermediate in size (10nm in diameter) and the toughest and most durable filaments. They form a

network from the nucleus to the cell periphery and are most abundant in cells that experience great

mechanical stress (epithelia, muscle, nerve). They connect neighboring cells using a specialized junction

called a desmosome and to the extracellular matrix by a junction called a hemi-desmosome (hemi=half)

(Figure 17-3).

The subunits that form IFs are members of a large gene family (>70 IF genes in human genome). Your

book identifies four major classes, the keratins, vimentin & vimentin-related, neurofilaments, and the

nuclear lamins (Figure 17-5). The typical subunit is an elongate fibrous protein with a central rod domain

and unstructured domains at its amino and carboxy-termini. The central rod domain is similar in size and

amino acid sequence among different IFs, and it packs together with other subunits to form a coiled coil

dimer. Dimers then form staggered, anti-parallel tetramers. Eight tetramers twist together to form a

ropelike filament (Figure 17-4).

The globular amino and carboxy termini vary greatly in size and sequence between different IFs and

confer IF and cell specific functions. The most typical function is to stretch and distribute the effect of

locally applied forces, like carbon fibers in fiberglass or steel bars in concrete. This function is most obvious

with the keratin IFs of the skin (epidermis). Defects in keratins and their associated proteins in skin can

lead to cell rupture and blistering of the skin (Figure 17-6). Different keratins are expressed in different

layers of the skin, and so the effect of a keratin mutation depends on where the gene is expressed and the

nature of the mutation. When the mutation affects a keratin or associated junctional protein located in the

basal layer of the skin (which attaches the skin to the underlying connective tissue), the consequences can

be devastating. Epidermolysis bullosa simplex leads to blisters in response to even slight mechanical

stress, and some children die within 1-2 years. There is no treatment beyond wound care, although efforts

are now being made to treat some patients with stem cell therapies, depending on the nature of the

mutation.

There are numerous intermediate filament associated proteins (IFAPs), including plectin, which is

expressed in several tissues and links several different kinds of IFs to other cellular structures, such

as cell junctions, microtubules, and actin filaments (Figure 17-9). Defects in plectin can lead to intermediate

filament-associated defects in several tissues.

Vimentin and vimentin related IFs (desmin, glial fibrillary acid protein) play a similar role in connective

tissue cells (fibroblasts), muscle cells, and glial cells. Neurofilaments are the IFs of nerve cells, where

they are especially important in mechanical support of the nerve axon.

The fourth and most unusual class of IFs are the nuclear lamins (lamin A, B and C). They do not form

true filaments but instead form a 2D fibrous network that lines the nuclear envelope of the interphase

nucleus (Figure 17-7). The lamins provide mechanical support to the nucleus and function to anchor

chromatin to the nuclear envelope. Assembly of the nuclear lamina and nuclear envelope is controlled

during the cell cycle by phosphorylation of the nuclear lamins. Lamin phosphorylation drives disassembly

of the nuclear envelope at the beginning of mitosis. Lamin dephosphorylation drives reassembly of the

nuclear envelope at the end of mitosis. Certain types of lamin mutations are associated with a raredisease known as progeria or premature aging (Figure 17-8). The mutation affects the normal post-

translational modifications of lamin that regulate its association with the nuclear envelope, leading to defects

in nuclear shape. Children with progeria usually die of heart disease or stroke by their early teens.

Microtubules (MTs)

MTs are the largest filaments in the cell (25 nm in diameter) and play a crucial role in cell organization and

movement. They are long and relatively stiff hollow tubes that can rapidly assemble and disassemble in

different parts of the cell (Figure 17-12). In most animal cells, one end of a microtubule (the minus end) is

anchored in a structure known as the microtubule organizing center (MTOC) or centrosome (cell center,

often located adjacent to nucleus) (Figure 17-11). The other end (the plus end) extends outward to the cell

periphery to form a track for the movement of cellular components.

During mitosis, the cytoplasmic MT array reorganizes itself to form another structure, called the mitotic

spindle. The centrosome duplicates to form two MTOCs or spindle poles. MTs extend from the spindle

poles and attach to the mitotic chromosomes to separate them equally into two daughter cells (see Chapter

18 for more details). MTOCs known as basal bodies also promote the assembly of a different kind of MT

array, the 9+2 structures of eukaryotic cilia and flagella. Motile cilia or flagella beat rhythmically to

move fluids over the cell surface or to propel single cells such as sperm through fluid environments.

Another type of cilia, the primary cilium, is composed of a 9+0 MT array. Primary cilia are typically non-

motile but function as sensory structures on the surfaces of cells.

MTs are built from subunits of alpha and beta tubulin dimers that assemble in a head-to-tail fashion into

13 parallel protofilaments (Figure 17-12). The MTs have an intrinsic structural polarity, with one end,

the beta tubulin end, known as the plus end, and the other end, the alpha tubulin end, known as the minus

end. Plus and minus do NOT refer to electrical charge, but instead refer to the relative rates of subunit

addition and loss at the two ends of the MT. Subunits add more rapidly to the plus ends. This structural

polarity is critical for MT function.

In most animal cells, MT assembly is organized by the centrosome, which contains two centrioles and

hundreds of ring-shaped structures called gamma tubulin ring complexes. The gamma tubulin ring

complex is formed from another kind of tubulin, called gamma tubulin, and accessory proteins. This

complex binds to alpha tubulin subunits (the minus end of tubulin dimers) and nucleates MT assembly,

stabilizing the minus end against disassembly. The plus end of the microtubule extends outward from the

centrosome (Figure 17-13), and MTs grow by addition of subunits to the plus ends.

MTs exhibit an important behavior known as dynamic instability, in which an individual MT can grow or

shrink rapidly independent of its neighbors (Figure 17-15, Movie 17-2). Dynamic instability depends on

GTP hydrolysis. Each tubulin dimer contains a bound GTP molecule. Tubulin-GTP will bind rapidly to the

end of a growing MT to form a GTP cap (Figure 17-16). Eventually the GTP will be hydrolyzed to GDP,

and the MT will contain predominantly GDP-tubulin subunits. These MTs are less stable and will begin to

depolymerize. Some MTs will depolymerize completely. The released tubulin-GDP dimers will rapidly

exchange GDP for GTP and can then add again to the tip of a growing MT (see Movie 17-3). The instability

of MTs allows the cell to reorganize its cytoskeleton rapidly in response to both extracellular and

intracellular signals. In addition, if a growing MT makes contact with another protein (a capping protein)

that stabilizes the plus end, the MT can be protected from disassembly. This allows the cell the ability to

change its shape and move organelles to specific positions, setting up cell polarity (Figure 17-14).

Drugs that promote the assembly or disassembly of MTs have profound effects on the organization of

MTs, especially MTs of the mitotic spindle, which are especially dynamic (see Table 17-1). Colchicine

(from crocus bulbs) binds tubulin dimers and blocks MT assembly, whereas Taxol (from the Pacific yew)

binds MT polymers and blocks disassembly. Both arrest dividing cells in mitosis. These and other related

anti-mitotic drugs are sometimes used to treat cancer, especially breast and ovarian cancersDuring cell differentiation, various microtubule-associated proteins or MAPs will bind to MTs and modify

them, such as the capping proteins mentioned above, or stabilizing proteins that protect MTs against

disassembly (Figure 17-19). These are especially abundant in post-mitotic cells of the nervous system,

which use MTs to generate their highly polarized cell shapes and to generate and maintain long cellular

processes such as axons and dendrites (Figure 17-17).

All cell types, but especially neurons, depend on MTs to serve as railroad tracks for the movement of

intracellular cargoes (Figure 17-18). Transport along these MTs by motor proteins is much faster and

more efficient than diffusion. Motor proteins use the energy derived from ATP binding and hydrolysis to

move intracellular cargoes. Two large families of MT motor proteins have been described, the kinesins

and the dyneins (Figure 17-20). In general, kinesins move their cargoes toward the plus ends of MTs

(towards the cell periphery), and dyneins move their cargoes toward the minus ends of MTs (towards

the cell center). Each motor contains a globular head domain that binds ATP and the MT track and a tail

domain that binds a specific cargo (Figure 17-20). There are >45 different kinesin motors and 16 different

dynein motors in the human genome, many of which are specialized for specific cargoes in specific tissues.

In addition, MTs and motor proteins play a critical role in the positioning of membrane organelles such as

the ER, Golgi, and other organelles at their appropriate locations (Figures 17-21, 17-22).

The discovery and characterization of motor proteins did not happen until the late 1980s, due to

breakthroughs in computer-enhanced, video light microscopy and its application to the study of

organelle transport in squid axoplasm (see How we know, Figures 17-23 and 17-24, and movies on

website).

Cilia and flagella are membrane-enclosed projections from the cell surface constructed from a stable MT

array known as the 9+2 axoneme. They contain 9 outer doublet microtubules that surround 2 singlet

microtubules known as the central pair. These MTs are associated with >300 accessory proteins that form

several additional structures attached to the microtubules (see Figure 17-27). In general, cilia are shorter,

but they are found in larger numbers on the surfaces of epithelial cells, where they function to move fluids

and/or mucus over the surfaces of the respiratory and reproductive tracts (Figure 17-25). They generate

a 3D waveform that drives movement in one direction to clear particles out of the airway or to move an egg

down the oviduct. Flagella are longer and usually found on a single cell such as sperm, where they propel

the cell through a fluid environment towards an egg using a 2D planar waveform (Figure 17-26).

Movement of cilia and flagella depends on dynein motors firmly attached to the outer doublet

microtubules by their tail domains, such that the MT is itself a cargo. The globular head domains of the

ciliary dyneins reach across to form ATP-driven cross-bridges with the neighboring outer doublet

microtubule (see Figure 17-28). If the doublet MTs were completely free to move, the two outer doublet

MTs would slide apart. The dynein arms on one MT “walk” toward the minus end of the neighboring MT,

and push it towards its plus end. However, other accessory proteins in the axoneme (nexin linkages

and radial spokes) link all the MTs together and limit MT sliding. As a result, the axoneme bends (see

Figure 17-28). Defects in the ciliary dyneins and other axonemal proteins are associated with a disease

known as Kartagener’s syndrome. XY individuals with this disorder are infertile because their sperm

are immotile, and all patients experience chronic respiratory infections because they cannot clear

bacteria and other debris from their airway. In addition, ~50% of the patients exhibit situs inversus

(reversal of the left-right body axis in their internal organs). This is because the movement of cilia in the

early embryo is critical for determining the left-right body axis during vertebrate development. In the

absence of ciliary movement, determination of the body axis is randomActin filaments/microfilaments

Actin filaments are the smallest filaments of the cytoskeleton (~7nm in diameter). They are flexible

strands with great tensile strength. They are especially abundant in the cell cortex where they are

involved in stabilization and movement of the cell membrane. They also form contractile filaments in

nearly all cell types. These contractile filaments are most organized in muscle cells. Actin filaments are

highly dynamic structures (like MTs) but they can be organized and stabilized by a large variety (>200)

actin binding proteins (ABPs) to form different kinds of actin-based networks (Figures 17-29, 17-32).

These include relatively stable structures like microvilli, which increase the surface area for absorption in

the gut, and contractile bundles, which generate and maintain tension in a variety of cells. Actin-based

membrane protrusions can also be very dynamic, such as the finger-like structures known as filopodia or

the sheet-like structures known as lamellipodia on the leading edge of migratory cells. Finally,

contractile rings can form at specific stages in the cell cycle in order to pinch dividing cells into two

daughter cells.

By EM, the actin filaments look relatively uniform, but the globular actin monomers (G-actin) are actually

structurally asymmetric, and they assemble into the filament in a head-to-tail fashion to form structurally

polarized filaments (F-actin). Like MTs, actin filaments have a plus end and a minus end, and the rate of

growth is faster at the plus end than at the minus end (Figures 17-30, 17-31). Each actin monomer

contains a bound ATP molecule. ATP-actin binds more readily to a growing actin filament than ADP-actin

to form an ATP cap. Over time, ATP hydrolysis destabilizes the filament, and filaments with an ADP-cap

will depolymerize (Figures 17-31).

The rates of subunit addition to the plus and minus ends will vary depending on the concentration

of free actin monomers. At very high concentrations, subunits can add to both ends. At more intermediate

concentrations, subunits will add to the plus end faster than the rate of ATP hydrolysis, and the plus end will

grow. However, the rate of subunit addition to the minus end can be slower than the rate of ATP hydrolysis,

and the minus end will shrink. Under these conditions, actin monomers will move through the filament from

the plus end to the minus end in a behavior known as treadmilling (Figure 17-31). The actin filaments are

“dynamic” but their filament lengths may not change very dramatically. However, the nucleotide state of the

actin monomers in the filament make them very sensitive to perturbations brought upon by toxins or the

action of actin binding proteins (ABPs).

Polymerization can be affected by toxins that bind to actin monomers or actin filaments (Table 17-2).

Cytochalasin (from sea sponges) binds to actin monomers and blocks polymerization. Phalloidin (from

mushrooms) binds to actin filaments and prevents depolymerization. Both will freeze cell movement, which

depends on a dynamic equilibrium between monomer and polymer.

About 5% of the total cellular protein is actin (the % is much higher in muscle cells), and about 50% is

monomer and 50% is polymer. The high concentration of monomer is prevented from spontaneous

assembly into filaments by actin binding to monomer sequestering proteins such as thymosin and

profilin. Other actin-binding proteins promote the assembly of filaments at the appropriate time and place

(Figure 17-32). These include the formins (especially abundant in filopodia) and the actin-related proteins

or ARPs (especially abundant in lamellopodia). Formins promote the assembly of relatively long actin

bundles, whereas ARPs promote the assembly of highly branched actin networks (Figure 17-36).

There are many other kinds of actin-binding proteins that bind to polymerized actin filaments and promote

their organization into different structures (Figure 17-32). These include capping proteins, severing

proteins, cross-linking proteins (to form gels), bundling proteins, side-binding proteins, and motor

proteins.

Actin is most highly concentrated in the cell cortex, where actin filaments are linked by ABPs in a

meshwork that supports cell membrane. We discussed a relatively simple cell cortex in red blood cells that

depends on the association of short actin filaments with spectrin and ankryin (Chapter 11). In other celltypes, the cell cortex is typically much thicker and more complex and supports a greater diversity of cell

shape changes and movements. This is most clearly illustrated by the activity of the cell cortex during

cell locomotion, such as nerve growth cone movement during development, cell sheet movement during

development and wound healing, or the migration of white blood cells in response to infection. There

is also a great deal of interest in cell locomotion because of the role that it plays in migration of tumor cells

during cancer metastasis.

There are important and subtle differences in cell locomotion between cell types, but in general, the cell

pushes out protrusions at its front leading edge. Protrusion is typically driven by controlled actin

polymerization gently pushing the plasma membrane forward (Figure 17-34). Many of these protrusion

structures are very dynamic, and they form and retract rapidly (~1  m/sec). The type of protrusion made

depends on the associated actin-binding proteins (Figures 17-34, 17-35).

Once a cell protrusion makes contact with the appropriate target, the proteins in the protrusion reorganize to

form a focal contact (Figure 17-34). The actin filaments are stabilized and organized with other actin

binding proteins (talin, vinculin, alpha-actinin) that link to the filaments to a transmembrane protein known

as integrin. The integrin makes contact on its extracellular surface with proteins located in the extracellular

matrix (Figure 20-15). The stabilized actin filaments can now exert force on the cell by associating with a

motor protein localized in the contractile bundles (Figure 17-34).

The activity of the actin-binding proteins that control the polymerization and organization of the actin

filaments is regulated by extracellular signals that allow the cell to respond rapidly to changes in its

environment. The binding of signaling molecules to transmembrane receptors activates several different

signaling pathways (discussed in Chapter 16). Many of these pathways converge on group of closely-

related GTP-binding proteins known as the Rho protein family (Figure 17-37). The Rho family of

monomeric G-proteins (Rho, Rac, Cdc42) is similar to Ras. Activation stimulates GDP-GTP exchange, and

then the monomeric G-proteins with bound GTP signal to various ABPs to form specific actin networks.

Active Rho promotes the assembly of long, unbranched contractile bundles. Active Rac stimulates ARP

and the formation of lamellipodia. Active Cdc42 stimulates formin and the formation of filopodia.

The motor protein that interacts with actin filaments is myosin. Myosins are members of another large

gene family (>40 genes in the human genome). Your book describes two major subfamilies, myosin-I and

myosin-II. Myosin-II was discovered first, as it is the major form of myosin found in muscle cells and also in

contractile actin networks of non-muscle cells. The myosin-I subfamily was discovered later, and it is much

simpler. Myosin-I molecules contain a single head or motor domain that contains the binding sites for

ATP and actin and a small tail domain that attaches to cargo (Figure 17-33 and Movie 17-8). The tail

domains can attach myosins to vesicles for short-range transport on actin filaments. The tail domains can

also attach myosins to the plasma membrane for movement over cortical actin filaments. Myosins walk

towards the plus ends of the actin filaments, which is the end that is usually anchored into the cell

membrane.

Muscle contraction

Actin filaments and myosin II filaments are the major protein components of muscle cells, including

skeletal, smooth, and cardiac muscle. Again there are important and subtle differences between the

various muscle types, but the mechanism of muscle contraction is fundamentally the same. Skeletal

muscle moves bones and is under voluntary control by motor neurons. Cardiac muscle beats

continuously and spontaneously throughout the lifetime of an individual, but the rate of contraction is under

involuntary control. Smooth muscle underlies the epithelia of many organs and controls the diameter of

blood vessels, peristalsis in the gut, and the contraction of the uterus in response to signals from the

autonomic nervous system and hormones.

Myosin II molecules are dimers with two head or motor domains and a long, coiled-coil tail domain

(Figure 17-38). Tail domains can associate with one another to form a bipolar thick filament. The size ofthe thick filament can vary in different muscle types, but in all cases, there is a bare zone in the middle, and

the heads on either size of the bare zone are oriented in opposite directions. The myosin heads on

either side will walk towards the plus ends of any actin filament that they encounter. Depending on the

arrangement of actin filaments in the cell, the myosin II filaments can cause oppositely oriented actin

filaments to slide past each other (Figure 17-39). In non-muscle cells, the myosin activity can generate

tension in contractile bundles (Figure 17-29) or pinch a dividing cell in two using the contractile ring

(Figure 17-29)

In skeletal muscle, the organization of actin filaments and myosin-II filaments is highly ordered and

designed for rapid contraction. Skeletal muscle cells are generated by cell fusion of precursor cells to

form extremely large cells. As a result, they are post-mitotic and cannot regenerate. (Skeletal muscle

regeneration requires recruitment and activation of precursor cells to make new muscle tissue. The number

of precursor cells is limited). Cell fusion stimulates a transcription factor, MyoD to direct expression of

skeletal muscle proteins that assemble into myofibrils (1-2  m in diameter). Myofibrils are long strings of

contractile proteins organized into sarcomeres. The sarcomere is the smallest unit of contraction, about

2.5  m long, and there are 100-1000s of sarcomeres per myofibril (Figures 17-40, 17-41).

The myosin II thick filament occupies a central position in each sarcomere (Figures 17-41, 17-42). Actin

thin filaments are attached to Z-lines or Z-discs at each end of the sarcomere. When the muscle is

stimulated to contract, the myosin heads on either side of each thick filament start to move on the thin

filaments. Each myosin head moves ~5 nm on the actin filament per molecule of ATP hydrolyzed.

With repeated cross-bridge cycles, the two sets of filaments slide past one another at speeds of 15  m/sec.

The sarcomere shortens in less than 1/10 of a second (Figure 17-42, movie 17.9). Details of the

cross-bridge cycle are shown in Figure 17-43 and Movie 17.10.

Skeletal muscle contraction only occurs in response to the release of acetylcholine from a nerve

terminal and its binding the Ach receptor in the plasma membrane of the skeletal muscle cells (remember

Chapter 12). The resulting action potential spreads quickly in the plasma membrane into a series of

membrane invaginations called the T-Tubules or transverse tubules (Figure 17-44). The T-tubules make

direct physical contact with the endoplasmic reticulum of the skeletal muscle cell known as the

sarcoplasmic reticulum (SR). The action potential stimulates the opening of a voltage gated Ca2+

channel in the T-tubule membrane, and extracellular Ca2+ flows in, binding to a Ca2+ activated release

channel in the sarcoplasmic reticulum (Figure 17-45). Ca2+ is then released from the sarcoplasmic

reticulum into the cytosol of the muscle cell.

The rise in intracellular calcium triggers muscle contraction. In skeletal muscle, Ca2+ binds to

troponin, a molecule distantly related to calmodulin. The tropinin complex interacts with an actin binding

protein called tropomyosin. In the absence of calcium, tropomyosin blocks the interaction between

actin and myosin. However, in the presence of calcium, troponin induces tropomyosin to move on

the actin filament, exposing myosin binding sites (Figure 17-46). Calcium is released near every

sarcomere, and so all sarcomeres contract at the same time. As soon as the nerve signal and action

potential stops, the Ca2+ ATPase in the SR membrane pumps calcium back into the SR. Troponin and

tropomyosin shift their position on the actin filament and block myosin binding, ending contraction.

In smooth muscle and non-muscle cells, a rise in intracellular Ca2+ also stimulates myosin II mediated

contraction, but the mechanism is slightly different. First, sarcomeres are not clearly organized, but

contractile bundles are organized in various orientations. Second, Ca++ binds calmodulin, and calmodulin

binds a kinase called myosin light chain kinase (MLCK). This kinase phosphorylates the myosin II

molecule and activates it, allowing it to interact with actin. Contraction is terminated by myosin

dephosphorylation. This type of regulation is slower, because it depends on cycles of phosphorylation and

dephosphorylation. However, it can be driven by a variety of different signals that influence intracellular

calcium levels (remember chapter 16!)Cardiac muscle uses sarcomeres, calcium, and the troponin/tropomyosin system to control sarcomere

contraction, similar to skeletal muscle. However, the speed of contraction can be modified by inputs from

the autonomic nervous system that also alter myosin phosphorylation and activity. Defects in sarcomere

proteins can lead to different forms of cardiomyopathy. These are often associated with sudden death

in young athletes