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Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000.

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The Cell: A Molecular Approach. 2nd edition.

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Structure and Organization of Actin Filaments

The major cytoskeletal protein of most cells is actin, which polymerizes to form actin filaments—thin, flexible fibers approximately 7 nm in diameter and up to several micrometers in length (Figure 11.1). Within the cell, actin filaments (also called microfilaments) are organized into higher-order structures, forming bundles or three-dimensional networks with the properties of semisolid gels. The assembly and disassembly of actin filaments, their crosslinking into bundles and networks, and their association with other cell structures (such as the plasma membrane) are regulated by a variety of actin-binding proteins, which are critical components of the actin cytoskeleton. Actin filaments are particularly abundant beneath the plasma membrane, where they form a network that provides mechanical support, determines cell shape, and allows movement of the cell surface, thereby enabling cells to migrate, engulf particles, and divide.

Figure 11.1. Actin filaments.

Figure 11.1

Actin filaments. Electron micrograph of actin filaments. (Courtesy of Roger Craig, University of Massachusetts Medical Center.)

Assembly and Disassembly of Actin Filaments

Actin was first isolated from muscle cells, in which it constitutes approximately 20% of total cell protein, in 1942. Although actin was initially thought to be uniquely involved in muscle contraction, it is now known to be an extremely abundant protein (typically 5 to 10% of total protein) in all types of eukaryotic cells. Yeasts have only a single actin gene, but higher eukaryotes have several distinct types of actin, which are encoded by different members of the actin gene family. Mammals, for example, have at least six distinct actin genes: Four are expressed in different types of muscle and two are expressed in nonmuscle cells. All of the actins, however, are very similar in amino acid sequence and have been highly conserved throughout the evolution of eukaryotes. Yeast actin, for example, is 90% identical in amino acid sequence to the actins of mammalian cells.

The three-dimensional structures of both individual actin molecules and actin filaments were determined in 1990 by Kenneth Holmes, Wolfgang Kabsch, and their colleagues. Individual actin molecules are globular proteins of 375 amino acids (43 kd). Each actin monomer (globular [G] actin) has tight binding sites that mediate head-to-tail interactions with two other actin monomers, so actin monomers polymerize to form filaments (filamentous [F] actin) (Figure 11.2). Each monomer is rotated by 166o in the filaments, which therefore have the appearance of a double-stranded helix. Because all the actin monomers are oriented in the same direction, actin filaments have a distinct polarity and their ends (called the plus and minus ends) are distinguishable from one another. This polarity of actin filaments is important both in their assembly and in establishing a unique direction of myosin movement relative to actin, as discussed later in the chapter.

Figure 11.2. Assembly and structure of actin filaments.

Figure 11.2

Assembly and structure of actin filaments. (A) Actin monomers (G actin) polymerize to form actin filaments (F actin). The first step is the formation of dimers and trimers, which then grow by the addition of monomers to both ends. (B) Structure of an (more...)

The assembly of actin filaments can be studied in vitro by regulation of the ionic strength of actin solutions. In solutions of low ionic strength, actin filaments depolymerize to monomers. Actin then polymerizes spontaneously if the ionic strength is increased to physiological levels. The first step in actin polymerization (called nucleation) is the formation of a small aggregate consisting of three actin monomers. Actin filaments are then able to grow by the reversible addition of monomers to both ends, but one end (the plus end) elongates five to ten times faster than the minus end. The actin monomers also bind ATP, which is hydrolyzed to ADP following filament assembly. Although ATP is not required for polymerization, actin monomers to which ATP is bound polymerize more readily than those to which ADP is bound. As discussed below, ATP binding and hydrolysis play a key role in regulating the assembly and dynamic behavior of actin filaments.

Because actin polymerization is reversible, filaments can depolymerize by the dissociation of actin subunits, allowing actin filaments to be broken down when necessary (Figure 11.3). Thus, an apparent equilibrium exists between actin monomers and filaments, which is dependent on the concentration of free monomers. The rate at which actin monomers are incorporated into filaments is proportional to their concentration, so there is a critical concentration of actin monomers at which the rate of their polymerization into filaments equals the rate of dissociation. At this critical concentration, monomers and filaments are in apparent equilibrium.

Figure 11.3. Reversible polymerization of actin monomers.

Figure 11.3

Reversible polymerization of actin monomers. Actin polymerization is a reversible process, in which monomers both associate with and dissociate from the ends of actin filaments. The rate of subunit dissociation (koff) is independent of monomer concentration, (more...)

As noted earlier, the two ends of an actin filament grow at different rates, with monomers being added to the fast-growing end (the plus end) five to ten times faster than to the slow-growing (minus) end. Because ATP-actin dissociates less readily than ADP-actin, this results in a difference in the critical concentration of monomers needed for polymerization at the two ends. This difference can result in the phenomenon known as treadmilling, which illustrates the dynamic behavior of actin filaments (Figure 11.4). For the system to be at an overall steady state, the concentration of free actin monomers must be intermediate between the critical concentrations required for polymerization at the plus and minus ends of the actin filaments. Under these conditions, there is a net loss of monomers from the minus end, which is balanced by a net addition to the plus end. Treadmilling requires ATP, with ATP-actin polymerizing at the plus end of filaments while ADP-actin dissociates from the minus end. Although the role of treadmilling in the cell is unclear, it may reflect the dynamic assembly and disassembly of actin filaments required for cells to move and change shape.

Figure 11.4. Treadmilling.

Figure 11.4

Treadmilling. The minus ends grow less rapidly than the plus ends of actin filaments. This difference in growth rate is reflected in a difference in the critical concentration for addition of monomers to the two ends of the filament. Actin bound to ATP (more...)

It is noteworthy that several drugs useful in cell biology act by binding to actin and affecting its polymerization. For example, the cytochalasins bind to the plus ends of actin filaments and block their elongation. This results in changes in cell shape as well as inhibition of some types of cell movements (e.g., cell division following mitosis), indicating that actin polymerization is required for these processes. Another drug, phalloidin, binds tightly to actin filaments and prevents their dissociation into individual actin molecules. Phalloidin labeled with a fluorescent dye is frequently used to visualize actin filaments by fluorescence microscopy.

Within the cell, both the assembly and disassembly of actin filaments are regulated by actin-binding proteins (Figure 11.5). The turnover of actin filaments is about 100 times faster within the cell than it is in vitro, and this rapid turnover of actin plays a critical role in a variety of cell movements. The key protein responsible for actin filament disassembly within the cell is cofilin, which binds to actin filaments and enhances the rate of dissociation of actin monomers from the minus end. In addition, cofilin can sever actin filaments, generating more ends and further enhancing filament disassembly.

Figure 11.5. Effects of actin-binding proteins on filament turnover.

Figure 11.5

Effects of actin-binding proteins on filament turnover. Cofilin binds to actin filaments and increases the rate of dissociation of actin monomers (bound to ADP) from the minus end. Cofilin remains bound to the ADP-actin monomers, preventing their reassembly (more...)

Cofilin preferentially binds to ADP-actin, so it remains bound to actin monomers following filament disassembly and sequesters them in the ADP-bound form, preventing their reincorporation into filaments. However, another actin-binding protein, profilin, can reverse this effect of cofilin and stimulate the incorporation of actin monomers into filaments. Profilin acts by stimulating the exchange of bound ADP for ATP, resulting in the formation of ATP-actin monomers, which dissociate from cofilin and are then available for assembly into filaments. Other proteins (Arp2/3 proteins) can serve as nucleation sites to initiate the assembly of new filaments, so cofilin, profilin, and the Arp2/3 proteins (as well as other actin-binding proteins) can act together to promote the rapid turnover of actin filaments and remodeling of the actin cytoskeleton which is required for a variety of cell movements and changes in cell shape. As might be expected, the activities of cofilin, profilin, and Arp2/3 proteins are controlled by a variety of cell signaling mechanisms (discussed in Chapter 13), allowing actin polymerization to be appropriately regulated in response to environmental stimuli.

Organization of Actin Filaments

Individual actin filaments are assembled into two general types of structures, called actin bundles and actin networks, which play different roles in the cell (Figure 11.6). In bundles, the actin filaments are crosslinked into closely packed parallel arrays. In networks, the actin filaments are loosely crosslinked in orthogonal arrays that form three-dimensional meshworks with the properties of semisolid gels. The formation of these structures is governed by a variety of actin-binding proteins that crosslink actin filaments in distinct patterns.

Figure 11.6. Actin bundles and networks.

Figure 11.6

Actin bundles and networks. (A) Electron micrograph of actin bundles (arrowheads) projecting from the actin network (arrows) underlying the plasma membrane of a macrophage. The bundles support cell surface projections called microspikes or filopodia (see (more...)

All of the actin-binding proteins involved in crosslinking contain at least two domains that bind actin, allowing them to bind and crosslink two different actin filaments. The nature of the association between these filaments is then determined by the size and shape of the crosslinking proteins (see Figure 11.6). The proteins that crosslink actin filaments into bundles (called actin-bundling proteins) usually are small rigid proteins that force the filaments to align closely with one another. In contrast, the proteins that organize actin filaments into networks tend to be large flexible proteins that can crosslink perpendicular filaments. These actin-crosslinking proteins appear to be modular proteins consisting of related structural units. In particular, the actin-binding domains of many of these proteins are similar in structure. They are separated by spacer sequences that vary in length and flexibility, and it is these differences in the spacer sequences that are responsible for the distinct crosslinking properties of different actin-binding proteins.

There are two structurally and functionally distinct types of actin bundles, involving different actin-bundling proteins (Figure 11.7). The first type of bundle, containing closely spaced actin filaments aligned in parallel, supports projections of the plasma membrane, such as microvilli (see Figures 11.15 and 11.16). In these bundles, all the filaments have the same polarity, with their plus ends adjacent to the plasma membrane. An example of a bundling protein involved in the formation of these structures is fimbrin, which was first isolated from intestinal microvilli and later found in surface projections of a wide variety of cell types. Fimbrin is a 68-kd protein, containing two adjacent actin-binding domains. It binds to actin filaments as a monomer, holding two parallel filaments close together.

Figure 11.7. Actin-bundling proteins.

Figure 11.7

Actin-bundling proteins. Actin filaments are associated into two types of bundles by different actin-bundling proteins. Fimbrin has two adjacent actin-binding domains (ABD) and crosslinks actin filaments into closely packed parallel bundles in which the (more...)

Figure 11.15. Electron micrograph of microvilli.

Figure 11.15

Electron micrograph of microvilli. The microvilli (arrows) of intestinal epithelial cells are fingerlike projections of the plasma membrane. They are supported by actin bundles anchored in a dense region of the cortex called the terminal web. (Fred E. (more...)

Figure 11.16. Organization of microvilli.

Figure 11.16

Organization of microvilli. The core actin filaments of microvilli are crosslinked into closely packed bundles by fimbrin and villin. They are attached to the plasma membrane along their length by lateral arms, consisting of myosin I and calmodulin. The (more...)

The second type of actin bundle is composed of filaments that are more loosely spaced and are capable of contraction, such as the actin bundles of the contractile ring that divides cells in two following mitosis. The looser structure of these bundles (which are called contractile bundles) reflects the properties of the crosslinking protein α-actinin. In contrast to fimbrin, α-actinin binds to actin as a dimer, each subunit of which is a 102-kd protein containing a single actin-binding site. Filaments crosslinked by α-actinin are consequently separated by a greater distance than those crosslinked by fimbrin (40 nm apart instead of 14 nm). The increased spacing between filaments allows the motor protein myosin to interact with the actin filaments in these bundles, which (as discussed later) enables them to contract.

The actin filaments in networks are held together by large actin-binding proteins, such as filamin (Figure 11.8). Filamin (also called actin-binding protein or ABP-280) binds actin as a dimer of two 280-kd subunits. The actin-binding domains and dimerization domains are at opposite ends of each subunit, so the filamin dimer is a flexible V-shaped molecule with actin-binding domains at the ends of each arm. As a result, filamin forms cross-links between orthogonal actin filaments, creating a loose three-dimensional meshwork. As discussed in the next section, such networks of actin filaments underlie the plasma membrane and support the surface of the cell.

Figure 11.8. Actin networks and filamin.

Figure 11.8

Actin networks and filamin. Filamin is a dimer of two large (280-kd) subunits, forming a flexible V-shaped molecule that crosslinks actin filaments into orthogonal networks. The carboxy-terminal dimerization domain is separated from the amino-terminal (more...)

Association of Actin Filaments with the Plasma Membrane

Actin filaments are highly concentrated at the periphery of the cell, where they form a three-dimensional network beneath the plasma membrane (see Figure 11.6). This network of actin filaments and associated actin-binding proteins (called the cell cortex) determines cell shape and is involved in a variety of cell surface activities, including movement. The association of the actin cytoskeleton with the plasma membrane is thus central to cell structure and function.

Red blood cells (erythrocytes) have proven particularly useful for studies of both the plasma membrane (discussed in the next chapter) and the cortical cytoskeleton. The principal advantage of red blood cells for these studies is that they contain no nucleus or internal organelles, so their plasma membrane and associated proteins can be easily isolated without contamination by the various internal membranes that are abundant in other cell types. In addition, human erythrocytes lack other cytoskeletal components (microtubules and intermediate filaments), so the cortical cytoskeleton is the principal determinant of their distinctive shape as biconcave discs (Figure 11.9).

Figure 11.9. Morphology of red blood cells.

Figure 11.9

Morphology of red blood cells. Scanning electron micrograph of red blood cells illustrating their biconcave shape. (Omikron/Photo Researchers, Inc.)

The major protein that provides the structural basis for the cortical cytoskeleton in erythrocytes is the actin-binding protein spectrin, which is related to filamin (Figure 11.10). Erythrocyte spectrin is a tetramer consisting of two distinct polypeptide chains, called α and β, with molecular weights of 240 and 220 kd, respectively. The β chain has a single actin-binding domain at its amino terminus. The α and β chains associate laterally to form dimers, which then join head to head to form tetramers with two actin-binding domains separated by approximately 200 nm. The ends of the spectrin tetramers then associate with short actin filaments, resulting in the spectrin-actin network that forms the cortical cytoskeleton of red blood cells (Figure 11.11). The major link between the spectrin-actin network and the plasma membrane is provided by a protein called ankyrin, which binds both to spectrin and to the cytoplasmic domain of an abundant transmembrane protein called band 3. An additional link between the spectrin-actin network and the plasma membrane is provided by protein 4.1, which binds to spectrin-actin junctions as well as recognizing the cytoplasmic domain of glycophorin (another abundant transmembrane protein).

Figure 11.10. Structure of spectrin.

Figure 11.10

Structure of spectrin. Spectrin is a tetramer consisting of two α and two β chains. Each β chain has a single actin-binding domain (ABD) at its amino terminus. Both α and β chains contain multiple repeats of α-helical (more...)

Figure 11.11. Association of the erythrocyte cortical cytoskeleton with the plasma membrane.

Figure 11.11

Association of the erythrocyte cortical cytoskeleton with the plasma membrane. The plasma membrane is associated with a network of spectrin tetramers crosslinked by short actin filaments in association with protein 4.1. The spectrin-actin network is linked (more...)

Other types of cells contain linkages between the cortical cytoskeleton and the plasma membrane that are similar to those observed in red blood cells. Proteins related to spectrin (nonerythroid spectrin is also called fodrin), ankyrin, and protein 4.1 are expressed in a wide range of cell types, where they fulfill functions analogous to those described for erythrocytes. For example, a family of proteins related to protein 4.1 (the ERM proteins) link actin filaments to the plasma membranes of many different kinds of cells and the spectrin-related protein filamin (see Figure 11.8) constitutes a major link between actin filaments and the plasma membrane of blood platelets. Another member of this group of spectrin-related proteins is dystrophin, which is of particular interest because it is the product of the gene responsible for two types of muscular dystrophy (Duchenne's and Becker's). These X-linked inherited diseases result in progressive degeneration of skeletal muscle, and patients with the more severe form of the disease (Duchenne's muscular dystrophy) usually die in their teens or early twenties. Molecular cloning of the gene responsible for this disorder revealed that it encodes a large protein (427 kd) that is either absent or abnormal in patients with Duchenne's or Becker's muscular dystrophy, respectively. The sequence of dystrophin further indicated that it is related to spectrin, with a single actin-binding domain at its amino terminus and a membrane-binding domain at its carboxy terminus. Like spectrin, dystrophin forms dimers that link actin filaments to transmembrane proteins of the muscle cell plasma membrane. These transmembrane proteins in turn link the cytoskeleton to the extracellular matrix, which plays an important role in maintaining cell stability during muscle contraction.

In contrast to the uniform surface of red blood cells, most cells have specialized regions of the plasma membrane that form contacts with adjacent cells, tissue components, or other substrates (such as the surface of a culture dish). These regions also serve as attachment sites for bundles of actin filaments that anchor the cytoskeleton to areas of cell contact. These attachments of actin filaments are particularly evident in fibroblasts maintained in tissue culture (Figure 11.12). Such cultured fibroblasts secrete extracellular matrix proteins (discussed in Chapter 12) that stick to the plastic surface of the culture dish. The fibroblasts then attach to the culture dish via the binding of transmembrane proteins (called integrins) to the extracellular matrix. The sites of attachment are discrete regions (called focal adhesions) that also serve as attachment sites for large bundles of actin filaments called stress fibers.

Figure 11.12. Stress fibers and focal adhesions.

Figure 11.12

Stress fibers and focal adhesions. Fluorescence microscopy of a human fibroblast in which actin filaments have been been stained with a fluorescent dye. Stress fibers are revealed as bundles of actin filaments anchored at sites of cell attachment to the (more...)

Stress fibers are contractile bundles of actin filaments, crosslinked by α-actinin, that anchor the cell and exert tension against the substratum. They are attached to the plasma membrane at focal adhesions via interactions with integrin. These associations, which are complex and not well understood, may be mediated by several other proteins, including talin and vinculin (Figure 11.13). For example, both talin and α-actinin bind to the cytoplasmic domains of integrins. Talin also binds to vinculin, which in turn interacts with actin. Other proteins found at focal adhesions may also participate in the attachment of actin filaments, and a combination of these interactions may be responsible for the linkage of actin filaments to the plasma membrane.

Figure 11.13. Attachment of stress fibers to the plasma membrane at focal adhesions.

Figure 11.13

Attachment of stress fibers to the plasma membrane at focal adhesions. Focal adhesions are mediated by the binding of integrins to proteins of the extracellular matrix. Stress fibers (bundles of actin filaments crosslinked by α-actinin) are then (more...)

The actin cytoskeleton is similarly anchored to regions of cell-cell contact called adherens junctions (Figure 11.14). In sheets of epithelial cells, these junctions form a continuous beltlike structure (called an adhesion belt) around each cell in which an underlying contractile bundle of actin filaments is linked to the plasma membrane. Contact between cells at adherens junctions is mediated by transmembrane proteins called cadherins, which are discussed further in Chapter 12. The cadherins form a complex with cytoplasmic proteins called catenins, which associate with actin filaments.

Figure 11.14. Attachment of actin filaments to adherens junctions.

Figure 11.14

Attachment of actin filaments to adherens junctions. Cell-cell contacts at adherens junctions are mediated by cadherins, which serve as sites of attachment of actin bundles. In sheets of epithelial cells, these junctions form a continuous belt of actin (more...)

Protrusions of the Cell Surface

The surfaces of most cells have a variety of protrusions or extensions that are involved in cell movement, phagocytosis, or specialized functions such as absorption of nutrients. Most of these cell surface extensions are based on actin filaments, which are organized into either relatively permanent or rapidly rearranging bundles or networks.

The best-characterized of these actin-based cell surface protrusions are microvilli, fingerlike extensions of the plasma membrane that are particularly abundant on the surfaces of cells involved in absorption, such as the epithelial cells lining the intestine (Figure 11.15). The microvilli of these cells form a layer on the apical surface (called a brush border) that consists of approximately a thousand microvilli per cell and increases the exposed surface area available for absorption by 10- to 20-fold. In addition to their role in absorption, specialized forms of microvilli, the stereocilia of auditory hair cells, are responsible for hearing by detecting sound vibrations.

Their abundance and ease of isolation have facilitated detailed structural analysis of intestinal microvilli, which contain closely packed parallel bundles of 20 to 30 actin filaments (Figure 11.16). The filaments in these bundles are crosslinked in part by fimbrin, an actin-bundling protein (discussed earlier) that is present in surface projections of a variety of cell types. However, the major actin-bundling protein in intestinal microvilli is villin, a 95-kd protein present in microvilli of only a few specialized types of cells, such as those lining the intestine and kidney tubules. Along their length, the actin bundles of microvilli are attached to the plasma membrane by lateral arms consisting of the calcium-binding protein calmodulin in association with myosin I, which may be involved in movement of the plasma membrane along the actin bundle of the microvillus. At their base, the actin bundles are anchored in a spectrin-rich region of the actin cortex called the terminal web, which crosslinks and stabilizes the microvilli.

In contrast to microvilli, many surface protrusions are transient structures that form in response to environmental stimuli. Several types of these structures extend from the leading edge of a moving cell and are involved in cell locomotion (Figure 11.17). Pseudopodia are extensions of moderate width, based on actin filaments crosslinked into a three-dimensional network, that are responsible for phagocytosis and for the movement of amoebas across a surface. Lamellipodia are broad, sheetlike extensions at the leading edge of fibroblasts, which similarly contain a network of actin filaments. Many cells also extend microspikes or filopodia, thin projections of the plasma membrane supported by actin bundles. The formation and retraction of these structures is based on the regulated assembly and disassembly of actin filaments, as discussed in the following section.

Figure 11.17. Examples of cell surface projections involved in phagocytosis and movement.

Figure 11.17

Examples of cell surface projections involved in phagocytosis and movement. (A) Scanning electron micrograph showing pseudopodia of a macrophage engulfing a tumor cell during phagocytosis. (B) An amoeba with several extended pseudopodia. (C) A tissue (more...)

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Molecular Medicine: Muscular Dystrophy and the Cytoskeleton. The muscular dystrophies are a group of hereditary diseases characterized by the progressive loss of muscle cells. Duchenne's muscular dystrophy (DMD) is the most common and severe form, affecting (more...)

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

Copyright © 2000, Geoffrey M Cooper.
Bookshelf ID: NBK9908


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