2. MICROFILAMENTS

2.1. Estructura i Acoblament

Monòmer d’Actina-G

Filaments d’Actina (Actina-F)

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 nm) divided by a central cleft into two approximately equal-sized lobes and four subdomains, numbered I – IV. ATP (red) binds at the bottom of the cleft and contacts both lobes (the yellow ball represents Mg2+). The N- and C-termini lie in subdomain I. (b) In the electron microscope, negatively stained actin filaments appear as long, flexible, and twisted strands of beaded subunits. Because of the twist, the filament appears alternately thinner (7 nm diameter) and thicker (9 nm diameter) (arrows). (c) In one model of the arrangement of subunits in an actin filament, the subunits lie in a tight helix along the filament, as indicated by the arrow. One repeating unit consists of 28 subunits (13 turns of the helix), covering a distance of 72 nm. Only 14 subunits are shown in the figure. The ATP-binding cleft is oriented in the same direction (top) in all actin subunits in the filament. As discussed later, this end of a filament is designated the (−) end; the opposite end is the (+) end. [Part (a) adapted from C. E. Schutt et al., 1993, Nature 365:810, courtesy of M. Rozycki; part (b) courtesy of R. Craig; part (c) see M. F. Schmid et al., 1994, J. Cell Biol. 124:341, courtesy of M. Schmid.] (Fuente: Lodish et al., 2000)

Model d’Organització de les Subunitats d’Actina G als Filaments d’Actina-F

Relació entre la Polimerització de l’Actina G i la Hidròlisi de l’ATP

Figure 16-49. Actin filaments. (A) Electron micrographs of negatively stained actin filaments. (B) The helical arrangement of actin molecules in an actin filament. (A, courtesy of Roger Craig.) (Fuente: Alberts et al., 1993)

Figure 16-51. The trapping of ADP in an actin filament. An actin molecule has a structure that is related to that of the ubiquitous enzyme hexokinase, with two domains that are hinged around an ATP-binding site. The bound ATP is hydrolyzed to ADP immediately after the molecule becomes incorporated into an actin filament. In order for the ADP to be replaced by ATP, the hinge would have to open. But in the actin filament the two domains in each actin molecule are held together by interactions with neighboring subunits, thereby keeping the hinge closed and trapping the ADP in the actin filament until the filament depolymerises. (Fuente: Alberts et al., 1993)

Nucleació Allargament Estat Estacionari

Polimerització d’Actina-G in vitro

Figure 18-11. The three phases of G-actin polymerization in vitro. (a) During the initial nucleation phase, ATP – G-actin monomers (pink) slowly form stable complexes of actin (purple). These nuclei are more rapidly elongated in the second phase by addition of subunits to both ends of the filament. In the third phase, the ends of actin filaments are in a steady state with monomeric ATP – G-actin. After their incorporation into a filament, subunits slowly hydrolyze ATP and become stable ADP – F-actin (white). Note that the ATP-binding clefts (black triangles) of all the subunits are oriented in the same direction in F-actin. (b) Time course of the in vitro polymerization reaction (pink curve) reveals the initial lag period. If some actin filament fragments are added at the start of the reaction to act as nuclei, elongation proceeds immediately without any lag period (purple curve). (Fuente: Lodish et al., 2000)

Demostració Experimental de la Polaritat dels Filaments d’Actina

La polimerització es produeix molt més ràpidament a l’extrem positiu

del filament

Figure 18-13. Experimental demonstration of unequal growth rates at the two ends of an actin filament. (a) When short myosin-decorated filaments are the nuclei for actin polymerization, the resulting elongated filaments have a much longer undecorated (+) end than (−) end. This result indicates that ATP – G-actin monomers are added much faster at the (+) end than at the (−) end. (b) Blocking the (+) or (−) ends of a filament with actin-capping proteins permits growth only at the opposite end. In polymerization assays with capped filaments, the critical concentration (Cc) is determined by the unblocked growing end. Such assays show that the Cc at the (+) end is much lower than the Cc at the (−) end. (c) At G-actin concentrations intermediate between the Cc values for the (−) and (+) ends, actin subunits can flow through the filaments by attaching preferentially to the (+) end and dissociating preferentially from the (−) end of the filament, a phenomenon known as “treadmilling.” The oldest subunits in a treadmilling filament lie at the (−) end. [Part (a) courtesy of T. Pollard.] (Fuente: Lodish et al., 2000)

2.2. Proteïnes d’Unió a l’Actina Proteïnes que Formen Feixos

Proteïnes que Formen Xarxes

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 to form the spikelike filopodia. In the lamellar region of the cell, the actin filaments form a network that fills the cytosol. In contrast to the roughly parallel alignment of bundled filaments, the filaments in networks lie at various angles approaching 90°. (Fuente: Lodish et al., 2000)

Figure 16-66. The formation of two types of actin filament bundles. (A) α-actinin, which is a homodimer, cross-links actin filaments into loose bundles, which allow the motor protein myosin-II (not shown) to participate in the assembly. Fimbrin cross-links actin filaments into tight bundles, which exclude this motor protein. Fimbrin and α-actinin tend to exclude each other because of the very different spacing of the actin filament bundles that they form. (Fuente: Alberts et al., 1993)

Figure 18-5. Long cross-linking proteins such as filamin are flexible and thus can cross-link pairs of filaments lying at various angles (Fuente: Lodish et al., 2000)

+ Calci

Proteïnes Fragmentadores: la Gelsolina

Figure 18-16. Action of gelsolin in severing actin filaments. At cytosolic Ca2+ levels below 10−6 M, gelsolin does not bind to actin filaments. At higher Ca2+ concentrations, binding of gelsolin to filaments causes a distortion that disrupts the noncovalent bonds between subunits. One consequence of severing is that actin networks are broken apart and new ends are created. Gelsolin remains bound to the (+) end of the fragments, preventing addition of monomers, while the uncapped (−) ends rapidly disassemble. Thus severing contributes to the turnover of actin filaments. Note that even though the filaments are shortened, they still remain cross-linked. The other major severing protein, cofilin, functions in a similar way, but it is regulated by reversible phosphorylation and dephosphorylation. (Fuente: Lodish et al., 2000)

Figure 16-79. Some of the major classes of actin-binding proteins found in most vertebrate cells. Actin is shown in red,while the actin-binding proteins are shown in green. The molecular mass of each protein is given in kilodaltons (kD). (Fuente: Alberts et al., 1993)

MICROVELLOVITATS INTESTINALS

2.3. Microvellositats

Figure 16-77. Freeze-etch electron micrograph of an intestinal epithelial cell, showing the terminal web beneath the apical plasma membrane. Bundles of actin filaments forming the core of microvilli extend into the terminal web, where they are linked together by a complex set of cytoskeletal proteins that includes spectrin and myosin-II. Beneath the terminal web is a layer of intermediate filaments. (From N. Hirokawa and J.E. Heuser, J. Cell Biol. 91:399-409, 1981, by copyright permission of the Rockefeller University Press.) (Fuente: Alberts et al., 1993)

2.4. Proteïnes Motores: la Miosina

Estructura dels Diversos Tipus de Miosina

La Proteolisi de la Miosina II Revela els Diferents Dominis Estructurals

Figure 18-20. Structure of various myosin molecules. (a) The three major myosin proteins are organized into head, neck, and tail domains, which carry out different functions. The head domain binds actin and has ATPase activity. The light chains, bound to the neck domain, regulate the head domain. The tail domain dictates the specific role of each myosin in the cell. Note that myosin II, the form that functions in muscle contraction, is a dimer with a long rigid coiled-coil tail. (b) Proteolysis of myosin II reveals its domain structure. For example, most proteases cleave myosin II at the base of the neck domain to generate a paired-head and neck fragment, called heavy meromyosin (HMM), and a rodlike tail fragment, called light meromyosin (LMM). Further digestion of HMM with papain splits off the neck region (S2 fragment) and leads to separation of the two head domains into single myosin head fragments (S1 fragments). (Fuente: Lodish et al., 2000)

Funcions dels Dominis de la Cua de la Miosina

L’Estructura del Sarcòmer Muscular

Figure 18-21. Functions of the myosin tail domain. (a) Myosin I and myosin V are localized to cellular membranes by undetermined sites in their tail domains. As a result, these myosins are associated with intracellular membrane vesicles or the cytoplasmic face of the plasma membrane. (b) In contrast, the coiled-coil tail domains of myosin II molecules pack side by side, forming a thick filament from which the heads project. In a skeletal muscle, the thick filament is bipolar. Heads are at the ends of the thick filament and are separated by a bare zone, which consists of the side-by-side tails. (Fuente: Lodish et al., 2000)

Figure 16-89. The myosin and actin filaments of a sarcomere overlap with the same relative polarity on either side of the midline. (Fuente: Alberts et al., 1993)

Organització de les Fibres d’Actina i Miosina al Múscul Estriat i al Llis

Figure 18-26. General structure of skeletal and smooth muscle. (a) Skeletal muscle tissue is composed of bundles of multinucleated muscle cells, or myofibers. Each muscle cell is packed with bundles of actin and myosin filaments, organized into myofibrils that extend the length of the cell. Packed end to end in a myofibril is a chain of sarcomeres, the functional units of contraction. The internal organization of the filaments gives skeletal muscle cells a striated appearance. (b) Smooth muscle is composed of loosely organized spindle-shaped cells that contain a single nucleus. Loose bundles of actin and myosin filaments pack the cytoplasm of smooth muscle cells. These bundles are connected to dense bodies in the cytosol and to the membrane at attachment plaques. (Fuente: Lodish et al., 2000)

Cicle de la Miosina

Figure 16-91. The cycle of changes by which a myosin molecule walks along an actin filament. (Based on I. Rayment et al., Science261:50-58, 1993. © 1993 the AAAS.) (Fuente: Alberts et al., 1993)

Formacions transitòries semblants a les musculars

Relació entre els contactes focals i les fibres d'estrès a fibroblasts en cultiu

Figure 16-72. Musclelike contractile assemblies in nonmuscle cells. Each assembly contains myosin-II filaments in addition to actin filaments(Fuente: Alberts et al., 1993)

Figure 16-74. The relation between focal contacts and stress fibers in cultured fibroblasts. Focal contacts are best seen in living cells by reflection-interference microscopy (A). In this technique, light is reflected from the lower surface of a cell attached to a glass slide, and the focal contacts appear as dark patches. (B) Immunofluorescence staining of the same cell (after fixation) with antibodies to actin shows that most of the cell's actin filament bundles (or stress fibers) terminate at or close to a focal contact. (Fuente: Alberts et al., 1993)

Interaccions Competitives i Cooperatives entre les Proteïnes d’Unió a l’Actina: el Comportament de

l’Escorça Cel·lular

Figure 16-78. Some examples of competitive and cooperative interactions between actin-binding proteins. The arrowhead at the end of each actin filament indicates the minus end. Tropomyosin and filamin both bind strongly to actin filaments, but their binding is competitive. Because tropomyosin binds cooperatively to actin filaments, either tropomyosin or filamin will predominate over large regions of the actin filament network. Other actin-binding proteins, such as α-actinin or myosin-II, will be excluded from specific sites by a competitive interaction; thus, for example, α-actinin binds all along pure actin filaments in vitro, but it binds relatively weakly to actin filaments in cells, where it is largely confined to sites near the plus ends because of competition with other proteins. Alternatively, binding can be enhanced through cooperative interaction; thus tropomyosin appears to enhance the binding of myosin-II to actin filaments. Multiple interactions of these types between the many different types of actin-binding proteins are thought to be responsible for the complex variety of actin networks found in all eucaryotic cells (see Figure16-79 for key to symbols). (Fuente: Alberts et al., 1993)

3. MIGRACIÓ CEL·LULAR

4. FILAMENTS INTERMEDIS

Estructura i Organització dels

Filaments Intermedis

Figure 19-51. Levels of organization and assembly of intermediate filaments. IF proteins form parallel homo- or heterodimers with a highly conserved coiled-coil core domain and nonhelical tails and heads, which are variable in length and sequence. The central core domain contains three nonhelical spacer elements. A tetramer is formed by antiparallel, staggered side-by-side aggregation of two identical dimers. Tetramers aggregate end to end, forming a protofilament; pairs of protofilaments then laterally associate into a protofibril. Lateral association of four protofibrils form a cylindrical filament 10 nm thick. [Adapted from D. A. D. Parry and P. M. Steinert, 1995, Intermediate Filament Structure, Springer-Verlag, pp. 27 and 101.] (Fuente: Lodish et al., 2000)

Figure 19-52. Electron micrograph of a neurofilament purified from spinal cord. After the filament is rotary-shadowed with metals, long whiskers are seen to project from its walls. These projections correspond to the N- and C-terminal domains of the neurofilament-protein tetramers. (Fuente: Lodish et al., 2000)

Figure 16-12. The intermediate filaments in the cytoplasm of a tissue culture cell. Rat kangaroo epithelial cells (Ptk2 cells) in interphase were labeled with antibodies to one class of intermediate filaments (called keratin filaments) and examined by fluorescence microscopy. (Fuente: Alberts et al., 1993)

Figure 19-56. Intermediate filaments anchored to desmosomes and hemidesmosomes. A schematic diagram of cells in the basal layer of the epidermis shows the keratin filaments crisscrossing the cell and making connections to desmosomes (blue) between cells, hemidesmosomes (gray) at the basement membrane, and peripheral proteins (orange) at the apical membrane. [Adapted from E. Fuchs and D. Cleveland, 1998, Science, 279:514.] (Fuente: Lodish et al., 2000)

Figure 16-19. Blistering of the skin caused by a mutant keratin gene. A mutant gene encoding a truncated keratin protein (lacking both the amino- and carboxyl-terminal domains) was expressed in a transgenic mouse. The defective protein assembles with the normal keratins and thereby disrupts the keratin filament network in the basal cells. Light micrographs of normal (A) and mutant (B) skin show that the blistering results from the rupturing of cells in the basal layer of the mutant epidermis. The sketch in (C) of three cells observed by electron microscopy in the basal layer of the mutant epidermis shows that the cells rupture between the nucleus and the hemidesmosomes, which connect the keratin filaments to the underlying basal lamina. (From P.A. Coulombe, M.E. Hutton, R. Vassar, and E. Fuchs, J. Cell Biol. 115:1661-1674, 1991, by copyright permission of the Rockefeller University Press.) (Fuente: Alberts et al., 1993)

La Queratina i les Malalties de la Pell