Molecular Biology of the Cell, 3rd edn. Part III. Internal Organization of the Cell Chapter 16. The Cytoskeleton

Actin Filaments 31

Introduction

All eucaryotic species contain actin. This cytoskeletal protein is the most abundant protein in many eucaryotic cells, often constituting 5% or more of the total cell protein. Vertebrate skeletal muscle cells are the usual source of actin for experiments done in vitro, as about 20% of their mass is actin. If dry powdered muscle is treated with a very dilute salt solution, the actin filaments dissociate into their actin subunits. Each actin molecule is a single polypeptide 375 amino acids long that has a molecule of ATP tightly associated with it.

Actin filaments can form both stable and labile structures in cells. Stable actin filaments form the core of microvilli and are a crucial component of the contractile apparatus of muscle cells. Many cell movements, however, depend on labile structures constructed from actin filaments. In this section we focus on the question of how the cell controls the assembly of dynamic actin filaments from pools of soluble actin subunits in the cytosol.

Actin Filaments Are Thin and Flexible 32

Actin filaments appear in electron micrographs as threads about 8 nm wide. They consist of a tight helix of uniformly oriented actin molecules (also known as globular actin, or G actin) ( Figure16-49). Like a microtubule, an actin filament is a polar structure, with two structurally different ends - a relatively inert and slow-growing minus end and a faster-growing plus end. Because of the oriented "arrowhead" appearance of the complex formed between actin filaments and the motor protein myosin, which we describe later, the minus end is also referred to as the "pointed end" and the plus end as the "barbed end." The three-dimensional structure of the actin molecule has been solved by x-ray diffraction analysis, and this information has been used to deduce the structure of an actin filament at the level of individual amino acids ( Figure16-50).

Some lower eucaryotes, such as yeasts, have only one actin gene, encoding a single protein. All higher eucaryotes, however, have several isoforms encoded by a family of actin genes. At least six types of actin are present in mammalian tissues; these fall into three classes, depending on their isoelectric point. Alpha actins are found in various types of muscle, whereas β and γ actins are the principal constituents of nonmuscle cells. Although there are subtle differences in the properties of different forms of actin, the amino acid sequences have been highly conserved in evolution, and all assemble into filaments that are essentially identical in most tests performed in vitro.

The total length of all of the actin filaments in a cell is at least 30 times greater than the total length of the microtubules, reflecting a fundamental difference in the way these two cytoskeletal polymers are organized and function in cells. Actin filaments are thinner and more flexible, and usually much shorter, than microtubules. We shall see that actin filaments rarely occur in isolation in the cell but rather in cross-linked aggregates and bundles, which are much stronger than the individual filaments.

Actin and Tubulin Polymerize by Similar Mechanisms 33

Polymerization of pure actin in vitro requires ATP as well as both monovalent and divalent cations, which are usually K⁺ and Mg²⁺. The reaction is often studied either by observing the change in the light emission from a fluorescent probe that has been covalently attached to the actin or by monitoring the large increase in viscosity caused by the polymerization. When K⁺ and Mg²⁺are added to monomeric actin in the presence of ATP, there is initially a lag phase, as new filaments are nucleated, and then a rapid polymerization phase, as the short filaments elongate. The lag in polymerization with pure actin is due to the same kinetic barrier to nucleation that we discussed for tubulin polymerization (see Figure 16-23). For actin the rate of nucleation is proportional to the cube of the actin concentration, suggesting that the nucleating structure for the spontaneous polymerization of pure actin is a trimer of actin molecules. By contrast, the rate at which each filament elongates is proportional, as for microtubules, to the concentration of the free subunit, indicating that the filament elongates by the addition of one actin molecule at a time.

The polymerization rate is different at the two ends of the actin filament, and this difference is greater than for microtubules: the plus (or barbed) end of actin filaments polymerizes at up to 10 times the rate of the minus (or pointed) end. The critical concentration for actin polymerization - that is, the free actin monomer concentration at which the proportion of actin in polymer stops increasing - is around 0.2 micromolar (about 8 µg/ml). This concentration is very much lower than the concentration of unpolymerized actin in a cell, and the cell has evolved special mechanisms to prevent most of its monomeric actin from assembling into filaments, as we discuss later.

Shortly after polymerization, the terminal phosphate of the ATP bound to the actin molecule is hydrolyzed, leaving the resulting ADP trapped in the polymer. The hydrolysis of ATP during actin polymerization is analogous to the GTP hydrolysis that accompanies microtubule assembly, but in the case of actin we can understand the conformational changes involved because the three-dimensional structure of actin is known. The actin molecule is clam-shaped and binds ATP in the crevice between its two halves; like a clam shell, it can open and close. When actin polymerizes, the shell is clamped shut by interactions between amino acids on both lips of the shell and the back side of the next subunit in the polymer. It is thought that ATP hydrolysis is triggered by the closing of the clam shell as each actin molecule is incorporated into the filament, leaving ADP trapped inside ( Figure 16-51).

ATP Hydrolysis Is Required for the Dynamic Behavior of Actin Filaments 34

The role of ATP hydrolysis in actin polymerization is similar to the role of GTP hydrolysis in tubulin polymerization, as explained in Panel16-1 (pp. 824-825). In neither case is hydrolysis required to form the filament; instead, it serves to weaken the bonds in the polymer and thereby promote depolymerization. There are, however, important differences in the behavior of the bound nucleotide in the subunits of these two polymers. An especially interesting difference is that ATP-ADP exchange (the replacement of bound ADP by ATP) is relatively slow for free actin (half-time of minutes), while GTP-GDP exchange is very rapid for free tubulin (half-time of seconds); thus, when actin molecules are released by disassembly of a filament, there is a relatively long delay before they can be re-used in filament assembly. In principle, this property of actin allows the cell to maintain a high cytosolic concentration of unpolymerized actin molecules in the form of ADP actin; furthermore, the ADP-actin monomer in a cell can be stabilized by binding to another protein, and this could provide a way to regulate actin polymerization.

The effect of ATP hydrolysis on actin is subtle, and there are still many questions about its precise consequences for the cell. Actin filaments, unlike microtubules, do not seem to show drastic dynamic instability in vitro. Instead, they can engage in an interesting dynamic behavior called treadmilling, which occurs when actin molecules are added continually to the plus end of the filament and are lost continually from the minus end, with no net change in filament length (see Panel 16-1, pp. 824-825). Treadmilling, like dynamic instability, is a nonequilibrium behavior that requires an input of energy, which is provided by the ATP hydrolysis that accompanies polymerization. This phenomenon is thought to contribute to the rapid exchange of the subunits of actin filaments that takes place in cells.

It is remarkable that actin and tubulin have both evolved nucleoside tri-phosphate hydrolysis for the same basic reason - to enable them, having polymerized, to depolymerize readily. Actin and tubulin are completely unrelated in amino acid sequence: actin is distantly related in structure to the glycolytic enzyme hexokinase, whereas tubulin is distantly related to a large family of GTPases that includes the heterotrimeric G proteins and monomeric GTPases such as Ras. (Both types of structures are discussed in detail in Chapter 5.) The convergent evolution of the capacity for nucleotide hydrolysis in actin and tubulin demonstrates just how important it is to microtubule and actin filament function: the dynamic assembly and disassembly of these cytoskeletal polymers that hydrolysis makes possible lies at the heart of cytoplasmic organization.

The Functions of Actin Filaments Are Inhibited by Both Polymer-stabilizing and Polymer-destabilizing Drugs 35

Drugs that stabilize or destabilize actin filaments provide important tools to investigate their dynamic behavior in cells. The cytochalasins are fungal products that prevent actin from polymerizing by binding to the plus end of actin filaments. The phalloidins are toxins isolated from the Amanita mushroom that bind tightly all along the side of actin filaments and stabilize them against depolymerization. (One remedy for Amanita mushroom poisoning is to eat a large quantity of raw meat: the high concentration of actin filaments in the muscle tissue binds the phalloidin and thereby reduces its toxicity.) Both of these drugs cause dramatic changes in the actin cytoskeleton. We saw earlier for microtubules that both polymer-destabilizing drugs such as colchicine and polymer-stabilizing drugs such as taxol are toxic to cells, and the same is true for drugs affecting the stability of actin filaments, indicating that the function of actin filaments also depends on a dynamic equilibrium between the filaments and actin monomer.

Cytochalasin has found its greatest use in studying cell locomotion. In particular, the leading edge of a moving cell contains actin filaments that are continually polymerizing and are therefore very sensitive to cytochalasin. In most moving cells cytochalasin causes the leading edge rapidly to retract. If the plasma membrane of the leading edge is very firmly attached to the substratum, however, cytochalasin causes the actin filaments to retract but leaves the membrane behind, stuck to the substratum ( Figure16-52).

Phalloidin is widely used, as a fluorescent derivative, to stain actin filaments in fixed cells, and it also has a profound effect on living cells. When it is microinjected into a living fibroblast, for example, it drives all of the actin monomer into filaments at random positions in the cytoplasm, causing a drastic blebbing and contraction that often destroys the cell.

The Actin Molecule Binds to Small Proteins That Help to Control Its Polymerization 36

In a fibroblast cell approximately 50% of the actin is in filaments and 50% is in monomer. The monomer concentration is typically 50-200 micromolar (2-8 mg/ml) in a variety of cell types; this is surprisingly high, given the low critical concentration of pure actin (less than 1 micromolar), and it reflects the presence of special proteins that bind to the actin molecule and inhibit its addition to the ends of actin filaments. The most abundant of these actin-monomer-binding proteins in many cells is thymosin, an unusually small protein with a molecular weight of about 5000. In the cells in which it has been most carefully studied (blood platelets and neutrophils), it is present in concentrations that are sufficient to sequester all of the monomeric actin. It is not clear how this protein inhibits actin polymerization: it could sterically block polymerization by covering a site where one monomer binds to another, or it could trap ADP on actin by inhibiting ADP-ATP exchange, thereby making the actin molecule unlikely to polymerize ( Figure 16-53).

Another actin-monomer-binding protein is profilin, which is present in all cells and is thought to play a part in controlling actin polymerization in response to extracellular stimuli. Profilin, which in many cells is largely associated with the plasma membrane, accelerates the exchange of ATP for ADP when bound to actin monomers and is thought to play a part in promoting the regulated polymerization of actin during cell movement, although this is still controversial. A mutant yeast cell that is deficient in profilin has a deficit of actin filaments, which supports a role for this molecule in stimulating the polymerization of actin.

In addition to thymosin and profilin, cells contain other abundant proteins that are able to bind actin monomers, and some of these, such as actin-depolymerizing factor (ADF), inhibit the assembly of actin into filaments. Evidently cells have a variety of mechanisms, the details of which are not yet understood, by which they hold stocks of actin monomer in reserve in order to assemble actin filaments only when and where they are needed.

Many Cells Extend Dynamic Actin-containing Microspikes and Lamellipodia from Their Leading Edge 37

Dynamic surface extensions containing actin filaments are a common feature of animal cells, especially when the cells are moving or changing shape. The large, free-living cells of Amoeba proteus,for example, produce pseudopodia - stubby distensions of the actin cortex - with which they walk over surfaces. Many cells in vertebrate tissues are also capable of independent migration over surfaces, especially when put into tissue culture. The leading edge of a crawling fibroblast regularly extends a thin, sheetlike process known as a lamellipodium, which contains a dense meshwork of actin filaments. Many cells also extend thin, stiff protrusions called microspikes, which are about 0.1 µ wide and 5 to 10 µ long and contain a loose bundle of about 20 actin filaments oriented with their plus ends pointing outward (see Figure 16-9). The growing tip (growth cone) of a developing nerve cell axon extends even longer microspikes, called filopodia, which can be up to 50 µ long.

A lamellipodium can be viewed as a two-dimensional version of a micro-spike; indeed, short microspikes often project from the edges of a lamellipodium. When carefully fixed and stained for examination in an electron microscope, the actin filaments in the lamellipodium of a moving cell appear to be more organized than they are in other regions of the cell cortex. Many of the filaments project outward in an orderly array, with their plus ends inserted into the leading edge of the plasma membrane ( Figure16-54). The lamellipodium behaves as a structural unit; if it fails to adhere to the substratum, it is usually swept rapidly backward over the top of the cell as a "ruffle" ( Figure16-55).

Both lamellipodia and microspikes are motile structures that can form and retract with great speed. As we discuss next, it is thought that microspikes and lamellipodia are generated by local actin polymerization at the plasma membrane and that such actin polymerization can rapidly push out the plasma membrane without tearing it.

The Leading Edge of Motile Cells Nucleates Actin Polymerization 38

When the behavior of actin filaments at the leading edge is studied by labeling a small patch of actin and following its movement, it is seen that actin is continually moving back toward the cell body at a speed of about 1 µm/minute, suggesting that actin is continuously polymerizing near the tip of the leading edge and continuously depolymerizing at more internal sites ( Figure16-56). This highly dynamic behavior of actin filaments at the leading edge is thought to be crucial for such processes as directed cell locomotion and chemotaxis. It gives the impression that the leading edge is propelling itself forward by pushing actin filaments backward.

The leading edge of a cell seems to organize actin filaments much as a centrosome organizes microtubules but with one crucial difference: it not only nucleates the growth of new filaments, but also seems to be the site at which monomers are added subsequently to enable the filaments to elongate. This role can be demonstrated by gently lysing a fibroblast and then adding rhodamine-tagged actin monomers, which are seen to polymerize preferentially at the tip of the leading edge ( Figure 16-57). Moreover, if the actin filaments in a cell are decorated to reveal their polarity, the fast-growing plus end of each actin filament is found to be attached to the membrane at the leading edge.

There are many unanswered questions about the mechanism by which the leading edge nucleates actin filament polymerization. Does the leading edge hold on to the plus end of a filament that it nucleates, for example, or does it nucleate a new filament and then quickly release it? Because of the continuous backward movement of actin (see Figure 16-56), any model that postulates that the leading edge holds onto actin filament ends would require that the filaments in the lamellipodium undergo continuous treadmilling by insertion of actin monomers at the site where the filaments are held by the membrane. In an alternative model, individual actin filaments are released and move away from the membrane (presumably as a cross-linked meshwork) soon after they form ( Figure16-58).

The rapid assembly of actin filaments at the leading edge of a moving cell requires that actin monomers be released from the actin-monomer-binding proteins that normally restrain their polymerization into filaments. We discuss below how signals in the cell's environment may regulate the release of actin monomers for polymerization at the tip of the leading edge.

Some Pathogenic Bacteria Use Actin to Move Within and Between Cells 39

Listeria monocytogenes, a bacterium that causes a severe form of food poisoning, has provided unexpected insights into the mechanism by which the local polymerization of actin is controlled in cells. This pathogenic bacterium enters cells by being phagocytosed; it then escapes into the host cell cytosol by secreting enzymes that break down the membrane of the phagosome. Once in the cytosol, the bacteria not only grow and divide, but they also spread to adjoining cells by mobilizing the actin-based motility system of the host cell. By nucleating actin filaments at one region of its surface, an individual bacterium moves through the cytosol at rates of 10 µm/minute or more, laying down a tail of actin filaments behind it. When it collides with the plasma membrane of the host cell, it keeps moving outward, inducing the formation of a long, thin microspike with a bacterium at its tip. This projection is often engulfed by a neighboring cell, allowing the bacterium to enter its cytoplasm without exposure to the extracellular environment, thereby avoiding recognition by antibodies produced by the host ( Figure 16-59).

This form of movement suggests that the bacterium may be using actin to propel itself forward in the same way that the plasma membrane of a eucaryotic cell uses actin to propel itself forward during the formation of a normal microspike or lamellipodium.

If the actin filaments in the tail behind a Listeria bacterium migrating in the cytosol are marked with a fluorescent tag and observed by fluorescence microscopy, they are found to be stationary. The filaments form at the rear of the bacterium and are left behind like a rocket trail as the bacterium advances, depolymerizing again within a minute or so as they encounter depolymerizing factors in the cytosol. Assembly is induced by a specific protein on the surface of the bacterium that acts indirectly by sequestering host-cell proteins, including profilin. Since bacterium-induced movement can be reproduced in a concentrated cell-free extract, details of the mechanism should emerge from biochemical studies. These details should help us to understand how actin nucleation and polymerization occur in the microspikes and lamellipodia of a normal, uninfected cell and how these processes power the forward movement of the cell.

Polymerization of Actin in the Cell Cortex Is Controlled by Cell-Surface Receptors 40

The production of movement is of little use unless it is properly directed according to the environment. As discussed earlier, the dynamic cortical meshwork of actin filaments rearranges rapidly in response to signals from outside the cell that impinge on the plasma membrane. The actin cytoskeleton can therefore be considered to be an integral part of the cell's signal-transduction systems, discussed in Chapter 15: when certain growth factors are added to the medium bathing quiescent cells in culture, for example, they immediately cause actin-containing lamellipodia to form and move over the cell surface.

The response of the actin cortex to external signals conveying spatial information can be highly localized. We considered one example earlier when we discussed the polarization of a cytotoxic T cell that is induced by contact with the target cell it subsequently kills (see Figure16-11). A signal-induced polarization of the actin cortex also occurs in animal cells that are capable of chemotaxis, which is defined as movement in a direction controlled by a gradient of a diffusible chemical sensed by the cell. One well-studied example is the chemotactic movement of certain white blood cells ( neutrophils) toward a source of bacterial infection. Neutrophils have receptor proteins on their surface that enable them to detect the very low concentrations of the N-formylated peptides derived from bacterial proteins (only procaryotes begin protein synthesis with N-formyl methionine). The neutrophils can be guided to their targets by a difference of only 1% in the concentration of these diffusible peptides on one side of the cell versus the other.

Another example of chemotaxis is provided by the cellular slime mold Dictyostelium discoideum. These eucaryotes live on the forest floor as independent motile cells called amoebae, which feed on bacteria and yeast and, under optimal conditions, divide every few hours. When their food supply is exhausted, the amoebae stop dividing and gather together to form tiny (1-2 mm), multicellular, wormlike structures, which crawl about as glistening slugs and leave trails of slime behind them ( Figure 16-60). As the slug migrates, the cells begin to differentiate, initiating a process that ends with the production of a tiny plantlike structure consisting of a stalk and a fruiting body some 30 hours after the beginning of aggregation ( Figure 16-61). The fruiting body contains large numbers of spores, which can survive for long periods of time even in extremely hostile environments. Only when conditions are favorable do the spores germinate to produce the free-living amoebae that start the cycle again.

The Dictyostelium amoebae aggregate by chemotaxis, migrating toward a source of cyclic AMP, which is secreted by the starved amoebae. Like neutrophils, the amoebae reorient their leading edge in order to migrate up a shallow chemoattractant gradient. And when they are exposed to a local source of cyclic AMP leaking from a micropipette, they extend actin-containing processes directly toward the pipette ( Figure 16-62). This experiment shows that eucaryotic chemotaxis involves detecting a spatial gradient of attractant concentration directly, in contrast to bacterial chemotaxis, which uses a time-dependent variation in concentration to detect gradients, as discussed in Chapter 15.

The cytoskeletal reaction of Dictyostelium amoebae to cyclic AMP can be examined by making lysates of these cells very shortly after bulk stimulation with cyclic AMP in solution. As shown in Figure16-63, a dramatic burst of actin polymerization occurs 5-10 seconds after adding cyclic AMP, which corresponds to the time required for flattening of the cells on the substratum. Between 20 and 40 seconds after the pulse of cyclic AMP, actin depolymerizes and the cells round up. Then there is a more prolonged burst of actin polymerization as actin-binding proteins are recruited into the cytoskeleton from soluble pools; during this latter period the cells that respond to cyclic AMP begin to extend lamellipodia and other actin-rich processes.

Heterotrimeric G Proteins and Small GTPases Relay Signals from the Cell Surface to the Actin Cortex 41

How does cyclic AMP binding to its receptor in Dictyostelium amoebae trigger massive actin polymerization? The receptor is known to activate a heterotrimeric G protein. The cytoplasm contains a reservoir of actin monomers, which, as we saw earlier, are stabilized by actin-monomer-binding proteins. Stimulation of actin polymerization requires that these actin molecules be made available in a form that can polymerize and also that nucleation sites for actin filaments be provided to overcome the kinetic barrier to nucleation. The actin-monomer-binding protein profilin binds tightly to the inositol phospholipids in the plasma membrane that generate intracellular signals in response to extracellular ligands (see Figure 15-30). According to one hypothesis, activation of this signaling pathway (which occurs via a heterotrimeric G protein) could release profilin from the plasma membrane into the cytosol. Profilin can catalyze ATP-ADP exchange on actin in vitro, and so when it is released from the plasma membrane, it may rapidly convert inactive ADP actin to active ATP actin to induce the local formation of actin filaments.

G proteins have also been implicated in the signaling processes that activate the actin cortex during the chemotactic response of neutrophils and the activation of blood platelets. There is evidence that two Ras-related small GTPases known as Rho and Rac act downstream; these proteins have been shown to have distinct effects on the actin cytoskeleton in fibroblasts. Microinjection of Rac protein into cultured cells causes a dramatic increase in the formation of lamellipodia within 5 minutes. Moreover, a dominant-negative mutant form of Rac inhibits the formation of lamellipodia normally induced by various growth factors, indicating that this response to growth factors depends on Rac. Microinjection of Rho protein leads to the appearance of large bundles of actin filaments known as stress fibers and to the enhancement of focal contacts, where the cell is attached to the substratum externally and stress fibers are anchored internally (as we discuss later). Rho is also thought to be needed to assemble the contractile ring during cell division. Thus Rac and Rho not only control the polymerization of actin into filaments but also govern the organization of these filaments into specific types of structures.

Mechanisms of Cell Polarization Can Be Analyzed in Yeast Cells 42

Further clues to how cells may orient the activities of their cytoskeleton have come from the behavior of yeast cells. The ease of genetic analysis in yeasts has made them an important source of fundamental information about biological mechanisms that are common to all eucaryotic cells. In particular, studies on the interactions between yeast cells during mating have begun to identify mechanisms by which eucaryotic cells become structurally polarized. In the budding yeast Saccharomyces cerevisiae, cells of two mating types, a and α, secrete hormones, known as a-factor and α-factor, respectively. These hormones act by binding to cell-surface receptors that belong to the large G-protein-linked receptors discussed in Chapter 15. One consequence of the binding of α-factor to its receptors on an a-cell is to cause the cell to become polarized so that it adopts a shape known as a "shmoo" ( Figure16-64). If an α-factor gradient is present, the shmoo tip is directed toward the highest concentration of this signaling molecule.

During this polarization response the yeast cell undergoes cytoskeletal reorganizations that parallel those of an animal cell that is becoming polarized. Actin filaments congregate at the pointed shmoo tip, where they are thought to direct the local secretion of cell-wall components - possibly by directing the transport vesicles carrying these components to the shmoo tip. At the same time, the microtubule organizing center (in this case the spindle pole body, see Figure 17-24) moves to the side of the nucleus that is closest to the shmoo tip, and microtubules extend from it toward the tip. By screening for mutant cells that fail to form a shmoo during mating, many of the genes involved in yeast-cell polarization are being identified. It is likely that some of the proteins that these genes encode will also be involved in polarizing an animal cell.

Summary

Actin is a highly conserved cytoskeletal protein that is present at high concentrations in nearly all eucaryotic cells. Purified actin exists as a monomer in low ionic strength solutions and spontaneously assembles into actin filaments on addition of salt provided ATP is present. As with tubulin, the polymerization of actin is a dynamic process that is regulated by the hydrolysis of a tightly bound nucleotide (ATP in this case). In cells, approximately half of the actin is kept in a monomeric form through its binding to small proteins such as thymosin. In the cortex of animal cells, actin molecules continually polymerize and depolymerize to generate cell-surface protrusions such as lamellipodia and microspikes. Polymerization can be regulated by extracellular signals binding to cell-surface receptors that act through heterotrimeric G proteins and the small GTPases Rac and Rho.

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About this book III. Internal Organization of the Cell 16. The Cytoskeleton Introduction The Nature of the Cytoskeleton Intermediate Filaments Microtubules Cilia and Centrioles Actin Filaments Actin-binding Proteins Muscle References

Figures

Figure 16-49. Actin filaments... Figure 16-50. The structure of... Figure 16-51. The trapping of... Figure 16-52. The effect of... Figure 16-53. Two possible mechanisms... Figure 16-54. Actin filaments at... Figure 16-55. Lamellipodia and microspikes... Figure 16-56. Actin filament dynamics... Figure 16-57. The tip of... Figure 16-58. Two models that... Figure 16-59. The actin-based movement... Figure 16-60. Light micrograph of... Figure 16-61. Light micrograph of... Figure 16-62. A chemotactic response... Figure 16-63. The effect of... Figure 16-64. Morphological polarization of...