Cytoskeletal Elements The ability of eukaryotic cells to adopt a variety of shapes, organize the many components in their interior, interact mechanically with the environment, and carry out coordinated movements depends on the cytoskeleton—an intricate network of protein filaments that extends throughout the cytoplasm. The cytoskeleton is built on a framework of three types of protein filaments: • intermediate filaments microtubules, and actin filaments. Each type of filament has distinct mechanical properties and is formed from a different protein subunit. A family of fibrous proteins forms the intermediate filaments; globular tubulin subunits form microtubules; and globular actin subunits form actin filaments

Each cytoskeletal element has distinct properties. Microtubules are long, hollow, unbranched tubes composed of subunits of the protein tubulin. Microfilaments are solid, thinner structures, often organized into a branching network and composed of the protein actin. Intermediate filaments are tough, ropelike fibers composed of a variety of related proteins. Intermediate filaments are ropelike fibers Microtubules are hollow Actin filaments (also known as with a diameter of about 10 nm; they are cylinders made of the protein microfilaments) are helical polymers made of fibrous intermediate filament tubulin. They are long and of the protein actin. They are flexible proteins. One type of intermediate filament straight and typically have one structures, with a diameter of about 7 forms a meshwork called the nuclear lamina end attached to a single nm, that are organized into a variety just beneath the inner nuclear membrane. microtubule-organizing center of linear bundles, two-dimensional Other types extend across the cytoplasm, called a centrosome. With an networks, and three-dimensional gels. giving cells mechanical strength and outer diameter of 25 nm, Although actin filaments are dispersed distributing the mechanical stresses in an microtubules are more rigid than throughout the cell, they are most epithelial tissue by spanning the cytoplasm actin filaments or intermediate highly concentrated in the cortex, the from one cell–cell junction to another. filaments, and they rupture layer of cytoplasm just beneath the Intermediate filaments are very flexible and when stretched. plasma membrane. have great tensile strength. They deform under stress but do not rupture. 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. 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. 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). 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. 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. Because actin polymerization is reversible, filaments can depolymerize by the dissociation of actin subunits, allowing actin filaments to be broken down when necessary. 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 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.

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. Organization of Actin Filaments

Individual actin filaments are assembled into two general types of structures which play different roles in the cell. Actin bundles • In bundles, the actin filaments are crosslinked into closely packed parallel arrays. • 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.

Actin networks

▪ In networks, the actin filaments are loosely crosslinked in orthogonal arrays that form three-dimensional meshworks with the properties of semisolid gels. ▪ The proteins that organize actin filaments into networks tend to be large flexible proteins that can crosslink perpendicular filaments. There are two structurally and functionally distinct types of actin bundles, involving different actin-bundling proteins.

The first type of bundle, containing closely spaced actin filaments aligned in parallel, supports projections of the plasma membrane, such as microvilli. 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 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 40 nm apart distance than those crosslinked by fimbrin of 14 nm). The increased spacing between filaments allows the motor protein myosin to interact with the actin filaments in these bundles, which enables them to contract. The actin filaments in networks are held together by large actin-binding proteins , such as filamin. 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. Association of Actin Filaments with Plasma Membrane Actin filaments are highly concentrated at the periphery of the cell, where they form a three-dimensional network beneath the plasma membrane. 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) used for plasma membrane as 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.

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. 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).

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) 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. 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. 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. The actin cytoskeleton is similarly anchored to regions of cell-cell contact called adherens junctions. 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. The cadherins form a complex with cytoplasmic proteins called catenins, which associate with actin filaments.

Intermediate Filaments • Intermediate filaments are strong, flexible, ropelike fibers that provide mechanical strength to cells that are subjected to physical stress, including neurons, muscle cells, and the epithelial cells that line the body’s cavities. • They are solid, unbranched filaments with a diameter of 10–12 nm. • The filaments are called “intermediate” because, in the smooth muscle cells where they were first discovered, their diameter (about 10 nm) is between that of the thinner actin filaments and the thicker myosin filaments. Intermediate filaments are the toughest and most durable of the cytoskeletal filaments: when cells are treated with concentrated salt solutions and nonionic detergents, the intermediate filaments survive, while most of the rest of the cytoskeleton is destroyed. • They typically form a network throughout the cytoplasm, surrounding the nucleus and extending out to the cell periphery. There they are often anchored to the plasma membrane at cell–cell junctions called desmosomes, where the plasma membrane is connected to that of another cell. • Intermediate filaments are also found within the nucleus of all eukaryotic cells. There they form a meshwork called the nuclear lamina, which underlies and strengthens the nuclear envelope. Unlike microfilaments and microtubules, IFs are a chemically heterogeneous group of structures that, in humans, are encoded by approximately 70 different genes. The polypeptide subunits of IFs can be divided based on the type of cell in which they are found. Intermediate Filament Assembly and Disassembly The basic building block of IF assembly is thought to be a rodlike tetramer formed by two dimers that become aligned side by side in a staggered fashion with their N- and C-termini pointing in opposite (antiparallel) directions. Because the dimers point in opposite directions, the tetramer itself lacks polarity. 8 tetramers associate with one another in a side-by-side (lateral) arrangement to form a filament that is one unit in length (about 60 nm). Filaments associate with one another in an end-to-end fashion to form the highly elongated intermediate filament. No direct involvement of either ATP or GTP. Because the tetrameric building blocks lack polarity, so too does the assembled filament, which is another important feature that distinguishes IFs from other cytoskeletal elements. Microtubules Microtubules are hollow, relatively rigid, tubular structures, and they occur in nearly every eukaryotic cell.

Microtubules are components of a diverse array of structures, including the mitotic spindle of dividing cells and the core of cilia and flagella. Microtubules have an outer diameter of 25 nm and a wall thickness of approximately 4 nm, and may extend across the length or breadth of a cell. The wall of a microtubule is composed of globular proteins arranged in longitudinal rows, termed protofilaments, that are aligned parallel to the long axis of the tubule. Microtubules are built from subunits—molecules of tubulin—each of which is itself a dimer composed of two very similar globular proteins called α-tubulin and β-tubulin, bound tightly together by noncovalent interactions. The tubulin dimers stack together, again by noncovalent bonding, to form the wall of the hollow cylindrical microtubule. This tubelike structure is made of 13 parallel protofilaments, each a linear chain of tubulin dimers with α- and β- tubulin alternating along its length. Each protofilament has a structural polarity, Microtubules prepared from living tissue typically contain additional proteins, called microtubule-associated proteins (or MAPs). MAPs comprise a heterogeneous collection of proteins. MAPs generally increase the stability of microtubules and promote their assembly. The microtubule-binding activity of the various MAPs is controlled primarily by the addition and removal of phosphate groups from particular amino acid residues. An abnormally high level of phosphorylation of one particular MAP, called tau, has been implicated in the development of several fatal neurodegenerative disorders, including Alzheimer’s disease. Microtubules grow from specialized organizing centers that control the location, number, and orientation of the microtubules. In animal cells, for example, the centrosome—which is typically close to the cell nucleus when the cell is not in mitosis—organizes an array of microtubules that radiates outward through the cytoplasm. The centrosome consists of a pair of centrioles, surrounded by a matrix of proteins. The centrosome matrix includes hundreds of ringshaped structures formed from a special type of tubulin, called γ-tubulin, and each γ-tubulin ring complex serves as the starting point, or nucleation site, for the growth of one microtubule. The αβ-tubulin dimers add to each γ-tubulin ring complex in a specific orientation, with the result that the minus end of each microtubule is embedded in the centrosome, and growth occurs only at the plus end that extends into the cytoplasm.

Cilia are hairlike structures about 0.25 μm in diameter, covered by plasma membrane, that extend from the surface of many kinds of eukaryotic cells; each cilium contains a core of stable microtubules, arranged in a bundle, that grow from a cytoplasmic basal body, which serves as an organizing center. Cilia beat in a whiplike fashion, either to move fluid over the surface of a cell or to propel single cells through a fluid. The flagella (singular flagellum) that propel sperm and many protozoa are much like cilia in their internal structure but are usually very much longer. They are designed to move the entire cell, rather than moving fluid across the cell surface. Flagella propagate regular waves along their length, propelling the attached cell along. The microtubules in cilia and flagella are slightly different from cytoplasmic microtubules; they are arranged in a curious and distinctive pattern, which was one of the most striking revelations of early electron microscopy. A cross section through a cilium shows nine doublet microtubules arranged in a ring around a pair of single microtubules.