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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 18.1The Actin Cytoskeleton

Cell locomotion results from the coordination of motions generated by different parts of a cell. These motions are complex and difficult to describe, but we can pick out their major features using powerful fluorescent antibody-labeling methods combined with fluorescence microscopy. To provide a background for more detailed discussions of the mechanism of cell locomotion later, we first briefly describe movement by two types of crawling animal cells. One feature that characterizes all moving cells is polarity; that is, certain structures always form at the front of the cell, whereas others are found at the rear.

In the micrograph shown at the opening to this chapter, a skin cell (keratinocyte) displays motions typical of a fast-moving cell like an ameba. Initially, the keratinocyte forms a large, broad membrane protrusion, the lamellipodium, which moves forward from the cell. After contacting the substratum and forming special attachment structures, called focal adhesions, the lamellipodium quickly fills with cytosol and the rear of the cell retracts forward toward the body of the cell.

Locomotion of a slow-moving cell like a fibroblast involves several structures not seen in a fast-moving keratinocyte. As a fibroblast moves forward, it extends slender “fingers” of membrane, called filopodia, as well as lamellipodia (Figure 18-1a). Where the cell membrane contacts the substratum, focal adhesions assemble on the ventral surface of the cell. As the cell continues to travel, its tail is pulled forward, often leaving behind small patches of the cell still firmly attached to the substratum through the focal adhesions. Some areas of the cell membrane do not form stable adhesions at the leading edge of the cell; these areas project upward as a thin veil, or ruffle, that moves as an undulating ridge back along the dorsal cell surface toward the cell body.

Figure 18-1. Actin structures in a fibroblast.

Figure 18-1

Actin structures in a fibroblast. (a) Scanning electron micrograph of a cultured fibroblast. At the front of the cell, filopodia, lamellipodia, and ruffles project from the cell membrane. At the (more...)

The machinery that powers cell migration is built from the actin cytoskeleton, which is larger than any organelle. When a fibroblast is observed by fluorescence microscopy after the actin filaments are stained with a fluorescent dye, radially oriented actin filament bundles can be seen at the leading edge, and axial bundles, called stress fibers, are visible underlying the cell body (Figure 18-1b). In addition, a network of actin filaments fills the rest of the cell, but the individual filaments of this network are difficult to resolve in the light microscope.

Much of the discussion in this section focuses on the ability of huge actin filament structures to control the shape of a cell. Because the actin cytoskeleton is so big, it can easily change cell morphology just by assembling or disassembling itself. In previous chapters, we have seen examples of large protein complexes in which the number and positions of the subunits are fixed. For example, all ribosomes have the same number of protein and RNA components, and their three-dimensional geometry is invariant. However, the actin cytoskeleton is different — the lengths of filaments vary greatly, the filaments are cross-linked into imperfect bundles and networks, and the ratio of cytoskeletal proteins is not rigidly maintained. This organizational flexibility of the actin cytoskeleton permits a cell to assume many shapes and to vary them easily. In moving cells, the cytoskeleton must assemble rapidly and does not always have the chance to form well-organized, highly ordered structures. Keeping this in mind, we examine actin as a model for understanding how polymeric proteins form the structural framework of a cell and how the cell tailors this framework to carry out various tasks involving motion of the entire cell or subcellular parts.

Eukaryotic Cells Contain Abundant Amounts of Highly Conserved Actin

Actin is the most abundant intracellular protein in a eukaryotic cell. In muscle cells, for example, actin comprises 10 percent by weight of the total cell protein; even in nonmuscle cells, actin makes up 1 – 5 percent of the cellular protein. A typical cytosolic concentration of actin in nonmuscle cells is 0.5 mM; in special structures like microvilli, however, the local actin concentration can be tenfold higher. To appreciate how much actin cells contain, consider a typical liver cell, which has 20,000 insulin receptor molecules but approximately half a billion (0.5 × 109) actin molecules. This predominance of actin compared with other cell proteins is a common feature of all cytoskeletal proteins. Because they form structures that must cover large spaces in a cell, these proteins are among the most abundant proteins in a cell.

A moderate-sized protein consisting of approximately 375 residues, actin is encoded by a large, highly conserved gene family. Some single-celled eukaryotes like yeasts and amebas have a single actin gene, whereas many multicellular organisms contain multiple actin genes. For instance, humans have six actin genes, which encode isoforms of the protein, and some plants have as many as 60. Sequencing of these actins has revealed that it is one of the most conserved proteins in a cell, comparable with histones, the structural proteins of chromatin (Chapter 9). Actin residues from amebas and from animals are identical at 80 percent of the positions. In vertebrates, the four α-actin isoforms present in various muscle cells and the β- and γ-actin isoforms present in nonmuscle cells differ at only four or five positions. Although these differences among isoforms seem minor, the isoforms have different functions: α-Actin is associated with contractile structures, and β-actin is at the front of the cell where actin filaments polymerize.

Recently, a family of actin-related proteins (Arps), exhibiting 50 percent homology with actin, has been identified in many eukaryotic organisms. As noted later, one group of Arps (Arp2/3) stimulates actin assembly; intriguingly, another group (Arp1) is associated with microtubules and a microtubule motor protein. They are discussed in the next chapter.

ATP Holds Together the Two Lobes of the Actin Monomer

Actin exists as a globular monomer called G-actin and as a filamentous polymer called F-actin, which is a linear chain of G-actin subunits. (The microfilaments visualized in a cell by electron microscopy are F-actin filaments plus any bound proteins.) Each actin molecule contains a Mg2+ ion complexed with either ATP or ADP. Thus there are four states of actin: ATP – G-actin, ADP – G-actin, ATP – F-actin, and ADP – F-actin. Two of these forms, ATP – G-actin and ADP – F-actin, predominate in a cell. We discuss later how the interconversion between the ATP and ADP forms of actin is important in the assembly of the cytoskeleton.

Although G-actin appears globular in the electron microscope, x-ray crystallographic analysis reveals that it is separated into two lobes by a deep cleft (Figure 18-2a). The lobes and the cleft compose the ATPase fold, the site where ATP and Mg2+ are bound. In actin, the floor of the cleft acts as a hinge that allows the lobes to flex relative to each other. When ATP or ADP is bound to G-actin, the nucleotide affects the conformation of the molecule. In fact, without a bound nucleotide, G-actin denatures very quickly.

Figure 18-2. Structures of monomeric G-actin and F-actin filament.

Figure 18-2

Structures of monomeric G-actin and F-actin filament. (a) Model of a β-actin monomer from a nonmuscle cell shows it to be a platelike molecule (measuring 5.5 × 5.5 × 3.5 (more...)

G-Actin Assembles into Long, Helical F-Actin Polymers

The addition of ions — Mg2+, K+, or Na+— to a solution of G-actin will induce the polymerization of G-actin into F-actin filaments. The process is also reversible: F-actin depolymerizes into G-actin when the ionic strength of the solution is lowered. The F-actin filaments that form in vitro are indistinguishable from microfilaments isolated from cells. This indicates that other factors such as accessory proteins are not required for polymerization in vivo. The assembly of G-actin into F-actin is accompanied by the hydrolysis of ATP to ADP and Pi; however, as we discuss later, ATP hydrolysis affects the kinetics of polymerization but is not necessary for polymerization to occur.

When negatively stained by uranyl acetate for electron microscopy, F-actin appears as twisted strings of beads whose diameter varies between 7 and 9 nm (Figure 18-2b). From x-ray diffraction studies of actin filaments and the actin monomer structure shown in Figure 18-2a, scientists have produced a model of an actin filament in which the subunits are organized as a tightly wound helix (Figure 18-2c). In this arrangement, each subunit is surrounded by four other subunits, one above, one below, and two to one side. Each subunit corresponds to a bead seen in electron micrographs of actin filaments.

The ability of G-actin to polymerize into F-actin and of F-actin to depolymerize into G-actin is an important property of actin. In this chapter, we will see how the reversible assembly of actin lies at the core of many cell movements.

F-Actin Has Structural and Functional Polarity

All subunits in a filament point toward the same filament end (i.e., they have the same polarity). Consequently, at one end of the filament, by convention designated the (−) end, the ATP-binding cleft of an actin subunit is exposed to the surrounding solution; at the opposite end, the cleft contacts the neighboring actin subunit (see Figure 18-2c).

Without the atomic resolution afforded by x-ray crystallography, the cleft in an actin subunit, and therefore the polarity of a filament, is not detectable. However, the polarity of actin filaments can be demonstrated by electron microscopy in so-called “decoration” experiments, which exploit the ability of myosin to bind specifically to actin filaments. In this type of experiment, an excess of myosin S1, the globular head domain of myosin, is mixed with actin filaments and binding is permitted to occur. Myosin attaches to the sides of a filament with a slight tilt. When all subunits are bound by myosin, the filament appears coated (“decorated”) with arrowheads that all point toward one end of the filament (Figure 18-3). Because myosin binds to actin filaments and not to microtubules or intermediate filaments, arrowhead decoration is one criterion by which actin filaments are identified among the other cytoskeletal fibers in electron micrographs of thin-sectioned cells.

Figure 18-3. Experimental demonstration of polarity of an actin filament by binding of myosin S1 head domains.

Figure 18-3

Experimental demonstration of polarity of an actin filament by binding of myosin S1 head domains. When bound to all the actin subunits, S1 appears to spiral around the filament. This coating (more...)

The Actin Cytoskeleton Is Organized into Bundles and Networks of Filaments

On first looking at an electron micrograph or immunofluorescence micrograph of a cell, one is struck by the dense, seemingly disorganized mat of filaments present in the cytosol (Figure 18-4). However, a keen eye will start to pick out areas, generally where the membrane protrudes from the cell surface, in which the filaments are concentrated into bundles. From these bundles the filaments continue into the cell interior, where they fan out and become part of a network of filaments. These two structures, bundles and networks, are the most common arrangements of actin filaments in a cell.

Figure 18-4. Micrograph revealing bundles and networks of actin filaments in the cytosol of a spreading platelet treated with detergent to remove the plasma membrane.

Figure 18-4

Micrograph revealing bundles and networks of actin filaments in the cytosol of a spreading platelet treated with detergent to remove the plasma membrane. Actin bundles project from the cell (more...)

Functionally, bundles and networks have identical roles in a cell: both provide a framework that supports the plasma membrane and, therefore, determines a cell’s shape. Structurally, bundles differ from networks mainly in the organization of actin filaments. In bundles the actin filaments are closely packed in parallel arrays, whereas in a network the actin filaments crisscross, often at right angles, and are loosely packed. Cells contain two types of actin networks. One type, associated with the plasma membrane, is planar or two-dimensional, like a net or a web; the other type, present within the cell, is three-dimensional, giving the cytosol gel-like properties.

In all bundles and networks, the filaments are held together by actin cross-linking proteins. To connect two filaments, a cross-linking protein must have two actin-binding sites, one site for each filament. The length and flexibility of a cross-linking protein critically determine whether bundles or networks are formed. Short cross-linking proteins hold actin filaments close together, forcing the filaments into the parallel alignment characteristic of bundles (Figure 18-5a). In contrast, long, flexible cross-linking proteins are able to adapt to any arrangement of actin filaments and tether orthogonally oriented actin filaments in networks (Figure 18-5b).

Figure 18-5. Actin cross-linking proteins bridging pairs of actin filaments.

Figure 18-5

Actin cross-linking proteins bridging pairs of actin filaments. (a) When cross-linked by fascin, a relatively short protein, actin filaments form a bundle. (b) Long cross-linking proteins (more...)

Many actin cross-linking proteins belong to the calponin homology – domain (CH-domain) superfamily (Table 18-1). Each of these proteins has a pair of actin-binding domains, whose sequence is homologous to calponin, a muscle protein. The actin-binding domains are separated by repeats of helical coiled-coil or β-sheet immunoglobulin motifs. Among the CH-domain proteins, the shortest (fimbrin and α-actinin) are found in actin bundles within cell extensions, and the longest (filamin, spectrin, and dystrophin) are found in actin networks in the cortical region adjacent to the plasma membrane. A smaller number of cross-linking proteins are classified into two other groups; these bind to different sites on actin than the CH-domain proteins.

Table 18-1. Actin Cross-Linking Proteins.

Table 18-1

Actin Cross-Linking Proteins.

Cortical Actin Networks Are Connected to the Membrane

The distinctive shape of a cell is dependent not only on the organization of actin filaments but also on proteins that connect the filaments to the membrane. These proteins, called membrane-microfilament binding proteins, act as spot welds that tack the membrane sheet to the underlying cytoskeleton framework. When attached to a bundle of filaments, the membrane acquires a fingerlike shape, as we discuss later. When attached to a planar network of filaments, the membrane is held flat. We focus on these network connections in this section. The simplest connections entail binding of integral membrane proteins directly to actin filaments. More common are complex linkages that connect actin filaments to integral membrane proteins through peripheral membrane proteins.

The richest area of actin filaments in a cell lies in the cortex, a narrow zone just beneath the plasma membrane. In this region, most actin filaments are arranged into a network that excludes most organelles from the cortical cytoplasm. Several ways to organize the cortical actin cytoskeleton are observed in different cell types. Perhaps the simplest cytoskeleton is the two-dimensional network of actin filaments adjacent to the erythrocyte plasma membrane. In more complicated cortical cytoskeletons, such as those in platelets, epithelial cells, and muscle, actin filaments are part of a three-dimensional network that fills the cytosol and anchors the cell to the substratum. We first describe the erythrocyte cytoskeleton and its linkage to the membrane and then examine the more complex cytoskeletons in platelets and muscle.

Erythrocyte Cytoskeleton

A red blood cell must squeeze through narrow blood capillaries without rupturing its membrane. The strength and flexibility of the erythrocyte plasma membrane depends on a dense cytoskeletal network that underlies the entire membrane and is attached to it at many points. The primary component of the erythrocyte cytoskeleton is spectrin, a long fibrous protein. Two dimeric subunits of spectrin, each composed of an α and β polypeptide chain, associate to form head-to-head tetramers, which are 200 nm long. The entire cytoskeleton is arranged in a spoke-and-hub network (Figure 18-6). Each spectrin tetramer comprises a spoke, extending from and cross-linking a pair of hubs, called junctional complexes. As illustrated in Figure 18-7, each junctional complex is composed of a short (14-subunit) actin filament plus adducin, tropomyosin, and tropomodulin. The last two proteins strengthen the network by preventing the actin filament from depolymerizing. Because several spectrin molecules can bind the same actin filament, the erythrocyte cytoskeletal network has a spoke-and-hub organization. This polygonal arrangement acts as a lamination of the membrane.

Figure 18-6. Electron micrograph of human erythrocyte cytoskeleton.

Figure 18-6

Electron micrograph of human erythrocyte cytoskeleton. The long “spokes” are composed mainly of spectrin and can be seen to intersect at the “hubs,” (more...)

Figure 18-7. The organization of the major erythrocyte cytoskeletal proteins and their interactions with integral membrane proteins.

Figure 18-7

The organization of the major erythrocyte cytoskeletal proteins and their interactions with integral membrane proteins. (Inset) Hypothetical arrangement of the (more...)

To ensure that the erythrocyte retains its characteristic shape, the spectrin-actin cytoskeleton is firmly attached to the overlying erythrocyte membrane by two peripheral membrane proteins, each of which binds to a specific integral membrane protein (see Figure 18-7). Ankyrin connects the center of spectrin to band 3 protein, the anion-transporter protein in the membrane. Band 4.1 protein, a component of the junctional complex, binds to the integral membrane protein glycophorin. This dual binding ensures that the membrane is connected to both the spokes and the hubs of the spectrin-actin cytoskeleton.

Platelet Cytoskeleton

Isoforms of spectrin and actin have been found in various nonerythroid cells, suggesting that these cell types have a cortical spectrin-actin cytoskeleton like that present in the erythrocyte. In nonerythroid cells, however, the actin cytoskeleton is more complicated than in erythrocytes. An example of this more complicated structure is seen in the platelet, a small, nonnucleated cell that is important in blood clotting and wound repair. The platelet cytoskeleton must undergo complicated rearrangements that are responsible for a repertoire of changes in cell shape during a blood clotting reaction (Figure 18-8).

Figure 18-8. Changes in shape of platelets during blood clotting.

Figure 18-8

Changes in shape of platelets during blood clotting. Resting cells have a discoid shape (left). When exposed to clotting agents, the cells settle and spread out on the substratum (more...)

These changes in platelet shape could not be generated by the simple cytoskeleton seen in the erythrocyte; thus additional components are needed in the platelet cytoskeleton. The cytoskeleton of an unactivated platelet consists of a rim of microtubules (the marginal band), a cortical actin network, and a cytosolic actin network. The cortical actin network in platelets consists of actin filaments cross-linked into a two-dimensional network by a nonerythroid isoform of spectrin and linked through ankyrin to an anion transporter, the Na/K ATPase, in the membrane. This network thus is somewhat similar to the erythrocyte cytoskeleton. A critical difference between the erythrocyte and platelet cytoskeletons is the presence in the platelet of the second network of actin filaments, which are organized by filamin cross-links into a three-dimensional gel (see Figure 18-5b). The gel fills the cytosol of a platelet and is anchored by filamin to the glycoprotein 1b-IX complex (Gp1b-IX) in the platelet membrane. Although both spectrin and filamin are CH-domain actin cross-linking proteins, filamin also can anchor the cytoskeleton to the plasma membrane.

In order to close a wound, a platelet must be able to transmit cytoskeletal changes within the cell to the blood clot outside the cell. This is effectively accomplished by linking the cytoskeleton to the same proteins in the membrane that also bind to the clot. For example, Gp1b-IX not only binds filamin but also is the membrane receptor for two blood-clotting proteins (Figure 18-9a). Contraction of the cell tightens the clot and closes the wound. The direct connection of the cytoskeleton to the extracellular matrix through shared membrane proteins also occurs in muscle and many other cells.

Figure 18-9. Cross-linkage of actin filament networks to the plasma membrane in platelets, muscle cells, and epithelial cells.

Figure 18-9

Cross-linkage of actin filament networks to the plasma membrane in platelets, muscle cells, and epithelial cells. (a) In platelets a three-dimensional network of actin filaments (more...)

Membrane-Cytoskeleton Linkage in Muscle Cells

Image med.jpgThe critical role of membrane-cytoskeleton linkages in muscle contraction was uncovered by studies on Duchenne muscular dystrophy (DMD), a fatal, degenerative sex-linked genetic disease of muscle that affects about 1 of every 3500 males born. Patients with this disease have a defect in the gene that encodes dystrophin, a very large protein that constitutes 5 percent of the membrane-associated cytoskeleton in muscle cells.

Like filamin in platelets, dystrophin cross-links actin filaments into a supportive cortical network and attaches this network to a glycoprotein complex in the muscle cell membrane (Figure 18-9b). This membrane complex also binds to proteins in the extracellular matrix. Thus the internal cytoskeleton is connected to the external matrix via the dystrophin – membrane glycoprotein linkage. In individuals with DMD, who lack functional dystrophin, the membrane of a muscle cell is not supported by a cortical cytoskeleton and presumably is easily damaged by the stress of repeated muscle contraction.

ERMs: Conformationally Regulated Membrane-Microfilament Proteins

In addition to the membrane linkages by CH domain proteins, several proteins in the apical membrane are cross-linked to microfilaments through the ERM (Ezrin, Radixin, and Moesin) family of proteins. Found in a variety of cell types, ERMs bind membrane proteins, including the cystic fibrosis transmembrane conductance regulator (CFTR) and β2-adrenergic receptor. This interaction with different membrane proteins requires an adapter protein, EBP50, and a Band 4.1 homology domain located at the N-terminus. The crosslink to the cytoskeleton is completed through an actin-binding site located in a C-terminal domain (Figure 18-9c).

Recent evidence suggests that a membrane-microfilament linkage represents an open conformation of the molecule that is regulated by several signaling pathways possibly through both serine/threonine and tyrosine kinases. In the inactive state, the membrane and microfilament binding sites are masked by an intermolecular association of the N- and C-terminal domains. In this closed conformation, ezrin is unable to bind the cytoskeleton, and most of the inactive protein is found in the cytosol. However, the membrane-binding function is correlated with phosphorylation of specific serine and threonine residues in the C-terminal domain.

Actin Bundles Support Projecting Fingers of Membrane

The surfaces of cells in multicellular organisms are studded with numerous membrane projections. Two types of fingerlike membrane projections, microvilli and filopodia, are supported by an internal actin bundle to which the phospholipid membrane is anchored. Because it is held together by protein cross-links, the actin bundle is stiff and provides a rigid structure that reinforces the fragile projecting membrane, enabling it to maintain its long, slender shape.

Microvilli range in length from 0.5 to 10 μm and are found where the cell membrane faces the fluid environment. As discussed in Chapter 15, a dense carpet of microvilli, the brush border, covers the surface of intestinal epithelial cells, greatly increasing the surface area of these cells, whose primary function is to transport nutrients. The cross-linking proteins that hold actin filaments in the core bundle differ in microvilli found on different cell types. Figure 18-10 shows the structure of intestinal microvilli and the various connections supporting them. The membrane is attached to the core bundle by membrane-microfilament linkages of myosin I.

Figure 18-10. Micrograph of intestinal cell showing microvilli.

Figure 18-10

Micrograph of intestinal cell showing microvilli. At the core of each 2-μm-long microvillus, a bundle of actin filaments, cross-linked by fimbrin and villin, stabilizes the fingerlike (more...)

Filopodia, which are much less common than microvilli, attach cells to a solid surface. These projections of the membrane typically are found at the edge of moving or spreading cells (see Figure 18-1). Filopodia are transient structures, present only during the time required to establish a stable contact with the underlying substratum.


  •  A major component of the cytoskeleton, actin is highly conserved in all eukaryotes. F-Actin is a helical filamentous polymer of globular G-actin subunits (see Figure 18-2). An actin polymer, along with bound proteins, constitutes a microfilament, one of the three types of fibers that form the cytoskeleton.
  •  Actin filaments are organized into bundles and networks by a variety of bivalent cross-linking proteins, including spectrin in erythrocytes, filamin in platelets, dystrophin in muscle, and fimbrin and fascin in microvilli (see Table 18-1 and Figures 18-5, 18-7, and 18-9).
  •  Cortical actin networks are attached to the cell membrane by bivalent membrane-microfilament binding proteins. These link cortical actin filaments to integral membrane proteins, which in some cells also bind to the extracellular matrix (see Figures 18-7 and 18-9).
  •  Actin bundles form the core of microvilli and filopodia, fingerlike projections of the plasma membrane. A complex structure of cytoskeletal elements provides support for these membrane extensions.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
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