Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Cytoskeleton
Intermediate filaments

Intermediate filaments (IFs) are tough, durable protein fibers found in the Cytoplasm of most higher Eukaryotic Cells. Their Structure resembles interwoven ropes, with a diameter of 8-10 nm—intermediate between the thick and thin filaments in Muscle, where IFs were first described; in thickness, they also occupy a middle ground between Actin filaments and microtubules. In most animal cells, they form a characteristic "basket" around The Nucleus, from which they extend along slightly curved pathways toward The Cell periphery. IFs are particularly abundant in regions subject to mechanical stress, such as epithelia, where these filaments help anchor cells to one another (via desmosomes, see Section 14.1.4), in nerve fibers, and throughout the cytoplasm of smooth muscle cells. When cells are extracted with high- and low-ionic-strength solutions and solubilized with nonionic detergents, IFs remain intact, whereas most other cytoskeletal structures are destroyed. In fact, the term "Cytoskeleton" was originally coined to describe these exceptionally resilient and insoluble fibers.

11.5.1. Intermediate filaments are formed from four types of fibrillar Polypeptides [51, 52]

Unlike actin monomers and tubulin, which are Globular Proteins, IF subunits have an elongated, fibrillar shape. They assemble into longitudinal bundles with staggered overlaps, forming long threads of high mechanical strength. The lateral interactions that build up IFs often involve only a portion of the protein subunit's molecule, allowing The structure of the remaining part to vary considerably without altering the overall architecture of the filament. Consequently, unlike actin filaments and microtubules, IFs are constructed from polypeptides with a wide range of molecular weights—from 40 to 130 kDa, depending on the cell type.

Based on their Primary Structure, intermediate filaments are divided into four major groups (Table 11-5). Type I IF proteins are most characteristic of epithelial cells and include two subfamilies of Keratins: acidic keratins and neutral or basic keratins. Keratin filaments are always heteropolymers composed of an equal number of subunits from each of these two subfamilies. In fact, keratins represent the largest group of IF proteins; at least 19 different forms are already known in human epithelia, with another 8 in Hair and Nails. Many types of epithelia that differ morphologically and functionally also synthesize distinct forms of keratins.

Class="center">Table 11-5. Major Types of intermediate filament proteins

¹⁾ Because these proteins migrate abnormally slowly in SDS-polyacrylamide gels, their molecular mass was previously overestimated.

Fig. 11-73. Immunofluorescence micrograph of interphase kangaroo rat epithelial cells (PtK2). The cells were double-labeled with Antibodies against vimentin (A) and keratin (B). Note that the cellular systems of vimentin and keratin filaments exist separately, despite having a similar distribution. (Courtesy of Mary Osborn.)

Type II IF proteins include 1) vimentin, 2) desmin, and 3) glial fibrillary acidic protein. Vimentin is widely distributed in cells of mesenchymal origin, including fibroblasts, Blood vessel endothelial cells, and leukocytes; it is frequently synthesized in cultured cells and appears transiently in various cell types during development. Desmin is found in both smooth and striated muscle cells, whereas glial fibrillary acidic protein forms glial filaments in specific types of glial cells (astrocytes and certain Schwann cells) within The Nervous system. All of these proteins are capable of self-assembly in vitro to form homopolymers, as well as forming heteropolymers with other Type II proteins. The latter capacity is also manifested in vivo: copolymers of vimentin with desmin and of vimentin with glial fibrillary acidic protein have been detected in certain cell types.

Type III IF proteins make up neurofilaments—an essential cytoskeletal component in the axons and dendrites of Nerve Cells. In vertebrates, there are three such proteins, collectively known as the "neurofilament triplet." Finally, Type IV IF proteins are the nuclear lamins (Section 11.5.5); they share sequence similarities with other IF proteins but possess several distinctive features. Most notably, they form highly ordered two-dimensional filamentous networks that undergo rapid disassembly and reassembly at specific stages of mitosis.

All eukaryotic cells synthesize nuclear lamins and at least one type of cytoplasmic IF protein. Some cells contain Two Types of cytoplasmic IFs that form separate structures. Examples include certain epithelial cells that harbor distinct networks of keratin and vimentin filaments (Fig. 11-73). 11-28

11.5.2. Intermediate filaments are formed from dimeric subunits with a rodlike central domain [53]

Despite significant differences in size, all cytoplasmic IF proteins are encoded by genes of a single multigene family. In the Introduction/19.html">Primary structure of all these polypeptides, There is a homologous central region of approximately 310 Amino Acids that forms an extended a-helix interrupted by three short non-a-helical linkers (Fig. 11-74). Furthermore, large stretches of this central domain exhibit a sequence characteristic of polypeptides capable of forming a coiled-coil (see Section 11.1.6). Similar to Tropomyosin or the tail of muscle Myosin, this two-stranded coil is a dimer composed of two identical IF polypeptides. The two chains in the IF homodimer are arranged parallel to one another, with globular domains flanking both ends of the central rod domain. During IF assembly, the rod domains interact with one another to form a uniform filament core, while the globular domains—which vary greatly in size among different IF proteins—protrude from the filament surface. One model of IF assembly from dimeric subunits is illustrated in Fig. 11-75.

11.5.3. Intermediate filaments extend from the nuclear envelope to the cell periphery [54]

When cultured cells are stained with antibodies against one of the cytoplasmic IF proteins (e.g., vimentin), a lace-like network of filaments surrounding the nucleus and permeating the cytoplasm is typically visible (see Fig. 11-73). Structurally, this network is distinct from other cytoskeletal components, although in places its filaments appear to run parallel to Cytoplasmic microtubules. The Organization of cytoplasmic IFs likely depends on interactions with microtubules, since depolymerization of microtubules by agents such as colchicine causes the entire IF network to collapse into a perinuclear "cap." It is thought that many cytoplasmic IFs are attached to the nuclear envelope and normally pulled outward toward the cell periphery by the microtubules to which they are connected.

The organization of IFs in the cytoplasm may also be determined by their interactions with The Plasma Membrane. In avian erythrocytes (which, unlike mammalian red Blood Cells, possess a nucleus and IFs), vimentin is believed to be linked to the plasma membrane via ankyrin (Section 6.2.4). In epithelial cells, keratin IFs are anchored to the plasma membrane at desmosomes—specialized Intercellular junctions that help hold neighboring cells together (Section 14.1.4). Because the keratin filaments of each cell are connected via desmosomes to corresponding filaments in adjacent cells, they form a continuous network spanning the entire epithelium.

Fig. 11-74. All intermediate filament proteins possess a homologous central region (about 310 amino acid residues) that forms an extended a-helix with three short non-helical interruptions. The N-terminal and C-terminal domains are non-helical and vary widely in size and Amino Acid Sequence among different intermediate filament proteins.

Fig. 11-75. One of the current models for intermediate filament (IF) assembly. A monomer (A) associates with an identical monomer to form a dimer (B) in which the conserved a-helical regions lie parallel, winding around each other. Two such dimers then align side-by-side to form a protofilament 48 nm long and 3 nm thick, consisting of four polypeptide chains (C). These protofilaments subsequently assemble into increasingly larger structures by staggering longitudinally (D and E). The final 10-nm-thick intermediate filament structure consists of eight protofilament rows (32 polypeptide chains) joined into a long, rope-like cable (F). At the top is an electron micrograph of such a "mature" filament. It remains unknown whether IFs are polar structures, like actin and tubulin, or nonpolar, like the DNA double helix (or equivalently, whether the two coiled strands within a protofilament are in a parallel or antiparallel orientation). (Micrograph courtesy of N. Geisler and K. Weber.)

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11.5.4. Intermediate filament assembly can be regulated by phosphorylation [55]

Isolated intermediate filaments (IFs) are extremely stable in an ionic environment mimicking the cytoplasm; furthermore, there is no significant pool of unpolymerized IF proteins in the cell (unlike the case for actin and tubulin). Yet the cell clearly can regulate the number, length, and distribution of its intermediate filaments, indicating an ability to control their assembly and disassembly. An important factor in this control is the phosphorylation of specific residues within IF proteins. Vimentin, for instance, exists in both unphosphorylated and phosphorylated forms. When isolated vimentin filaments are phosphorylated by protein kinase, they break down into smaller fragments. However, the most striking example of the crucial role phosphorylation plays in controlling IF disassembly is the nuclear lamins, which undergo depolymerization whenever a cell enters mitosis.

11.5.5. The nuclear lamina is formed by a specialized class of intermediate filaments [56]

The nuclear lamina is a protein meshwork (typically 10 to 20 nm thick) that lines the inner face of the nuclear envelope (see Fig. 9-1). It consists of an orthogonal lattice of intermediate filaments (Fig. 11-76D) built in mammals from three type V intermediate filament proteins: lamins A, B, and C (see Fig. 11-74 and Table 11-3). Lamins form dimers characterized by a central rod domain and two globular heads at one end (Fig. 11-76B). Under physiological pH and Ionic strength, these dimers spontaneously associate to form filaments that closely resemble cytoplasmic intermediate filaments in thickness and repeating structure.

However, nuclear lamins differ from cytoplasmic intermediate filaments in several respects. The most obvious difference is the ORGANIZATION OF THE resulting filaments into an orthogonal lattice (Fig. 11-76A), although this arrangement likely requires association with other proteins. In addition, the nuclear lamina is a highly dynamic structure. When mammalian cells enter mitosis, the transient phosphorylation of several Serine residues in the lamins triggers the reversible disassembly of the nuclear lamina into tetramers of hyperphosphorylated lamins A and C and membrane-associated lamin B. Upon the cell's return to interphase, the lamins are dephosphorylated, and an intact nuclear envelope re-forms around the separated Chromosomes (Section 13.5.11).

11.5.6. Keratin Filaments Are Remarkably Diverse [52]

Of all intermediate filament types, keratins are the most stable and long-lived, as well as the most diverse. Epithelia with primitive organization, such as those in the developing embryo, as well as certain mature tissues (like the Liver), contain two types of keratins—one acidic and one neutral. The epithelia of other Organs (such as the Tongue, Urinary Bladder, and Sweat Glands) express six or more different keratins, with the specific Complement varying according to anatomical Location. Because of their diversity and stability, keratin filaments can serve as a molecular "fingerprint" to trace THE ORIGIN OF certain epithelial tumors. Keratins are even more diverse in the epidermis, which forms a tough, Stratified Epithelium (Section 17.4.2). Different layers of the epidermis express distinct sets of keratins. As cells progress toward the outermost layers and eventually die, their keratin filaments become increasingly cross-linked to one another and to associated proteins, turning the cross-linked keratin cytoskeleton into a vital protective barrier on the body's surface. Specialized epithelial cells that produce surface structures such as hair, claws, and feathers provide additional local variations in the keratin repertoire. Thus, intermediate filaments protect animals against heat and Water loss, supply weapons and camouflage, or, conversely, serve to attract mates (via coloration).

Figure 11-76. A. Electron micrograph of a region of the nuclear lamina in a Xenopus oocyte (prepared by freeze-drying and metal shadowing). The lamina is formed by a highly ordered orthogonal meshwork of intermediate filaments composed of nuclear lamins. B. Isolated lamin dimers (L) (electron micrograph, metal shadowing). They resemble muscle myosin (M) in shape, featuring a rodlike tail and two globular heads, though they are much smaller. The globular heads are formed by two large C-terminal domains. (Courtesy of Ueli Aebi.)

11.5.7. What is the Function of Intermediate Filaments?

Animal cells can survive without intermediate filaments. In the Central Nervous System, myelin-producing glial cells are completely devoid of them. Microinjection of antibodies against intermediate filament proteins into cultured fibroblasts causes the filaments to collapse without any noticeable effect on cell organization or behavior. It appears likely that The primary function of most intermediate filaments is to provide mechanical support for the cell and its nucleus. In epidermal sheets, intermediate filaments form a "transcellular" network spanning multiple cells, whose role is presumably to withstand external mechanical stress. Neurofilaments in nerve fibers resist the mechanical deformations generated by animal movement; without them, these long, thin cytoplasmic cylinders would easily snap. Desmin filaments in muscle cells provide mechanical support for sarcomeres, whereas vimentin filaments surround (and likely support) large lipid droplets in fat cells. Yet, if the sole function of intermediate filaments is to resist tensile forces, why are there so many different variants of their protein subunits? What is the purpose of their variable domains, given that they do not appear to participate in building the filament backbone? Definitive Answers to these questions are not yet available, but it is clear that both the supportive role of intermediate filaments and the way they connect with other cellular components vary widely among cell types. For example, desmin filaments anchoring the Z-disk borders in striated muscle apparently possess binding sites for specific Z-disk proteins; neurofilaments experience lower mechanical loads, but they can bundle together laterally to form continuous cables up to a meter or more in length. This is likely why neurofilaments feature prominent side-arm projections along their entire length that are absent in other intermediate filaments (Figure 11-77).

Figure 11-77. Electron micrograph of two types of intermediate filaments found in Nervous Tissue (prepared by quick-freezing and deep-etching). A. Neurofilaments in an axon are linked by numerous cross-bridges, an organization thought to give this long cellular process high tensile strength. These cross-bridges are believed to be formed by the long, non-helical C-terminal tails of the largest neurofilament proteins (see Figure 11-74). B. Intermediate filaments (known as glial filaments) in an astrocyte. These experience lower mechanical stresses; their surface is relatively smooth, and few cross-bridges connect them. (Courtesy of N. Hirokawa.)

The variable domains of intermediate filament proteins may provide distinct binding capacities for other proteins. By modulating filament properties, these variable regions determine not only the self-assembly capacity of the filament but also how it interacts with other cellular components (such as microtubules and the plasma membrane). This strategy contrasts sharply with that of the other two major cytoskeletal networks—actin filaments and microtubules—whose polymers are structurally invariant, relying instead on distinct sets of accessory proteins (actin-binding proteins and microtubule-associated proteins) to execute diverse Functions. Thus, The Role of the variable domains in intermediate filament proteins is analogous to that of the accessory proteins of actin filaments and microtubules, with the sole difference that the former are covalently linked to the filament subunits, whereas the latter exist as separate molecules.

Summary

Intermediate filaments (IFs) are rope-like polymers built from elongated polypeptide subunits. They appear to provide mechanical support for cells or help them withstand tensile stress. There are numerous tissue-specific forms of IFs assembled from different polypeptides: keratin filaments in epithelial cells, neurofilaments in neurons, glial filaments in astrocytes and Schwann cells, desmin filaments in muscle fibers, and vimentin filaments in fibroblasts and many other cell types. A distinct family of IF proteins comprises the nuclear lamins, which form the fibrous meshwork (lamina) lining the inner nuclear envelope and are present in all eukaryotic cells.

The polypeptides that make up various types of intermediate filaments differ in amino acid sequence and, to a large extent, in molecular weight. However, all share a homologous central domain that forms a rigid coiled-coil structure upon dimerization. These dimeric subunits assemble into large, staggered bundles to form intermediate filaments. The rod domains of the subunits form the structural core of the IFs, while the globular domains at both ends project outward, conferring functional diversity on the IFs. It is this Variability that adapts the mechanical properties of IFs and their interactions with other cellular components to the specialized needs of specific cell types.