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Cell_Biology_Alberts. C. Jacobs-Wagner, Cell 115:705–713, 2003. With permission from Elsevier.) accessory proteins cross-link and bundle the filaments together, making large-Figure 16–11 The structures of an actin monomer and actin filament. (A) The scale actin structures that are much more rigid than an individual actin filament. ATP or ADP) bound in a deep cleft in the Nucleation Is the Rate-Limiting Step in the Formation of Actin center of the molecule. (B) Arrangement of two protofilaments, held together by lateral contacts, which wind around each


Cell_Biology_Alberts

Cell_Biology_Alberts. The regulation of actin filament formation is an important mechanism by which other as two parallel strands of a helix, cells control their shape and movement. Small oligomers of actin subunits can with a twist repeating every 37 nm. All the assemble spontaneously, but they are unstable and disassemble readily because subunits within the filament have the same each monomer is bound to only one or two other monomers. For a new actin fila-orientation. (C) Electron micrograph of ment to form, subunits must assemble into an initial aggregate, or nucleus, that is negatively stained actin filament. (C, courtesy of Roger Craig.) stabilized by multiple subunit–subunit contacts and can then elongate rapidly by addition of more subunits. This process is called filament nucleation.


Cell_Biology_Alberts

Cell_Biology_Alberts. (C, courtesy of Roger Craig.) stabilized by multiple subunit–subunit contacts and can then elongate rapidly by addition of more subunits. This process is called filament nucleation. Many features of actin nucleation and polymerization have been studied with purified actin in a test tube (Figure 16–13). The instability of smaller actin aggregates creates a kinetic barrier to nucleation. When polymerization is initiated, this barrier results in a lag phase during which no filaments are observed. During this lag phase, however, a few of the small, unstable aggregates succeed in making the transition to a more stable form that resembles an actin filament. This leads to a 0.5 µm


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 16–12 Structural polarity of the actin filament. (A) This electron micrograph shows an actin filament polymerized from a short actin filament seed that was decorated with myosin motor domains, resulting in an arrowhead pattern. The filament has grown much faster at the barbed (plus) end than at the pointed (minus) end. (B) Enlarged image and model showing the arrowhead pattern. (A, courtesy of Tom Pollard; B, adapted from M. Whittaker, B.O. Carragher and K.A. Milligan, Ultramicro. 54:245–260, 1995.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 16–13 The time course of actin polymerization in a test tube. (A) Polymerization of pure actin subunits into filaments occurs after a lag phase. (B) Polymerization occurs more rapidly in the presence of preformed fragments of actin filaments, which act as nuclei for filament growth. As indicated, the % free subunits after polymerization reflects the critical concentration (Cc), at which there is no net change in polymer. Actin polymerization is often studied by observing the change in the light emission from a fluorescent probe, called pyrene, that has been covalently attached to the actin. Pyrene-actin fluoresces more brightly when it is incorporated into actin filaments.


Cell_Biology_Alberts

Cell_Biology_Alberts. phase of rapid filament elongation during which subunits are added quickly to the ends of the nucleated filaments (Figure 16–13A). Finally, as the concentration of actin monomers declines, the system approaches a steady state at which the rate of addition of new subunits to the filament ends exactly balances the rate of subunit dissociation. The concentration of free subunits left in solution at this point is called the critical concentration, Cc. As explained in Panel 16–2, the value of the critical concentration is equal to the rate constant for subunit loss divided by the rate constant for subunit addition—that is, Cc = koff/kon, which is equal to the dissociation constant, Kd, and the inverse of the equilibrium constant, K (see Figure 3–44). In a test tube, the Cc for actin polymerization—that is, the free actin monomer concentration at which the fraction of actin in the polymer stops increasing—is about 0.2 μM. Inside the cell, the concentration of unpolymerized actin is much


Cell_Biology_Alberts

Cell_Biology_Alberts. is, the free actin monomer concentration at which the fraction of actin in the polymer stops increasing—is about 0.2 μM. Inside the cell, the concentration of unpolymerized actin is much higher than this, and the cell has evolved mechanisms to prevent most of its monomeric actin from assembling into filaments, as we discuss later.


Cell_Biology_Alberts

Cell_Biology_Alberts. The lag phase in filament growth is eliminated if preexisting seeds (such as fragments of actin filaments that have been chemically cross-linked) are added to the solution at the beginning of the polymerization reaction (Figure 16–13B). The cell takes great advantage of this nucleation requirement: it uses special proteins to catalyze filament nucleation at specific sites, thereby determining the location at which new actin filaments are assembled.


Cell_Biology_Alberts

Cell_Biology_Alberts. Due to the uniform orientation of asymmetric actin subunits in the filament, the structures at its two ends are different. This orientation makes the two ends of each polymer different in ways that have a profound effect on filament growth rates. The kinetic rate constants for actin subunit association and dissociation— kon and koff, respectively—are much greater at the plus end than the minus end. This can be seen when an excess of purified actin monomers is allowed to assemble onto polarity-marked filaments—the plus end of the filament elongates up to ten times faster (see Figure 16–12). If filaments are rapidly diluted so that the free subunit concentration drops below the critical concentration, the plus end also depolymerizes faster.


Cell_Biology_Alberts

Cell_Biology_Alberts. It is important to note, however, that the two ends of an actin filament have the same net affinity for actin subunits, if all of the subunits are in the same nucleotide state. Addition of a subunit to either end of a filament of n subunits results in a filament of n + 1 subunits. Thus, the free-energy difference, and therefore the equilibrium constant (and the critical concentration), must be the same for addition of subunits at either end of the polymer. In this case, the ratio of the rate constants, koff/kon, must be identical at the two ends, even though the absolute values of these rate constants are very different at each end (see Panel 16–2).


Cell_Biology_Alberts

Cell_Biology_Alberts. The cell takes advantage of actin filament dynamics and polarity to do mechanical work. Filament elongation proceeds spontaneously when the free-energy change (∆G) for addition of the soluble subunit is less than zero. This is the case when the concentration of subunits in solution exceeds the critical concentration. A cell can couple an energetically unfavorable process to this spontaneous process; thus, the cell can use free energy released during spontaneous filament polymerization to move an attached load. For example, by orienting the fast-growing plus ends of actin filaments toward its leading edge, a motile cell can push its plasma membrane forward, as we discuss later. ATP Hydrolysis Within Actin Filaments Leads to Treadmilling at Steady State


Cell_Biology_Alberts

Cell_Biology_Alberts. ATP Hydrolysis Within Actin Filaments Leads to Treadmilling at Steady State Thus far in our discussion of actin filament dynamics, we have ignored the critical fact that actin can catalyze the hydrolysis of the nucleoside triphosphate ATP. For free actin subunits, this hydrolysis proceeds very slowly; however, it is accelerated when the subunits are incorporated into filaments. Shortly after ATP hydrolysis occurs, the free phosphate group is released from each subunit, but the ADP remains trapped in the filament structure. Thus, two different types of filament structures can exist, one with the “T form” of the nucleotide bound (ATP), and one with the “D form” bound (ADP).


Cell_Biology_Alberts

Cell_Biology_Alberts. When the nucleotide is hydrolyzed, much of the free energy released by cleavage of the phosphate–phosphate bond is stored in the polymer. This makes the free-energy change for dissociation of a subunit from the D-form polymer more negative than the free-energy change for dissociation of a subunit from the T-form polymer. Consequently, the ratio of koff/kon for the D-form polymer, which is numerically equal to its critical concentration [Cc(D)], is larger than the corresponding ratio for the T-form polymer. Thus, Cc(D) is greater than Cc(T). At certain concentrations of free subunits, D-form polymers will therefore shrink while T-form polymers grow.


Cell_Biology_Alberts

Cell_Biology_Alberts. In living cells, most soluble actin subunits are in the T form, as the free concentration of ATP is about tenfold higher than that of ADP. However, the longer the time that subunits have been in the actin filament, the more likely they are to have hydrolyzed their ATP. Whether the subunit at each end of a filament is in the T or the D form depends on the rate of this hydrolysis compared with the rate of subunit addition. If the concentration of actin monomers is greater than the critical concentration for both the T-form and D-form polymer, then subunits will add to the polymer at both ends before the nucleotides in the previously added subunits are hydrolyzed; as a result, the tips of the actin filament will remain in the T form. On the other hand, if the subunit concentration is less than the critical concentrations for both the T-form and D-form polymer, then hydrolysis may occur before the next subunit is added and both ends of the filament will be in the D form and will shrink.


Cell_Biology_Alberts

Cell_Biology_Alberts. the critical concentrations for both the T-form and D-form polymer, then hydrolysis may occur before the next subunit is added and both ends of the filament will be in the D form and will shrink. At intermediate concentrations of actin subunits, it is possible for the rate of subunit addition to be faster than nucleotide hydrolysis at the plus end, but slower than nucleotide hydrolysis at the minus end. In this case, the plus end of the filament remains in the T conformation, while the minus end adopts the D conformation. The filament then undergoes a net addition of subunits at the plus end, while simultaneously losing subunits from the minus end. This leads to the remarkable property of filament treadmilling (Figure 16–14; see Panel 16–2).


Cell_Biology_Alberts

Cell_Biology_Alberts. At a particular intermediate subunit concentration, the filament growth at the plus end exactly balances the filament shrinkage at the minus end. Under these conditions, the subunits cycle rapidly between the free and filamentous states,


Cell_Biology_Alberts

Cell_Biology_Alberts. ON RATES AND OFF RATES A linear polymer of protein molecules, such as an actin flament or a microtubule, assembles (polymerizes) and disassembles (depolymerizes) by the addition and removal of subunits at the ends of the polymer. The rate of addition of these subunits (called monomers) is given by the rate constant kon, which has units of M–1 sec–1. The rate of loss is given by koff (units of sec–1). subunit + polymer (with n subunits) polymer (with n +1 subunits) kon koff NUCLEATION A helical polymer is stabilized by multiple contacts between adjacent subunits. In the case of actin, two actin molecules bind relatively weakly to each other, but addition of a third actin monomer to form a trimer makes the entire group more stable. Further monomer addition can take place onto this trimer, which therefore acts as a nucleus for polymerization. For tubulin, the nucleus is larger and has a more complicated structure (possibly a ring of 13 or more tubulin molecules)—but the principle is the


Cell_Biology_Alberts

Cell_Biology_Alberts. therefore acts as a nucleus for polymerization. For tubulin, the nucleus is larger and has a more complicated structure (possibly a ring of 13 or more tubulin molecules)—but the principle is the same. The assembly of a nucleus is relatively slow, which explains the lag phase seen during polymerization. The lag phase can be reduced or abolished entirely by adding premade nuclei, such as fragments of already polymerized microtubules or actin flaments. THE CRITICAL CONCENTRATION The number of monomers that add to the polymer (actin flament or microtubule) per second will be proportional to the concentration of the free subunit (konC), but the subunits will leave the polymer end at a constant rate (koff) that does not depend on C. As the polymer grows, subunits are used up, and C is observed to drop until it reaches a constant value, called the critical concentration (Cc). At this concentration, the rate of subunit addition equals the rate of subunit loss. At this equilibrium, kon C =


Cell_Biology_Alberts

Cell_Biology_Alberts. to drop until it reaches a constant value, called the critical concentration (Cc). At this concentration, the rate of subunit addition equals the rate of subunit loss. At this equilibrium, kon C = koff so that Cc = (where Kd is the dissociation constant; see Figure 3–44). koff kon = TIME COURSE OF POLYMERIZATION The assembly of a protein into a long helical polymer such as a cytoskeletal flament or a bacterial fagellum typically shows the following time course: The lag phase corresponds to time taken for nucleation. The growth phase occurs as monomers add to the exposed ends of the growing flament, causing flament elongation. The equilibrium phase, or steady state, is reached when the growth of the polymer due to monomer addition precisely balances the shrinkage of the polymer due to disassembly back to monomers. time amount of polymerLAG PHASE GROWTH PHASE EQUILIBRIUM PHASE PLUS AND MINUS ENDS The two ends of an actin flament or microtubule polymerize at different rates. The


Cell_Biology_Alberts

Cell_Biology_Alberts. to disassembly back to monomers. time amount of polymerLAG PHASE GROWTH PHASE EQUILIBRIUM PHASE PLUS AND MINUS ENDS The two ends of an actin flament or microtubule polymerize at different rates. The fast-growing end is called the plus end, whereas the slow-growing end is called the minus end. The difference in the rates of growth at the two ends is made possible by changes in the conformation of each subunit as it enters the polymer. free subunit subunit in polymer SLOW FAST minus end plus end This conformational change affects the rates at which subunits add to the two ends. Even though kon and koff will have different values for the plus and minus ends of the polymer, their ratio koff/kon—and hence Cc—must be the same at both ends for a simple polymerization reaction (no ATP or GTP hydrolysis). This is because exactly the same subunit interactions are broken when a subunit is lost at either end, and the fnal state of the subunit after dissociation is identical. Therefore, the ˜G for


Cell_Biology_Alberts

Cell_Biology_Alberts. This is because exactly the same subunit interactions are broken when a subunit is lost at either end, and the fnal state of the subunit after dissociation is identical. Therefore, the ˜G for subunit loss, which determines the equilibrium constant for its association with the end, is identical at both ends: if the plus end grows four times faster than the minus end, it must also shrink four times faster. Thus, for C > Cc, both ends grow; for C < Cc, both ends shrink. The nucleoside triphosphate hydrolysis that accompanies actin and tubulin polymerization removes this constraint. Kd PANEL 16–2: The Polymerization of Actin and Tubulin 902


Cell_Biology_Alberts

Cell_Biology_Alberts. Each actin molecule carries a tightly bound ATP molecule that is hydrolyzed to a tightly bound ADP molecule soon after its assembly into the polymer. Similarly, each tubulin molecule carries a tightly bound GTP that is converted to a tightly bound GDP molecule soon after the molecule assembles into the polymer. Hydrolysis of the bound nucleotide reduces the binding affnity of the subunit for neighboring subunits and makes it more likely to dissociate from each end of the flament (see Figure 16–44 for a possible mechanism). It is usually the form that adds to the flament and the form that leaves. Considering events at the plus end only: As before, the polymer will grow until C = Cc. For illustrative purposes, we can ignore kD and kToff since they are usually very small, so that polymer growth ceases when


Cell_Biology_Alberts

Cell_Biology_Alberts. As before, the polymer will grow until C = Cc. For illustrative purposes, we can ignore kD and kToff since they are usually very small, so that polymer growth ceases when This is a steady state and not a true equilibrium, because the ATP or GTP that is hydrolyzed must be replenished by a nucleotide exchange reaction of the free subunit ( ). One consequence of the nucleotide hydrolysis that accompanies polymer formation is to change the critical concentration at the two ends of the polymer. Since kDoff and kT refer to different reactions, their ratio on kDoff/kTon need not be the same at both ends of the polymer, so that:


Cell_Biology_Alberts

Cell_Biology_Alberts. Thus, if both ends of a polymer are exposed, polymerization proceeds until the concentration of free monomer reaches a value that is above Cc for the plus end but below Cc for the minus end. At this steady state, subunits undergo a net assembly at the plus end and a net disassembly at the minus end at an identical rate. The polymer maintains a constant length, even though there is a net fux of subunits through the polymer, known as treadmilling. Microtubules depolymerize about 100 times faster from an end containing GDP-tubulin than from one containing GTP-tubulin. A GTP cap favors growth, but if it is lost, then depolymerization ensues. The rate of addition of subunits to a can be faster than the rate at which their bound nucleotide is hydrolyzed. Under such conditions, the end has a “cap” of subunits containing the nucleoside flament or a GTP cap on a microtubule.


Cell_Biology_Alberts

Cell_Biology_Alberts. DYNAMIC INSTABILITY and TREADMILLING are two behaviors observed in cytoskeletal polymers. Both are associated with nucleoside triphosphate hydrolysis. Dynamic instability is believed to predominate in microtubules, whereas treadmilling may predominate in actin flaments. Individual microtubules can therefore alternate between a period of slow growth and a period of rapid disassembly, a phenomenon called dynamic instability. Figure 16–14 Treadmilling of an actin soluble subunits are in T form ( filament, made possible by the ATP hydrolysis that follows subunit addition.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 16–14 Treadmilling of an actin soluble subunits are in T form ( filament, made possible by the ATP hydrolysis that follows subunit addition. polymers are a mixture of T form ( (A) Explanation for the different critical concentrations (Cc) at the plus and minus at D minus a growing filament, and then undergo ends. Subunits with bound ATP (T-form nucleotide hydrolysis within the filament. As minus-end addition is slow— plus-end addition is fast— while the total length of the filament remains unchanged. This “steady-state tread-milling” requires a constant consumption of energy in the form of ATP hydrolysis. The Functions of Actin Filaments Are Inhibited by Both Polymer-stabilizing and Polymer-destabilizing Chemicals


Cell_Biology_Alberts

Cell_Biology_Alberts. The Functions of Actin Filaments Are Inhibited by Both Polymer-stabilizing and Polymer-destabilizing Chemicals Chemical compounds that stabilize or destabilize actin filaments are important tools in studies of the filaments’ dynamic behavior and function in cells. The cytochalasins are fungal products that prevent actin polymerization by binding to the plus end of actin filaments. Latrunculin prevents actin polymerization by binding to actin subunits. The phalloidins are toxins isolated from the Amanita mushroom that bind tightly all along the side of actin filaments and stabilize them against depolymerization. All of these compounds cause dramatic changes in the actin cytoskeleton and are toxic to cells, indicating that the function of actin filaments depends on a dynamic equilibrium between filaments and actin monomers (Table 16–1).


Cell_Biology_Alberts

Cell_Biology_Alberts. In a test tube, polymerization of actin is controlled simply by its concentration, as described above, and by pH and the concentrations of salts and ATP. Within a cell, however, actin behavior is also regulated by numerous accessory proteins that bind actin monomers or filaments (summarized in Panel 16–3). At steady state the filament grows, elongation is faster than hydrolysis at the plus end in this example, and the terminal subunits at this end are therefore always in the T form. However, hydrolysis is faster than elongation at the minus end, and so terminal subunits at this end are in the D form. (B) Treadmilling occurs at intermediate concentrations of free subunits. The critical concentration for polymerization on a filament end in the T form is lower than for a filament end in the D form. If the actual subunit concentration is somewhere between these two values, the plus end grows while the minus end shrinks, resulting in treadmilling.


Cell_Biology_Alberts

Cell_Biology_Alberts. nucleates assembly associated with the growing to form a web and remains plus end associated with the minus end binds subunits, prevents assembly proflin binds subunits, – + binds ADP-actin flaments, binds to plus end flament bundling, cross-linking, and attachment to membranes Some of the major accessory proteins of the actin cytoskeleton. Except for the myosin motor proteins, an example of each major type is shown. Each of these is discussed in the text. However, most cells contain more than a hundred different actin-binding proteins, and it is likely that there are important types of actin-associated proteins that are not yet recognized.


Cell_Biology_Alberts

Cell_Biology_Alberts. in vitro, when the monomer concentration is 0.2 μM, filament half-life, a measure of how long an individual actin monomer spends in a filament as it treadmills, is approximately 30 minutes. In a non-muscle vertebrate cell, actin half-life in filaments is only 30 seconds, demonstrating that cellular factors modify the dynamic behavior of actin filaments. Actin-binding proteins dramatically alter actin filament dynamics and organization through spatial and temporal control of monomer availability, filament nucleation, elongation, and depolymerization. In the following sections, we describe the ways in which these accessory proteins modify actin function in the cell.


Cell_Biology_Alberts

Cell_Biology_Alberts. In most non-muscle vertebrate cells, approximately 50% of the actin is in filaments and 50% is soluble—and yet the soluble monomer concentration is 50–200 μM, well above the critical concentration. Why does so little of the actin polymerize into filaments? The reason is that the cell contains proteins that bind to the actin monomers and make polymerization much less favorable (an action similar to that of the drug latrunculin). A small protein called thymosin is the most abundant of these proteins. Actin monomers bound to thymosin are in a locked state, where they cannot associate with either the plus or minus ends of actin filaments and can neither hydrolyze nor exchange their bound nucleotide.


Cell_Biology_Alberts

Cell_Biology_Alberts. How do cells recruit actin monomers from this buffered storage pool and use them for polymerization? The answer depends on another monomer-binding protein called profilin. Profilin binds to the face of the actin monomer opposite the ATP-binding cleft, blocking the side of the monomer that would normally associate with the filament minus end, while leaving exposed the site on the monomer that binds to the plus end (Figure 16–15). When the profilin–actin complex binds a free plus end, a conformational change in actin reduces its affinity for profilin and the profilin falls off, leaving the actin filament one subunit longer. Profilin competes with thymosin for binding to individual actin monomers. Thus, by regulating the local activity of profilin, cells can control the movement of actin subunits from the sequestered thymosin-bound pool onto filament plus ends.


Cell_Biology_Alberts

Cell_Biology_Alberts. Several mechanisms regulate profilin activity, including profilin phosphorylation and profilin binding to inositol phospholipids. These mechanisms can define the sites where profilin acts. For example, profilin is required for filament assembly at the plasma membrane, where it is recruited by an interaction with acidic membrane phospholipids. At this location, extracellular signals can activate profilin to produce local actin polymerization and the extension of actin-rich motile structures such as filopodia and lamellipodia.


Cell_Biology_Alberts

Cell_Biology_Alberts. In addition to the availability of active actin subunits, a second prerequisite for cellular actin polymerization is filament nucleation. Proteins that contain actin monomer binding motifs linked in tandem mediate the simplest mechanism of filament nucleation. These actin-nucleating proteins bring several actin subunits together to form a seed. In most cases, actin nucleation is catalyzed by one of two different types of factors: the Arp 2/3 complex or the formins. The first of these is a complex of proteins that includes two actin-related proteins, or ARPs, each of which is about 45% identical to actin. The Arp 2/3 complex nucleates actin filament growth from the minus end, allowing rapid elongation at the plus end (Figure 16–16A and B). The complex can attach to the side of another actin filament while remaining bound to the minus end of the filament that it has nucleated, thereby building individual filaments into a treelike web (Figure 16–16C and D).


Cell_Biology_Alberts

Cell_Biology_Alberts. Formins are dimeric proteins that nucleate the growth of straight, unbranched filaments that can be cross-linked by other proteins to form parallel bundles. Each formin subunit has a binding site for monomeric actin, and the formin dimer appears to nucleate actin filament polymerization by capturing two monomers.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 16–15 Effects of thymosin and profilin on actin polymerization. An actin monomer bound to thymosin is sterically prevented from binding to and elongating the plus end of an actin filament (left). An actin monomer bound to profilin, on the other hand, is capable of elongating a filament (right). Thymosin and profilin cannot both bind to a single actin monomer at the same time. In a cell in which most of the actin monomer is bound to thymosin, the activation of a small amount of profilin can produce rapid filament assembly. As indicated (bottom), profilin binds to actin monomers that are transiently released from the thymosin-bound monomer pool, shuttles them onto the plus ends of actin filaments, and is then released and recycled for further rounds of filament elongation.


Cell_Biology_Alberts

Cell_Biology_Alberts. As the newly nucleated filament grows, the formin dimer remains associated with the rapidly growing plus end while still allowing the addition of new subunits at that end (Figure 16–17). This mechanism of filament assembly is clearly different from that used by the Arp 2/3 complex, which remains stably bound to the filament minus end, preventing subunit addition or loss at that end. Formin-dependent actin filament growth is strongly enhanced by the association of actin monomers with profilin (Figure 16–18). Like profilin activation, actin filament nucleation by Arp 2/3 complexes and formins occurs primarily at the plasma membrane, and the highest density of actin filaments in most cells is at the cell periphery. The layer just beneath the plasma membrane is called the cell cortex, and the actin filaments in this region determine the shape and movement of the cell surface, allowing the cell to change its shape and stiffness rapidly in response to changes in its external environment.


Cell_Biology_Alberts

Cell_Biology_Alberts. Actin filament behavior is regulated by two major classes of binding proteins: those that bind along the side of a filament and those that bind to the ends (see Panel 16–3). Side-binding proteins include tropomyosin, an elongated protein that binds simultaneously to six or seven adjacent actin subunits along each of the two grooves of the helical actin filament. In addition to stabilizing and stiffening 908 Chapter 16: The Cytoskeleton the filament, the binding of tropomyosin can prevent the actin filament from interacting with other proteins; this aspect of tropomyosin function is important in the control of muscle contraction, as we discuss later. An actin filament that stops growing and is not specifically stabilized in the cell will depolymerize rapidly, particularly at its plus end, once the actin molecules


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 16–16 Nucleation and actin web formation by the Arp 2/3 complex. (A) The structures of Arp2 and Arp3 compared to the structure of actin. Although the face of the molecule equivalent to the plus end (top) in both Arp2 and Arp3 is very similar to the plus end of actin itself, differences on the sides and minus endprevent these actin-related proteins from forming filaments on their own or coassembling into filaments with actin. (B) A model for actin filament nucleation by the Arp 2/3 complex. In the absence of an activating factor, Arp2 and Arp3 are held by their accessory proteins in an orientation that prevents them from nucleating a new actin filament. When an activating factor (indicated by the blue triangle) binds the complex, Arp2 and Arp3 are brought together into a new configuration that resembles the plus end of an actin filament. Actin subunits can then assemble onto this structure, bypassing the rate-limiting step of filament nucleation. (C) The Arp 2/3 complex


Cell_Biology_Alberts

Cell_Biology_Alberts. configuration that resembles the plus end of an actin filament. Actin subunits can then assemble onto this structure, bypassing the rate-limiting step of filament nucleation. (C) The Arp 2/3 complex nucleates filaments most efficiently when it is bound to the side of a preexisting actin filament. The result is a filament branch that grows at a 70° angle relative to the original filament. Repeated rounds of branching nucleation result in a treelike web of actin filaments. (D) Top, electron micrographs of branched actin filaments formed by mixing purified actin subunits with purified Arp 2/3 complexes. Bottom, reconstructed image of a branch where the crystal structures of actin (pink) and the Arp 2/3 complex have been fitted to the electron density. The mother filament runs from top to bottom, and the daughter filament branches off to the right where the Arp 2/3 complex binds to three actin subunits in the mother filament. (D, top, from R.D. Mullins et al., Proc. Natl Acad. Sci. USA


Cell_Biology_Alberts

Cell_Biology_Alberts. and the daughter filament branches off to the right where the Arp 2/3 complex binds to three actin subunits in the mother filament. (D, top, from R.D. Mullins et al., Proc. Natl Acad. Sci. USA 95:6181–6186, 1998, with permission from National Academy of Sciences; botttom, from N. Volkmann et al., Science 293:2456–2459, 2001, with permission from AAAS.) have hydrolyzed their ATP. The binding of plus-end capping protein (also called CapZ for its location in the muscle Z band) stabilizes an actin filament at its plus end by rendering it inactive, greatly reducing the rates of filament growth and depolymerization (Figure 16–19). At the minus end, an actin filament may be capped by the Arp 2/3 complex that was responsible for its nucleation, although many minus ends in a typical cell are released from the Arp 2/3 complex and are uncapped.


Cell_Biology_Alberts

Cell_Biology_Alberts. Tropomodulin, best known for its function in the capping of exceptionally long-lived actin filaments in muscle, binds tightly to the minus ends of actin filaments that have been coated and thereby stabilized by tropomyosin. It can also transiently cap pure actin filaments and significantly reduce their elongation and depolymerization rates. A large family of tropomodulin proteins regulates actin filament length and stability in many cell types. For maximum effect, proteins that bind the side of actin filaments coat the filament completely, and must therefore be present in high amounts. In contrast, end-binding proteins can affect filament dynamics even when they are present at very low levels. Since subunit addition and loss occur primarily at filament ends, one molecule of an end-binding protein per actin filament (roughly one molecule per 200–500 actin subunits) can be enough to transform the architecture of an actin filament network.


Cell_Biology_Alberts

Cell_Biology_Alberts. Another important mechanism of actin filament regulation depends on proteins that break an actin filament into many smaller filaments, thereby generating a large number of new filament ends. The fate of these new ends depends on the presence of other accessory proteins. Under some conditions, newly formed ends nucleate filament elongation, thereby accelerating the assembly of new filament structures. Under other conditions, severing promotes the depolymerization of old filaments, speeding up the depolymerization rate by tenfold or more. In addition, severing changes the physical and mechanical properties of the cytoplasm: stiff, large bundles and gels become more fluid.


Cell_Biology_Alberts

Cell_Biology_Alberts. One class of actin-severing proteins is the gelsolin superfamily. These proteins are activated by high levels of cytosolic Ca2+. Gelsolin interacts with the side of the actin filament and contains subdomains that bind to two different sites: one that is exposed on the surface of the filament and one that is hidden between adjacent Figure 16–18 Profilin and formins. Some members of the formin protein family have unstructured domains or “whiskers” that contain several binding sites for profilin or the profilin–actin complex. These flexible domains serve as a staging area for addition of actin to the growing plus end of the actin filament when formin is bound. Under some conditions, this can enhance the rate of actin filament elongation so that filament growth is faster than that expected for a diffusion-controlled reaction, and faster in the presence of formin and profilin than the rate for pure actin alone (see also Figure 3–78).


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Cell_Biology_Alberts. Figure 16–17 Actin elongation mediated by formins. Formin proteins (green) form a dimeric complex that can nucleate the formation of a new actin filament (red) and remain associated with the rapidly growing plus end as it elongates. The formin protein maintains its binding to one of the two actin subunits exposed at the plus end as it allows each new subunit to assemble. Only part of the large dimeric formin molecule is shown here. Other regions regulate its activity and link it to particular structures in the cell. Many formins are indirectly connected to the cell plasma membrane and aid the insertional polymerization of the actin filament directly beneath the membrane surface. continued rapid growth of actin flament at plus end uncapped population of flaments: growth at plus and minus ends capped population of


Cell_Biology_Alberts

Cell_Biology_Alberts. continued rapid growth of actin flament at plus end uncapped population of flaments: growth at plus and minus ends capped population of Figure 16–19 Filament capping and its effects on filament dynamics. A population of uncapped filaments adds and loses subunits at both the plus and minus ends, resulting in rapid growth or shrinkage, depending on the concentration of available free monomers (green line). In the presence of a protein that caps the plus end (red line), only the minus end is able to add or lose subunits; consequently, flaments: growth filament growth will be slower at all at minus end only monomer concentrations above the critical concentration, and filament shrinkage will be slower at all monomer concentrations below the critical concentration. In addition, the critical concentration for the population shifts to that of the filament minus end.


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Cell_Biology_Alberts. subunits. According to one model, gelsolin binds the side of an actin filament until a thermal fluctuation creates a small gap between neighboring subunits, at which point gelsolin inserts itself into the gap to break the filament. After the severing event, gelsolin remains attached to the actin filament and caps the new plus end. Another important actin-filament destabilizing protein, found in all eukaryotic cells, is cofilin. Also called actin depolymerizing factor, cofilin binds along the length of the actin filament, forcing the filament to twist a little more tightly (Figure 16–20). This mechanical stress weakens the contacts between actin subunits in the filament, making the filament brittle and more easily severed by thermal motions, generating filament ends that undergo rapid disassembly. As a result, most of the actin filaments inside cells are shorter lived than are filaments formed from pure actin in a test tube.


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Cell_Biology_Alberts. Cofilin binds preferentially to ADP-containing actin filaments rather than to ATP-containing filaments. Since ATP hydrolysis is usually slower than filament assembly, the newest actin filaments in the cell still contain mostly ATP and are resistant to depolymerization by cofilin. Cofilin therefore tends to dismantle the older filaments in the cell. As we will discuss later, the cofilin-mediated disassembly of old but not new actin filaments is critical for the polarized, directed growth of the actin network that is responsible for unidirectional cell crawling and the intracellular motility of pathogens. Actin filaments can be protected from cofilin by tropomyosin binding. Thus, the dynamics of actin in different subcellular locations depends on the balance of stabilizing and destabilizing accessory proteins.


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Cell_Biology_Alberts. Figure 16–20 Twisting of an actin filament induced by cofilin. (A) Three-dimensional reconstruction from cryoelectron micrographs of filaments made of pure actin. The bracket shows the span of two twists of the actin helix. (B) Reconstruction of an actin filament coated with cofilin, which binds in a 1:1 stoichiometry to actin subunits all along the filament. Cofilin is a small protein (14 kD) compared to actin (43 kD), and so the filament appears only slightly thicker. The energy of cofilin binding serves to deform the actin filament, twisting it more tightly and reducing the distance spanned by each twist of the helix. (From A. McGough et al., J. Cell Biol. 138:771–781, 1997. With permission from the authors.)


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Cell_Biology_Alberts. Actin filaments in animal cells are organized into several types of arrays: dendritic networks, bundles, and weblike (gel-like) networks (Figure 16–21). Different structures are initiated by the action of distinct nucleating proteins: the actin filaments of dendritic networks are nucleated by the Arp 2/3 complex, while bundles are made of the long, straight filaments produced by formins. The proteins nucleating the filaments in the gel-like networks are not yet well defined.


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Cell_Biology_Alberts. The structural organization of different actin networks depends on specialized accessory proteins. As explained earlier, Arp 2/3 organizes filaments into dendritic networks by attaching filament minus ends to the side of other filaments. Other actin filament structures are assembled and maintained by two classes of proteins: bundling proteins, which cross-link actin filaments into a parallel array, and gel-forming proteins, which hold two actin filaments together at a large angle to each other, thereby creating a looser meshwork. Both bundling and gel-forming proteins generally have two similar actin-filament-binding sites, which can either be part of a single polypeptide chain or contributed by each of two polypeptide chains held together in a dimer (Figure 16–22). The spacing and arrangement of these two filament-binding domains determine the type of actin structure that a given cross-linking protein forms.


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Cell_Biology_Alberts. Each type of bundling protein also determines which other molecules can interact with the cross-linked actin filaments. Myosin II is the motor protein that enables stress fibers and other contractile arrays to contract. The very close packing of actin filaments caused by the small monomeric bundling protein fimbrin apparently excludes myosin, and thus the parallel actin filaments held together by fimbrin are not contractile. On the other hand, α-actinin cross-links oppositely Figure 16–21 Actin arrays in a cell.


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Cell_Biology_Alberts. Figure 16–21 Actin arrays in a cell. A fibroblast crawling in a tissue-culture dish is shown with four areas enlarged to show the arrangement of actin filaments. The actin filaments are shown in red, with arrowheads pointing toward the minus end. Stress fibers are contractile and exert tension. The actin cortex underlies the plasma membrane and consists of gel-like networks or dendritic actin networks that enable membrane protrusion at lamellopodia. Filopodia are spike-like projections of the plasma membrane that allow a cell to explore its environment.


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Cell_Biology_Alberts. Figure 16–22 The modular structures of four actin-cross-linking proteins. Each of the proteins shown has two actin-binding sites (red) that are related in sequence. Fimbrin has two directly adjacent actinbinding sites, so that it holds its two actin filaments very close together (14 nm apart), aligned with the same polarity (see Figure 16–23A). The two actin-binding sites in α-actinin are separated by a spacer around 30 nm long, so that it forms more loosely packed actin bundles (see Figure 16–23A). Filamin has two actin-binding sites with a V-shaped linkage between them, so that it cross-links actin filaments into a network with the filaments oriented almost at right angles to one another (see Figure 16–24). Spectrin is a tetramer of two α and two β subunits, and the tetramer has two actinbinding sites spaced about 200 nm apart (see Figure 10–38).


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Cell_Biology_Alberts. polarized actin filaments into loose bundles, allowing the binding of myosin and formation of contractile actin bundles (Figure 16–23). Because of the very different spacing and orientation of the actin filaments, bundling by fimbrin automatically discourages bundling by α-actinin, and vice versa, so that the two types of bundling protein are mutually exclusive.


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Cell_Biology_Alberts. The bundling proteins that we have discussed so far have straight, stiff connections between their two actin-filament-binding domains. Other actin cross-linking proteins have either a flexible or a stiff, bent connection between their two binding domains, allowing them to form actin filament webs or gels, rather than actin bundles. Filamin (see Figure 16–22) promotes the formation of a loose and highly viscous gel by clamping together two actin filaments roughly at right angles (Figure 16–24A). Cells require the actin gels formed by filamin to extend the thin, sheetlike membrane projections called lamellipodia that help them to crawl across solid surfaces. In humans, mutations in the filamin A gene cause defects in nerve-cell migration during early embryonic development. Cells in the periventricular region of the brain fail to migrate to the cortex and instead form nodules, causing a syndrome called periventricular heterotopia (Figure 16–24B). Interestingly, in addition to binding


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Cell_Biology_Alberts. region of the brain fail to migrate to the cortex and instead form nodules, causing a syndrome called periventricular heterotopia (Figure 16–24B). Interestingly, in addition to binding actin, filamins have been reported to interact with a large number of cellular proteins of great functional diversity, including membrane receptors for signaling molecules, and filamin mutations can also lead to defects in development of bone, the cardiovascular system, and other organs. Thus, filamins may also function as signaling scaffolds by connecting and coordinating a wide variety of cellular processes with the actin cytoskeleton.


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Cell_Biology_Alberts. A very different, well-studied web-forming protein is spectrin, which was first identified in red blood cells. Spectrin is a long, flexible protein made out of four elongated polypeptide chains (two α subunits and two β subunits), arranged so that the two actin-filament-binding sites are about 200 nm apart (compared with 14 nm for fimbrin and about 30 nm for α-actinin; see Figure 16–23). In the red blood cell, spectrin is concentrated just beneath the plasma membrane, where it forms a two-dimensional weblike network held together by short actin filaments whose precise lengths are tightly regulated by capping proteins at each end; spectrin links this web to the plasma membrane because it has separate binding sites for peripheral membrane proteins, which are themselves positioned near the lipid bilayer by integral membrane proteins (see Figure 10–38). The resulting network creates a strong, yet flexible cell cortex that provides mechanical support for the overlying plasma membrane,


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Cell_Biology_Alberts. lipid bilayer by integral membrane proteins (see Figure 10–38). The resulting network creates a strong, yet flexible cell cortex that provides mechanical support for the overlying plasma membrane, allowing the red blood cell to spring back to its original shape after squeezing through a capillary. Close relatives of spectrin are found in the cortex of most other vertebrate cell types, where they also help to shape and stiffen the surface membrane. A particularly striking example of spectrin’s role


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Cell_Biology_Alberts. Figure 16–23 The formation of two types of actin filament bundles. (A) Fimbrin cross-links actin filaments into tight bundles, which exclude the motor protein myosin II from participating in the assembly. In contrast, α-actinin, which is a homodimer, cross-links actin filaments into loose bundles, which allow myosin (not shown) to incorporate into the bundle. Fimbrin and α-actinin tend to exclude one another because of the very different spacing of the actin filament bundles that they form. (B) Electron micrograph of purified α-actinin molecules. (B, courtesy of John Heuser.) in promoting mechanical stability is the long, thin axon of neurons in the nematode worm Caenorhabditis elegans, where spectrin is required to keep them from breaking during the twisting motions the worms make during crawling.


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Cell_Biology_Alberts. The connections of the cortical actin cytoskeleton to the plasma membrane are only partially understood. Members of the ERM family (named for its first three members, ezrin, radixin, and moesin), help organize membrane domains through their ability to interact with transmembrane proteins and the underlying cytoskeleton. In so doing, they not only provide structural links to strengthen the cell cortex, but also regulate the activities of signal transduction pathways. Moesin also increases cortical stiffness to promote cell rounding during mitosis. Measurements by atomic force microscopy indicate that the cell cortex remains soft during mitosis when moesin is depleted. ERM proteins are thought to bind to and organize the cortical actin cytoskeleton in a variety of contexts, thereby affecting the shape and stiffness of the membrane as well as the localization and activity of signaling molecules. Bacteria Can Hijack the Host Actin Cytoskeleton


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Cell_Biology_Alberts. Bacteria Can Hijack the Host Actin Cytoskeleton The importance of accessory proteins in actin-based motility and force production is illustrated beautifully by studies of certain bacteria and viruses that use components of the host-cell actin cytoskeleton to move through the cytoplasm. The cytoplasm of mammalian cells is extremely viscous, containing organelles and cytoskeletal elements that inhibit diffusion of large particles like bacteria or viruses. To move around in a cell and invade neighboring cells, several pathogens, including Listeria monocytogenes (which causes a rare but serious form of food poisoning), overcome this problem by recruiting and activating the Arp 2/3 complex at their surface. The Arp 2/3 complex nucleates the assembly of actin filaments that generate a substantial force and push the bacterium through the


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Cell_Biology_Alberts. Figure 16–24 Filamin cross-links actin filaments into a three-dimensional network and is required for normal neuronal migration. (A) Each filamin homodimer is about 160 nm long when fully extended and forms a flexible, high-angle link between two adjacent actin filaments. A set of actin filaments cross-linked by filamin forms a mechanically strong web or gel. (B) Magnetic resonance imaging of a normal human brain (left) and of a patient with periventricular heterotopia (right) caused by mutation in the filamin A gene. In contrast to the smooth ventricular surface in the normal brain, a rough zone of cortical neurons (arrowheads) is seen along the lateral walls of the ventricles, representing neurons that have failed to migrate to the cortex during brain development. Remarkably, although many neurons are not in the right place, the intelligence of affected individuals is frequently normal or only mildly compromised, and the major clinical syndrome is epilepsy that often starts in the


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Cell_Biology_Alberts. many neurons are not in the right place, the intelligence of affected individuals is frequently normal or only mildly compromised, and the major clinical syndrome is epilepsy that often starts in the second decade of life. (B, adapted from


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Cell_Biology_Alberts. Y. Feng and C.A. Walsh, Nat. Cell Biol. 6:1034–1038, 2004. With permission from Macmillan Publishers Ltd.)


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Cell_Biology_Alberts. Figure 16–25 The actin-based movement of Listeria monocytogenes. (A) Fluorescence micrograph of an infected cell that has been stained to reveal bacteria in red and actin filaments in green. Note the cometlike tail of actin filaments behind each moving bacterium. Regions of overlap between red and green fluorescence appear yellow. (B) Listeria motility can be reconstituted in a test tube with ATP and just four purified proteins: actin, Arp 2/3 complex, capping protein, and cofilin. This micrograph shows the dense actin tails behind bacteria (black). (C) The ActA protein on the bacterial surface activates the Arp 2/3 complex to nucleate new filament assembly along the sides of existing filaments. Filaments grow at their plus end until capped by capping protein. Actin is recycled through the action of cofilin, which enhances depolymerization at the minus ends of the filaments. By this mechanism, polymerization is focused at the rear surface of the bacterium, propelling it forward (see


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Cell_Biology_Alberts. action of cofilin, which enhances depolymerization at the minus ends of the filaments. By this mechanism, polymerization is focused at the rear surface of the bacterium, propelling it forward (see Movie 23.7). (A, courtesy of Julie Theriot and Tim Mitchison; B, from T.P. Loisel et al., Nature 401:613–616, 1999. With permission from Macmillan Publishers Ltd.) cytoplasm at rates of up to 1 μm/sec, leaving behind a long actin “comet tail” (Figure 16–25; see also Figures 23–28 and 23–29). This motility can be reconstituted in a test tube by adding the bacteria to a mixture of pure actin, Arp 2/3 complex, cofilin, and capping protein, illustrating how actin polymerization dynamics generate movement through spatial regulation of filament assembly and disassembly. As we shall see, actin-based movement of this sort also underlies membrane protrusion at the leading edge of motile cells.


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Cell_Biology_Alberts. Actin is a highly conserved cytoskeletal protein that is present in high concentrations in nearly all eukaryotic cells. Nucleation presents a kinetic barrier to actin polymerization, but once formed, actin filaments undergo dynamic behavior due to hydrolysis of the bound nucleotide ATP. Actin filaments are polarized and can undergo treadmilling when a filament assembles at the plus end while simultaneously depolymerizing at the minus end. In cells, actin filament dynamics are regulated at every step, and the varied forms and functions of actin depend on a versatile repertoire of accessory proteins. Approximately half of the actin is kept in a monomeric form through association with sequestering proteins such as thymosin. Nucleation factors such as the Arp 2/3 complex and formins promote formation of branched and parallel filaments, respectively. Interplay between proteins that bind or cap actin filaments and those that promote filament severing or depolymerization can slow or


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Cell_Biology_Alberts. formation of branched and parallel filaments, respectively. Interplay between proteins that bind or cap actin filaments and those that promote filament severing or depolymerization can slow or accelerate the kinetics of filament assembly and disassembly. Another class of accessory proteins assembles the filaments into larger ordered structures by cross-linking them to one another in geometrically defined ways. Connections between these actin arrays and the plasma membrane of cells give an animal cell mechanical strength and permit the elaboration of cortical cellular structures such as lamellipodia, filopodia, and microvilli. By inducing actin filament polymerization at their surface, intracellular pathogens can hijack the host-cell cytoskeleton and move around inside the cell.


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Cell_Biology_Alberts. (A) light chains coiled-coil of two ˜ helices 100 nm N-terminus (B) Figure 16–26 Myosin II. (A) The two globular heads and long tail of a myosin II molecule shadowed with platinum can be seen in this electron micrograph. (B) A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long; green) and four light chains (blue). The light chains are of two distinct types, and one copy of each type is present on each myosin head. Dimerization occurs when the two α helices of the heavy chains wrap around each other to form a coiled-coil, driven by the association of regularly spaced hydrophobic amino acids (see Figure 3–9). The coiled-coil arrangement makes an extended rod in solution, and this part of the molecule forms the tail. (A, courtesy of David Shotton.)


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Cell_Biology_Alberts. A crucial feature of the actin cytoskeleton is that it can form contractile structures that cross-link and slide actin filaments relative to one another through the action of myosin motor proteins. In addition to driving muscle contraction, actin–myosin assemblies perform important functions in non-muscle cells. Actin-Based Motor Proteins Are Members of the Myosin Superfamily


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Cell_Biology_Alberts. Actin-Based Motor Proteins Are Members of the Myosin Superfamily The first motor protein to be identified was skeletal muscle myosin, which generates the force for muscle contraction. This protein, now called myosin II, is an elongated protein formed from two heavy chains and two copies of each of two light chains. Each heavy chain has a globular head domain at its N-terminus that contains the force-generating machinery, followed by a very long amino acid sequence that forms an extended coiled-coil that mediates heavy-chain dimerization (Figure 16–26). The two light chains bind close to the N-terminal head domain, while the long coiled-coil tail bundles itself with the tails of other myosin molecules. These tail–tail interactions form large, bipolar “thick filaments” that have several hundred myosin heads, oriented in opposite directions at the two ends of the thick filament (Figure 16–27).


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Cell_Biology_Alberts. Figure 16–27 The myosin II bipolar thick filament in muscle. (A) Electron micrograph of a myosin II thick filament isolated from frog muscle. Note the central bare zone, which is free of head domains. (B) Schematic diagram, not drawn to scale. The myosin II molecules aggregate by means of their tail regions, with their heads projecting to the outside of the filament. The bare zone in the center of the filament consists entirely of myosin II tails. (C) A small section of a myosin II filament as reconstructed from electron micrographs. An individual myosin molecule is highlighted in green. The cytoplasmic myosin II filaments in non-muscle cells are much smaller, although similarly organized (see Figure 16–39). (A, courtesy of Murray Stewart; C, based on R.A. Crowther, R. Padrón and R. Craig, J. Mol. Biol. 184:429–439, 1985. With permission from Academic Press.)


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Cell_Biology_Alberts. Each myosin head binds and hydrolyzes ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament (Figure 16–28). The opposing orientation of the heads in the thick filament makes the filament efficient at sliding pairs of oppositely oriented actin filaments toward each other, shortening the muscle. In skeletal muscle, in which carefully arranged actin filaments are aligned in “thin filament” arrays surrounding the myosin thick filaments, the ATP-driven sliding of actin filaments results in a powerful contraction. Cardiac and smooth muscle contain myosin II molecules that are similarly arranged, although different genes encode them. Myosin Generates Force by Coupling ATP Hydrolysis to Conformational Changes


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Cell_Biology_Alberts. Myosin Generates Force by Coupling ATP Hydrolysis to Conformational Changes Motor proteins use structural changes in their ATP-binding sites to produce cyclic interactions with a cytoskeletal filament. Each cycle of ATP binding, hydrolysis, and release propels them forward in a single direction to a new binding site along the filament. For myosin II, each step of the movement along actin is generated by the swinging of an 8.5-nm-long α helix, or lever arm, which is structurally stabilized by the binding of light chains. At the base of this lever arm next to the head, there is a pistonlike helix that connects movements at the ATP-binding cleft in the head to small rotations of the so-called converter domain. A small change at this point can swing the helix like a long lever, causing the far end of the helix to move by about 5.0 nm.


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Cell_Biology_Alberts. These changes in the conformation of the myosin are coupled to changes in its binding affinity for actin, allowing the myosin head to release its grip on the actin filament at one point and snatch hold of it again at another. The full mechanochemical cycle of nucleotide binding, nucleotide hydrolysis, and phosphate release (which causes the “power stroke”) produces a single step of movement (Figure 16–29). At low ATP concentrations, the interval between the force-producing step and the binding of the next ATP is long enough that single steps can be observed (Figure 16–30). Sliding of Myosin II Along Actin Filaments Causes Muscles to Contract Muscle contraction is the most familiar and best-understood form of movement in animals. In vertebrates, running, walking, swimming, and flying all depend on the rapid contraction of skeletal muscle on its scaffolding of bone, while involuntary


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Cell_Biology_Alberts. Figure 16–28 Direct evidence for the motor activity of the myosin head. In this experiment, purified myosin heads were attached to a glass slide, and then actin filaments labeled with fluorescent phalloidin were added and allowed to bind to the myosin heads. (A) When ATP was added, the actin filaments began to glide along the surface, owing to the many individual steps taken by each of the dozens of myosin heads bound to each filament. The video frames shown in this sequence were recorded about 0.6 second apart; the two actin filaments shown (one red and one green) were moving in opposite directions at a rate of about 4 μm/sec. (B) Diagram of the experiment. The large red arrows indicate the direction of actin filament movement (Movie 16.2). (A, courtesy of James Spudich.)


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Cell_Biology_Alberts. ATTACHED At the start of the cycle shown in this fgure, plus a myosin head lacking a bound nucleotide is locked end tightly onto an actin flament in a rigor confguration (so named because it is responsible for rigor mortis, the rigidity of death). In an actively contracting muscle, this state is very short-lived, being rapidly terminated by the binding of a molecule of ATP. RELEASED A molecule of ATP binds to the large cleft on the “back” of the head (that is, on the side furthest from the actin flament) and immediately causes a slight change in the conformation of the actin-binding site, reducing the affnity of the head for actin and allowing it to move along the flament. (The space drawn here between the head and actin emphasizes this change, although in reality the head probably remains very close to the actin.)


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Cell_Biology_Alberts. COCKED The cleft closes like a clam shell around the ATP molecule, triggering a movement in the lever arm that causes the head to be displaced along the flament by a distance of about 5 nm. Hydrolysis of ATP occurs, but the ADP and inorganic phosphate (Pi) remain tightly bound to the protein. FORCE-GENERATING Weak binding of the myosin head to a new site on the actin flament causes release of the inorganic phosphate produced by ATP hydrolysis, concomitantly with the tight binding of the head to actin. This release triggers the power stroke—the force-generating change in shape during which the head regains its original conformation. In the course of the power stroke, the head loses its bound ADP, thereby returning to the start of a new cycle. ATTACHED At the end of the cycle, the myosin head is again locked tightly to the actin flament in a rigor confguration. Note that the head has moved to a new position on the actin flament.


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Cell_Biology_Alberts. ATTACHED At the end of the cycle, the myosin head is again locked tightly to the actin flament in a rigor confguration. Note that the head has moved to a new position on the actin flament. Figure 16–29 The cycle of structural changes used by myosin II to walk along an actin filament. In the myosin II cycle, the head remains bound to the actin filament for only about 5% of the entire cycle time, allowing many myosins to work together to move a single actin filament (Movie 16.3). (Based on I. Rayment et al., Science 261:50–58, 1993.) movements such as heart pumping and gut peristalsis depend on the contraction of cardiac muscle and smooth muscle, respectively. All these forms of muscle contraction depend on the ATP-driven sliding of highly organized arrays of actin filaments against arrays of myosin II filaments.


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Cell_Biology_Alberts. Skeletal muscle was a relatively late evolutionary development, and muscle cells are highly specialized for rapid and efficient contraction. The long, thin muscle fibers of skeletal muscle are actually huge single cells that form during 918 Chapter 16: The Cytoskeleton development by the fusion of many separate cells. The large muscle cell retains the many nuclei of the contributing cells. These nuclei lie just beneath the plasma membrane (Figure 16–31). The bulk of the cytoplasm inside is made up of myofibrils, which is the name given to the basic contractile elements of the muscle cell. A myofibril is a cylindrical structure 1–2 μm in diameter that is often as long as the muscle cell itself. It consists of a long, repeated chain of tiny contractile units— called sarcomeres, each about 2.2 μm long—which give the vertebrate myofibril its striated appearance (Figure 16–32).


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Cell_Biology_Alberts. Each sarcomere is formed from a miniature, precisely ordered array of parallel and partly overlapping thin and thick filaments. The thin filaments are composed of actin and associated proteins, and they are attached at their plus ends to a Z disc at each end of the sarcomere. The capped minus ends of the actin filaments extend in toward the middle of the sarcomere, where they overlap with thick filaments, the bipolar assemblies formed from specific muscle isoforms of myosin II (see Figure 16–27). When this region of overlap is examined in cross section by electron microscopy, the myosin filaments are arranged in a regular hexagonal lattice, with the actin filaments evenly spaced between them (Figure 16–33). Cardiac muscle and smooth muscle also contain sarcomeres, although the organization is not as regular as that in skeletal muscle.


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Cell_Biology_Alberts. Figure 16–31 Skeletal muscle cells (also called muscle fibers). (A) These huge multinucleated cells form by the fusion of many muscle cell precursors, called myoblasts. Here, a single muscle cell is depicted. In an adult human, a muscle cell is typically 50 μm in diameter and can be up to several centimeters long. (B) Fluorescence micrograph of rat muscle, showing the peripherally located nuclei (blue) in these giant cells. Myofibrils are stained red. (B, courtesy of Nancy L. Kedersha.)


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Cell_Biology_Alberts. Figure 16–30 The force of a single myosin molecule moving along an actin filament measured using an optical trap. (A) Schematic of the experiment, showing an actin filament with beads attached at both ends and held in place by focused beams of light called optical tweezers (Movie 16.4). The tweezers trap and move the bead, and can also be used to measure the force exerted on the bead through the filament. In this experiment, the filament was positioned over another bead to which myosin II motors were attached, and the optical tweezers were used to determine the effects of myosin binding on movement of the actin filament.


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Cell_Biology_Alberts. These traces show filament movement in two separate experiments. Initially, when the actin filament is unattached to myosin, thermal motion of the filament produces noisy fluctuations in filament position. When a single myosin binds to the actin filament, thermal motion decreases abruptly and a roughly 10-nm displacement results from movement of the filament by the motor. The motor then releases the filament. Because the ATP concentration is very low in this experiment, the myosin remains attached to the actin filament for much longer than it would in a muscle cell. (Adapted from C. Rüegg et al., Physiology 17:213–218, 2002. With permission from the American Physiological Society.)


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Cell_Biology_Alberts. Figure 16–32 Skeletal muscle myofibrils. (A) Low-magnification electron micrograph of a longitudinal section through a skeletal muscle cell of a rabbit, showing the regular pattern of cross-striations. The cell contains many myofibrils aligned in parallel (see Figure 16–31). (B) Detail of the skeletal muscle shown in (A), showing portions of two adjacent myofibrils and the definition of a sarcomere (black arrow). (C) Schematic diagram of a single sarcomere, showing the origin of the dark and light bands seen in the electron micrographs. The Z discs, at each end of the sarcomere, are attachment sites for the plus ends of actin filaments (thin filaments); the M line, or midline, is the location of proteins that link adjacent myosin II filaments (thick filaments) to one another. (D) When the sarcomere contracts, the actin and myosin filaments slide past one another without shortening. (A and B, courtesy of Roger Craig.)


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Cell_Biology_Alberts. Sarcomere shortening is caused by the myosin filaments sliding past the actin thin filaments, with no change in the length of either type of filament (see Figure 16–32C and D). Bipolar thick filaments walk toward the plus ends of two sets of thin filaments of opposite orientations, driven by dozens of independent myosin heads that are positioned to interact with each thin filament. Because there is no coordination among the movements of the myosin heads, it is critical that they remain tightly bound to the actin filament for only a small fraction of each ATPase cycle so that they do not hold one another back. Each myosin thick filament has about 300 heads (294 in frog muscle), and each head cycles about five times per second in the course of a rapid contraction—sliding the myosin and actin filaments past one another at rates of up to 15 μm/sec and enabling the sarcomere to shorten by 10% of its length in less than one-fiftieth of a second. The rapid synchronized shortening of the


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Cell_Biology_Alberts. actin filaments past one another at rates of up to 15 μm/sec and enabling the sarcomere to shorten by 10% of its length in less than one-fiftieth of a second. The rapid synchronized shortening of the thousands of sarcomeres lying end-to-end in each myofibril enables skeletal muscle to contract rapidly enough for running and flying, or for playing the piano.


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Cell_Biology_Alberts. Accessory proteins produce the remarkable uniformity in filament organization, length, and spacing in the sarcomere (Figure 16–34). The actin filament plus ends are anchored in the Z disc, which is built from CapZ and α-actinin; the Z disc caps the filaments (preventing depolymerization), while holding them together in a regularly spaced bundle. The precise length of each thin filament is influenced by a protein of enormous size, called nebulin, which consists almost entirely of a repeating 35-amino-acid actin-binding motif. Nebulin stretches from the Z disc toward the minus end of each thin filament, which is capped and stabilized by tropomodulin. Although there is some slow exchange of actin subunits at both ends of the muscle thin filament, such that the components of the thin filament turn over with a half-life of several days, the actin filaments in sarcomeres are remarkably stable compared with those found in most other cell types, whose dynamic actin filaments turn over with


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Cell_Biology_Alberts. turn over with a half-life of several days, the actin filaments in sarcomeres are remarkably stable compared with those found in most other cell types, whose dynamic actin filaments turn over with half-lives of a few minutes or less.


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Cell_Biology_Alberts. Figure 16–33 Electron micrographs of an insect flight muscle viewed in cross section. The myosin and actin filaments are packed together with almost crystalline regularity. Unlike their vertebrate counterparts, these myosin filaments have a hollow center, as seen in the enlargement on the right. The geometry of the hexagonal lattice is slightly different in vertebrate muscle. (From J. Auber, J. de Microsc. 8:197–232, 1969. With permission from Societé Française de Microscopie Électronique.) plus end minus end of actin nebulin actin (thin flament)


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Cell_Biology_Alberts. J. de Microsc. 8:197–232, 1969. With permission from Societé Française de Microscopie Électronique.) plus end minus end of actin nebulin actin (thin flament) Opposing pairs of an even longer template protein, called titin, position the thick filaments midway between the Z discs. Titin acts as a molecular spring, with a long series of immunoglobulin-like domains that can unfold one by one as stress is applied to the protein. A springlike unfolding and refolding of these domains keeps the thick filaments poised in the middle of the sarcomere and allows the muscle fiber to recover after being overstretched. In C. elegans, whose sarcomeres are longer than those in vertebrates, titin is longer as well, suggesting that it serves also as a molecular ruler, determining in this case the overall length of each sarcomere.


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Cell_Biology_Alberts. The force-generating molecular interaction between myosin thick filaments and actin thin filaments takes place only when a signal passes to the skeletal muscle from the nerve that stimulates it. Immediately upon arrival of the signal, the muscle cell needs to be able to contract very rapidly, with all the sarcomeres shortening simultaneously. Two major features of the muscle cell make extremely rapid contraction possible. First, as previously discussed, the individual myosin motor heads in each thick filament spend only a small fraction of the ATP cycle time bound to the filament and actively generating force, so many myosin heads can act in rapid succession on the same thin filament without interfering with one another. Second, a specialized membrane system relays the incoming signal rapidly throughout the entire cell. The signal from the nerve triggers an action potential in the muscle cell plasma membrane (discussed in Chapter 11), and this electrical excitation spreads swiftly


Cell_Biology_Alberts

Cell_Biology_Alberts. rapidly throughout the entire cell. The signal from the nerve triggers an action potential in the muscle cell plasma membrane (discussed in Chapter 11), and this electrical excitation spreads swiftly into a series of membranous folds—the transverse tubules, or T tubules—that extend inward from the plasma membrane around each myofibril. The signal is then relayed across a small gap to the sarcoplasmic reticulum, an adjacent weblike sheath of modified endoplasmic reticulum that surrounds each myofibril like a net stocking (Figure 16–35A and B).


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Cell_Biology_Alberts. When the incoming action potential activates a Ca2+ channel in the T-tubule membrane, Ca2+ influx triggers the opening of Ca2+-release channels in the sarcoplasmic reticulum (Figure 16–35C). Ca2+ flooding into the cytosol then initiates the contraction of each myofibril. Because the signal from the muscle cell plasma membrane is passed within milliseconds (via the T tubules and sarcoplasmic reticulum) to every sarcomere in the cell, all of the myofibrils in the cell contract at once. The increase in Ca2+ concentration is transient because the Ca2+ is rapidly pumped back into the sarcoplasmic reticulum by an abundant, ATP-dependent Ca2+-pump (also called a Ca2+-ATPase) in its membrane (see Figure 11–13). Typically, the cytoplasmic Ca2+ concentration is restored to resting levels within 30 msec, allowing the myofibrils to relax. Thus, muscle contraction depends on two processes that consume enormous amounts of ATP: filament sliding, driven by the ATPase of the myosin motor domain, and


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Cell_Biology_Alberts. msec, allowing the myofibrils to relax. Thus, muscle contraction depends on two processes that consume enormous amounts of ATP: filament sliding, driven by the ATPase of the myosin motor domain, and Ca2+ pumping, driven by the Ca2+pump.


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Cell_Biology_Alberts. Figure 16–34 Organization of accessory proteins in a sarcomere. Each giant titin molecule extends from the Z disc to the M line—a distance of over 1 μm. Part of each titin molecule is closely associated with a myosin thick filament (which switches polarity at the M line); the rest of the titin molecule is elastic and changes length as the sarcomere contracts and relaxes. Each nebulin molecule is exactly the length of a thin filament. The actin filaments are also coated with tropomyosin and troponin (not shown; see Figure 16–36) and are capped at both ends. Tropomodulin caps the minus end of the actin filaments, and CapZ anchors the plus end at the Z disc, which also contains α-actinin (not shown).


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Cell_Biology_Alberts. The Ca2+-dependence of vertebrate skeletal muscle contraction, and hence its dependence on commands transmitted via nerves, is due entirely to a set of specialized accessory proteins that are closely associated with the actin thin filaments. One of these accessory proteins is a muscle form of tropomyosin, the elongated protein that binds along the groove of the actin filament helix. The other is troponin, a complex of three polypeptides, troponins T, I, and C (named for their tropomyosin-binding, inhibitory, and Ca2+-binding activities, respectively). Troponin I binds to actin as well as to troponin T. In a resting muscle, the troponin I–T complex pulls the tropomyosin out of its normal binding groove into a position along the actin filament that interferes with the binding of myosin heads, thereby preventing any force-generating interaction. When the level of Ca2+ is raised, troponin C—which binds up to four molecules of Ca2+—causes troponin I to release its hold on actin. This


Cell_Biology_Alberts

Cell_Biology_Alberts. heads, thereby preventing any force-generating interaction. When the level of Ca2+ is raised, troponin C—which binds up to four molecules of Ca2+—causes troponin I to release its hold on actin. This allows the tropomyosin molecules to slip back into their normal position so that the myosin heads can walk along the actin filaments (Figure 16–36). Troponin C is closely related to the ubiquitous Ca2+-binding protein calmodulin (see Figure 15–33); it can be thought of as a specialized form of calmodulin that has acquired binding sites for troponin I and troponin T, thereby ensuring that the myofibril responds extremely rapidly to an increase in Ca2+ concentration.


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Cell_Biology_Alberts. In smooth muscle cells, so called because they lack the regular striations of skeletal muscle, contraction is also triggered by an influx of calcium ions, but the regulatory mechanism is different. Smooth muscle forms the contractile portion of the stomach, intestine, and uterus, as well as the walls of arteries and many other structures requiring slow and sustained contractions. Smooth muscle is 0.5 µm


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Cell_Biology_Alberts. Figure 16–35 T tubules and the sarcoplasmic reticulum. (A) Drawing of the two membrane systems that relay the signal to contract from the muscle cell plasma membrane to all of the myofibrils in the cell. (B) Electron micrograph showing a cross section of a T tubule. Note the position of the large Ca2+-release channels in the sarcoplasmic reticulum membrane that connect to the adjacent T-tubule membrane. (C) Schematic diagram showing how a Ca2+-release channel in the sarcoplasmic reticulum membrane is thought to be opened by the activation of a voltage-gated Ca2+ channel (Movie 16.5). (B, courtesy of Clara Franzini-Armstrong.)


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Cell_Biology_Alberts. Figure 16–36 The control of skeletal muscle contraction by troponin. (A) A skeletal-muscle-cell thin filament, showing the positions of tropomyosin and troponin along the actin filament. Each tropomyosin molecule has seven evenly spaced regions with similar amino acid sequences, each of which is thought to bind to an actin subunit in the filament. (B) Reconstructed cryoelectron microscopy image of an actin filament showing the relative position of a superimposed tropomyosin strand in the presence (dark purple) or absence (light purple) of calcium. (A, adapted from G.N. Phillips, J.P. Fillers and C. Cohen, J. Mol. Biol. 192:111–131, 1986. With permission from Academic Press; B, adapted from C. Xu et al., Biophys. J. 77: 985–992, 1999. With permission from Elsevier.) composed of sheets of highly elongated spindle-shaped cells, each with a single nucleus. Smooth muscle cells do not express the troponins. Instead, elevated intracellular Ca2+ levels regulate contraction by a mechanism that


Cell_Biology_Alberts

Cell_Biology_Alberts. elongated spindle-shaped cells, each with a single nucleus. Smooth muscle cells do not express the troponins. Instead, elevated intracellular Ca2+ levels regulate contraction by a mechanism that depends on calmodulin (Figure 16–37). Ca2+-bound calmodulin activates myosin light-chain kinase (MLCK), thereby inducing the phosphorylation of smooth muscle myosin on one of its two light chains. When the light chain is phosphorylated, the myosin head can interact with actin filaments and cause contraction; when it is dephosphorylated, the myosin head tends to dissociate from actin and becomes inactive.


Cell_Biology_Alberts

Cell_Biology_Alberts. The phosphorylation events that regulate contraction in smooth muscle cells occur relatively slowly, so that maximum contraction often requires nearly a second (compared with the few milliseconds required for contraction of a skeletal muscle cell). But rapid activation of contraction is not important in smooth


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Cell_Biology_Alberts. Figure 16–37 Smooth muscle contraction. (A) Upon muscle stimulation by activation of cell-surface receptors, Ca2+ released into the cytoplasm from the sarcoplasmic reticulum (SR) binds to calmodulin (see Figure 15–29). Ca2+-bound calmodulin then binds myosin light-chain kinase (MLCK), which phosphorylates myosin light chain, stimulating myosin activity. Non-muscle myosin is regulated by the same mechanism (see Figure 16–39). (B) Smooth muscle cells in a cross section of cat intestinal wall. The outer layer of smooth muscle is oriented with the long axis of its cells extending parallel along the length of the intestine, and upon contraction will shorten the intestine. The inner layer is oriented circularly around the intestine and when contracted will cause the intestine to become narrower. Contraction of both layers squeezes material through the intestine, much like squeezing toothpaste out of a tube.


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Cell_Biology_Alberts. (C) A model for the contractile apparatus in a smooth muscle cell, with bundles of contractile filaments containing actin and myosin (red) oriented obliquely to the long axis of the cell. Their contraction greatly shortens the cell. Only a few of the many bundles are shown. (B, courtesy of Gwen V. Childs.) muscle: its myosin II hydrolyzes ATP about 10 times more slowly than skeletal muscle myosin, producing a slow cycle of myosin conformational changes that results in slow contraction. The heart is the most heavily worked muscle in the body, contracting about 3 billion (3 × 109) times during the course of a human lifetime (Movie 16.6). Heart cells express several specific isoforms of cardiac muscle myosin and cardiac muscle actin. Even subtle changes in these cardiac-specific contractile proteins—changes that would not cause any noticeable consequences in other tissues—can cause serious heart disease (Figure 16–38).


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Cell_Biology_Alberts. The normal cardiac contractile apparatus is such a highly tuned machine that a tiny abnormality anywhere in the works can be enough to gradually wear it down over years of repetitive motion. Familial hypertrophic cardiomyopathy is a common cause of sudden death in young athletes. It is a genetically dominant inherited condition that affects about two out of every thousand people, and it is associated with heart enlargement, abnormally small coronary vessels, and disturbances in heart rhythm (cardiac arrhythmias). The cause of this condition is either any one of over 40 subtle point mutations in the genes encoding cardiac β myosin heavy chain (almost all causing changes in or near the motor domain) or one of about a dozen mutations in other genes encoding contractile proteins— including myosin light chains, cardiac troponin, and tropomyosin. Minor mis-sense mutations in the cardiac actin gene cause another type of heart condition, called dilated cardiomyopathy, which can also result in


Cell_Biology_Alberts

Cell_Biology_Alberts. light chains, cardiac troponin, and tropomyosin. Minor mis-sense mutations in the cardiac actin gene cause another type of heart condition, called dilated cardiomyopathy, which can also result in early heart failure.


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Cell_Biology_Alberts. Actin and Myosin Perform a Variety of Functions in Non-Muscle Cells Most non-muscle cells contain small amounts of contractile actin–myosin II bundles that form transiently under specific conditions and are much less well organized than muscle fibers. Non-muscle contractile bundles are regulated by myosin phosphorylation rather than the troponin mechanism (Figure 16–39). These contractile bundles function to provide mechanical support to cells, for example, by assembling into cortical stress fibers that connect the cell to the extracellular bipolar flament of 15–20 molecules


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Cell_Biology_Alberts. Figure 16–38 Effect on the heart of a subtle mutation in cardiac myosin. Left, normal heart from a 6-day-old mouse pup. Right, heart from a pup with a point mutation in both copies of its cardiac myosin gene, changing Arg403 to Gln. The arrows indicate the atria. In the heart from the pup with the cardiac myosin mutation, both atria are greatly enlarged (hypertrophic), and the mice die within a few weeks of birth. (From D. Fatkin et al., J. Clin. Invest. 103:147–153, 1999. With permission from The American Society for Clinical Investigation.)


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Cell_Biology_Alberts. Figure 16–39 Light-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments. (A) The controlled phosphorylation by the enzyme myosin light-chain kinase (MLCK) of one of the two light chains (the so-called regulatory light chain, shown in light blue) on non-muscle myosin II in a test tube has at least two effects: it causes a change in the conformation of the myosin head, exposing its actinbinding site, and it releases the myosin tail from a “sticky patch” on the myosin head, thereby allowing the myosin molecules to assemble into short, bipolar, thick filaments. Smooth muscle is regulated by the same mechanism (see Figure 16–37). (B) Electron micrograph of negatively stained short filaments of myosin II that have been induced to assemble in a test tube by phosphorylation of their light chains. These myosin II filaments are much smaller than those found in skeletal muscle cells (see Figure 16–27). (B, courtesy of John Kendrick-Jones.) matrix through


Cell_Biology_Alberts

Cell_Biology_Alberts. by phosphorylation of their light chains. These myosin II filaments are much smaller than those found in skeletal muscle cells (see Figure 16–27). (B, courtesy of John Kendrick-Jones.) matrix through focal adhesions or by forming a circumferential belt in an epithelial cell, connecting it to adjacent cells through adherens junctions (discussed in Chapter 19). As described in Chapter 17, actin and myosin II in the contractile ring generate the force for cytokinesis, the final stage in cell division. Finally, as discussed later, contractile bundles also contribute to the adhesion and forward motion of migrating cells.


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Cell_Biology_Alberts. Non-muscle cells also express a large family of other myosin proteins, which have diverse structures and functions in the cell. Following the discovery of conventional muscle myosin, a second member of the family was found in the freshwater amoeba Acanthamoeba castellanii. This protein had a different tail structure and seemed to function as a monomer, and so it was named myosin I (for one-headed). Conventional muscle myosin was renamed myosin II (for two-headed). Subsequently, many other myosin types were discovered. The heavy chains generally start with a recognizable myosin motor domain at the N-terminus and then diverge widely with a variety of C-terminal tail domains (Figure 16–40). The myosin family includes a number of one-headed and two-headed varieties that are about equally related to myosin I and myosin II, and the nomenclature now reflects their approximate order of discovery (myosin III through at least myosin XVIII). Sequence comparisons among diverse eukaryotes indicate


Cell_Biology_Alberts

Cell_Biology_Alberts. to myosin I and myosin II, and the nomenclature now reflects their approximate order of discovery (myosin III through at least myosin XVIII). Sequence comparisons among diverse eukaryotes indicate that there are at least 37 distinct myosin families in the superfamily. All of the myosins except one move toward the plus end of an actin filament, although they do so at different speeds. The exception is myosin VI, which moves toward the minus end. The myosin tails (and the tails of motor proteins generally) have apparently diversified during evolution to permit the proteins to bind other subunits and to interact with different cargoes.


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Cell_Biology_Alberts. Some myosins are found only in plants, and some are found only in vertebrates. Most, however, are found in all eukaryotes, suggesting that myosins arose early in eukaryotic evolution. The human genome includes about 40 myosin genes. Nine of the human myosins are expressed primarily or exclusively in the hair cells of the inner ear, and mutations in five of them are known to cause hereditary deafness. These extremely specialized myosins are important for the construction and function of the complex and beautiful bundles of actin found in stereocilia that project from the apical surface of these cells (see Figure 9–51); these cellular protrusions tilt in response to sound and convert sound waves into electrical signals.


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Cell_Biology_Alberts. The functions of most of the myosins remain to be determined, but several are well characterized. The myosin I proteins often contain either a second actin-binding site or a membrane-binding site in their tails, and they are generally involved in intracellular organization—including the protrusion of actin-rich structures at the cell surface, such as microvilli (see Panel 16–1 and Figure 16–4), and endocytosis. Myosin V is a two-headed myosin with a large step size (Figure 16–41A) and is involved in organelle transport along actin filaments. In contrast to myosin II motors, which work in ensembles and are attached only transiently to actin filaments so as not to interfere with one another, myosin V moves continuously,


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Cell_Biology_Alberts. Figure 16–40 Myosin superfamily members. Comparison of the domain structure of the heavy chains of some myosin types. All myosins share similar motor domains (shown in dark green), but their C-terminal tails (light green) and N-terminal extensions (light blue) are very diverse. On the right are depictions of the molecular structure for these family members. Many myosins form dimers, with two motor domains per molecule, but a few (such as I, III, and XIV) seem to function as monomers, with just one motor domain. Myosin VI, despite its overall structural similarity to other family members, is unique in moving toward the minus end (instead of the plus end) of an actin filament. The small insertion within its motor head domain, not found in other myosins, is probably responsible for this change in direction.


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Cell_Biology_Alberts. or processively, along actin filaments without letting go. Myosin V functions are well studied in the yeast Saccharomyces cerevisiae, which undergoes a stereotypical pattern of growth and division called budding. Actin cables in the mother cell point toward the bud, where actin is found in patches that concentrate where cell wall growth is taking place. Myosin V motors carry a wide range of cargoes— including mRNA, endoplasmic reticulum, and secretory vesicles—along the actin cables and into the bud. In addition, myosin V mediates the correct partitioning of organelles such as peroxisomes and mitochondria between mother and daughter cells (see Figure 16–41B).


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Cell_Biology_Alberts. Using their neck domain as a lever arm, myosins convert ATP hydrolysis into mechanical work to move along actin filaments in a stepwise fashion. Skeletal muscle is made up of myofibrils containing thousands of sarcomeres assembled from highly ordered arrays of actin and myosin II filaments, together with many accessory proteins. Muscle contraction is stimulated by calcium, which causes the actin-filament-associated protein tropomyosin to move, uncovering myosin binding sites and allowing the filaments to slide past one another. Smooth muscle and non-muscle cells have less well-ordered contractile bundles of actin and myosin, which are regulated by myosin light-chain phosphorylation. Myosin V transports cargo by walking along actin filaments.


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Cell_Biology_Alberts. Microtubules are structurally more complex than actin filaments, but they are also highly dynamic and play comparably diverse and important roles in the cell. Microtubules are polymers of the protein tubulin. The tubulin subunit is itself a heterodimer formed from two closely related globular proteins called α-tubulin and β-tubulin, each comprising 445–450 amino acids, which are tightly bound together by noncovalent bonds (Figure 16–42A). These two tubulin proteins are found only in this heterodimer, and each α or β monomer has a binding site for one molecule of GTP. The GTP that is bound to α-tubulin is physically trapped at the dimer interface and is never hydrolyzed or exchanged; it can therefore be considered to be an integral part of the tubulin heterodimer structure. The nucleotide on the β-tubulin, in contrast, may be in either the GTP or the GDP form and is exchangeable within the soluble (unpolymerized) tubulin dimer.


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Cell_Biology_Alberts. Tubulin is found in all eukaryotic cells, and it exists in multiple isoforms. Yeast and human tubulins are 75% identical in amino acid sequence. In mammals, there are at least six forms of α-tubulin and a similar number of β-tubulins, each encoded by a different gene. The different forms of tubulin are very similar, and they generally copolymerize into mixed microtubules in the test tube. However, they can have distinct locations in cells and tissues and perform subtly different functions. As a striking example, mutations in a particular human β-tubulin gene give rise to a paralytic eye-movement disorder due to loss of ocular nerve function. Numerous human neurological diseases have been linked to specific mutations in different tubulin genes.


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Cell_Biology_Alberts. Figure 16–41 Myosin V carries cargo along actin filaments. (A) The lever arm of myosin V is long, allowing it to take a bigger step along an actin filament than myosin II (see Figure 16–29). (B) Myosin V transports cargo and organelles along actin cables, in this example moving a mitochondrion into the growing bud of a yeast cell. Microtubules Are Hollow Tubes Made of Protofilaments


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Cell_Biology_Alberts. A microtubule is a hollow cylindrical structure built from 13 parallel protofilaments, each composed of αβ-tubulin heterodimers stacked head to tail and then folded into a tube (Figure 16–42B–D). Microtubule assembly generates two new types of protein–protein contacts. Along the longitudinal axis of the microtubule, the “top” of one β-tubulin molecule forms an interface with the “bottom” of the α-tubulin molecule in the adjacent heterodimer. This interface is very similar to the interface holding the α and β monomers together in the dimer subunit, and the binding energy is high. Perpendicular to these interactions, neighboring protofilaments form lateral contacts. In this dimension, the main lateral contacts are between monomers of the same type (α–α and β–β). As longitudinal and lateral contacts are repeated during assembly, a slight stagger in lateral contacts gives rise to the helical microtubule lattice. Because multiple contacts within the lattice hold most of the subunits in a


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Cell_Biology_Alberts. contacts are repeated during assembly, a slight stagger in lateral contacts gives rise to the helical microtubule lattice. Because multiple contacts within the lattice hold most of the subunits in a microtubule in place, the addition and loss of subunits occurs almost exclusively at the microtubule ends (see Figure 16–5). These multiple contacts among subunits make microtubules stiff and difficult to bend. The persistence length of a microtubule is several millimeters, making microtubules the stiffest and straightest structural elements found in most animal cells.


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Cell_Biology_Alberts. The subunits in each protofilament in a microtubule all point in the same direction, and the protofilaments themselves are aligned in parallel (see Figure Figure 16–42 The structure of a microtubule and its subunit. (A) The subunit of each protofilament is a tubulin heterodimer, formed from a tightly linked pair of αand β-tubulin monomers. The GTP molecule in the α-tubulin monomer is so tightly bound that it can be considered an integral part of the protein. The GTP molecule in the β-tubulin monomer, however, is less tightly bound and has an important role in filament dynamics. Both nucleotides are shown in red. (B) One tubulin subunit (αβ-heterodimer) and one protofilament are shown schematically. Each protofilament consists of many adjacent subunits with the same orientation. (C) The microtubule is a stiff hollow tube formed from 13 protofilaments aligned in parallel.


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Cell_Biology_Alberts. (D) A short segment of a microtubule viewed in an electron microscope. (E) Electron micrograph of a cross section of a microtubule showing a ring of 13 distinct protofilaments. (D, courtesy of Richard Wade; E, courtesy of Richard Linck.) Figure 16–43 The preferential growth of microtubules at the plus end.


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Cell_Biology_Alberts. Figure 16–43 The preferential growth of microtubules at the plus end. Microtubules grow faster at one end than at the other. A stable bundle of microtubules obtained from the core of a cilium (called an axoneme) was incubated for a short time with tubulin subunits under polymerizing conditions. Microtubules grew fastest from the plus end of the microtubule bundle, the end at the top in this micrograph. (Courtesy of Gary Borisy.) 16–42). Therefore, the microtubule lattice itself has a distinct structural polarity, with α-tubulins exposed at the minus end and β-tubulins exposed at the plus end. As for actin filaments, the regular, parallel orientation of their subunits gives microtubules structural and dynamic polarity (Figure 16–43), with plus ends growing and shrinking more rapidly.