16-the cytoskeleton

Figure 16-10. Migratory keratocytes from a fish epidermis. (A) Light micrographs of a keratocyte in culture, taken about 15 sec apart. This cell is moving at about 15 m/sec. (B) Keratocyte seen by scanning electron microscopy, showing its broad, flat lamellipodium and small cell body, including the nucleus, carried up above the substratum at the rear. (C) Distribution of cytoskeletal filaments in this cell. Actin filaments (red) fill the large lamellipodium and are responsible for the cell's rapid movement. Microtubules (green) and intermediate filaments (blue) are restricted to the regions close to the nucleus. (A and B, courtesy of Juliet Lee.)

Figure 22-1. 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 rear of the cell, the tail is firmly attached to the surface. The arrow indicates the direction of movement. (b) Fluorescence micrograph of a fan-shaped fibroblast, stained with rhodamine phalloidin. Visible are numerous actin bundles in the lamellipodia and stress fibers in the cell body. [Part (a) courtesy of J. Heath; part (b) courtesy of B. Hollifield.]

Figure 22-9 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).

Figure 22-5. 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 such as filamin are flexible and thus can cross-link pairs of filaments lying at various angles.

Figure 22-6. 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 is attached to the integral membrane glycoprotein complex Gp1b-IX by filamin. Gp1b-IX also binds to proteins in a blood clot outside the platelet. Platelets also possess a two-dimensional cortical network of actin and spectrin similar to that underlying the erythrocyte membrane. (b) In muscle cells dystrophin attaches actin filaments to an integral membrane glycoprotein complex. This complex binds to laminin and agrin in the extracellular matrix (ECM). (c) In epithelial cells, the ERM protein, ezrin, and EBP50 crosslink an actin filament to the cystic fibrosis transmembrane conductance receptor. After activation, ezrin unfolds and oligomerizes to form head-to-tail dimers. The head domain binds EBP50, while the tail domain binds actin.

Figure 18-34. The circumferential belt is located near the apical surface of epithelial cells. In epithelial tissue, a belt of actin and myosin filaments rings the inner surface of the cell adjacent to the adherens junctions, where cell-cell contacts are maintained. The circumferential belt is attached by linker proteins to cell-adhesion molecules in the plasma membrane (Chapter 22).

Figure 22-38. Experimental demonstration that myosin II is required for cytokinesis. The activity of myosin II was inhibited either by deleting its gene or by microinjecting anti-myosin II antibodies into a cell. A cell that lacked myosin II was able to replicate its DNA and nucleus, but it failed to divide; this defect caused the cell to become large and multinucleate. In comparison, an untreated cell during the same period continued to divide and formed a multicellular ball of cells in which each cell

contained a single nucleus.

Figure 22-17. 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).

Figure 22-19. The sliding-filament assay. (a) Schematic diagram illustrates movement of actin filaments across myosin molecules attached to a coverslip. After myosin molecules are adsorbed onto the surface of a glass coverslip, excess myosin is removed; the coverslip then is placed myosin-side down on a glass slide to form a chamber through which solutions can flow. A solution of actin filaments, made visible by staining with rhodamine-labeled phalloidin, is allowed to flow into the chamber, and individual filaments are observed under a fluorescence light microscope. (The coverslip in the diagram is shown inverted from its orientation on the flow chamber to make it easier to see the positions of the molecules.) (b) Sliding movements of fluorescent actin filaments generated by the myosin head can be quantified by video microscopy. These photographs show the positions of three actin filaments (numbered 1, 2, 3) at 30-second intervals. In the presence of ATP, the actin filaments move at a velocity that can vary widely depending on the myosin tested and the assay conditions (ionic strength, temperature, ATP concentration, calcium concentration, etc.). [Part (b) courtesy of M. Footer and S. Kron.]

Figure 22-28. The titin-nebulin filament system stabilizes the alignment of thick and thin filaments in skeletal muscle. (a) A titin filament attaches at one end to the Z disk and spans the distance to the middle of the thick filament. Thick filaments are thus connected at both ends to Z disks through titin. Nebulin is associated with a thin filament from its (+) end at the Z disk to its (  ) end. The large titin and nebulin filaments remain connected to thick and thin filaments during muscle contraction and generate a passive tension when muscle is stretched. (b) To visualize the titin filaments in a sarcomere, muscle is treated with the actin-severing protein gelsolin, which removes the thin filaments. Without a supporting thin filament, nebulin condenses at the Z disk, leaving titin still attached to the Z disk and thick filament.

Figure 22-26. Schematic diagram showing location of capping proteins that stabilize the ends of actin thin filaments. CapZ (green) caps the (+), or barbed, ends of filaments at the Z disk, and tropomodulin (yellow) caps the ( - ), or pointed, ends of thin filaments. The presence of these two proteins at opposite ends of a thin filament prevents actin subunits from dissociating during muscle contraction.

Figure 22-27. The sliding-filament model of contraction in striated muscle. The arrangement of thick myosin and thin actin filaments in the relaxed state is shown in the top diagram. In the presence of ATP and Ca2+, the myosin heads extending from the thick filaments pivot, pulling the actin thin filaments toward the center of the sarcomere. Because the thin filaments are anchored at the Z disks (purple), this movement shortens the sarcomere length in

the contracted state (bottom).

Figure 22-22. The coupling of ATP hydrolysis to movement of myosin along an actin filament. In the absence of bound nucleotide, a myosin head binds actin tightly in a "rigor" state. When ATP binds (step 1 ), it opens the cleft in the head, disrupting the actin-binding site and weakening the interaction with actin. Freed of actin, the myosin head hydrolyzes ATP (step 2 ), causing a conformational change in the head that moves it to a new position, closer to the (+) end of the actin filament, where it rebinds to the filament. As phosphate (Pi) dissociates

from the ATP-binding pocket (step 3 ), the myosin head undergoes a second conformational change    the power stroke    which restores myosin to its rigor conformation. Because myosin is bound to actin, this conformational change exerts a force that causes myosin to move the actin filament. The diagram shows the cycle for a myosin II head that is part of a thick filament, but other myosins attached to a membrane are thought to operate according to the same mechanism. [Adapted from I. Rayment and H. M. Holden, 1994, Trends Biochem. Sci. 19:129.]

Figure 22-32. Three myosin-dependent mechanisms for regulating muscle contraction. (a) In invertebrate muscle, binding of Ca2+ to the myosin regulatory light chain (LC) activates contraction. (b) In vertebrate smooth muscle, phosphorylation of the myosin regulatory light chains on site X by Ca 2+-dependent myosin LC kinase activates contraction. At Ca2+concentrations <10  6 M, the myosin LC kinase is inactive, and a myosin LC phosphatase, which is not dependent on Ca2+ for activity,

dephosphorylates the myosin LC, causing muscle relaxation. (c) Activation of Rho kinase also leads to

phosphorylation of the myosin regulatory LC at ser 19.

Figure 18-42. Steps in keratinocyte movement. In a fast-moving cell such as a fish epidermal cell, movement begins with extension of one or more lamellipodia from the leading edge of the cell (step 1); some lamellipodia adhere to the substratum via focal adhesions (step 2). Then the bulk of the cytoplasm in the cell body flows forward (step 3). The trailing edge of the cell remains attached to the substratum until the tail eventually detaches and retracts into the cell body (step 4). See the text for

more discussion.

igure 22-43. A model of the molecular events at the leading edge of moving cells. The polymerization of actin filaments at the (+) end, stimulated by profilin located at the leading-edge membrane, pushes the membrane outward. Other proteins like Vasp and Arp2/3 may participate in directing assembly. Simultaneously, cofilin induces the loss of subunits from the (  ) ends of filaments. Arp2/3 and actin cross-linking proteins stabilize the actin filaments into networks and bundles. In addition, myosin I is thought to link actin filaments to the leading-edge plasma membrane.

Figure 22-12 Model of the complementary roles of profilin and thymosin 4 in regulating polymerization of G-actin. (a) At the cell membrane, profilin is bound to PIP2, a membrane

lipid, while most of the G-actin is complexed with thymosin 4 and thus cannot polymerize. (b) In response to an extracellular signal, such as chemotactic molecules that stimulate actin assembly, profilin is released from the membrane by hydrolysis of PIP2. The released profilin

displaces thymosin 4, forming profilin   G-actin complexes that can assemble into filaments. (c) The profilin-actin complexes interact with proline-rich proteins in the membrane, where profilin adds actin monomers to the (+) end of actin filaments. Eventually, the incorporation of monomers into filaments depletes the pools of profilin-actin and thymosin 4   actin complexes. (d) ADP   G-actin subunits that have dissociated from a filament are converted into ATP   G-actin by profilin, thus helping to replenish the cytoplasmic pool of ATP   G-actin.

Figure 22-39. Cytoplasmic streaming in cylindrical giant algae. (a) The center of a Nitella cell is filled with a single large water-filled vacuole, which is surrounded by a layer of moving cytoplasm (indicated by blue arrows). A nonmoving layer of cortical cytoplasm filled with chloroplasts lies just under the plasma membrane (enlarged lower figure). On the inner side of this layer are bundles of stationary actin filaments (red), all oriented with the same polarity. A myosinlike motor protein (blue dots) carries portions of the endoplasmic reticulum (ER) along the actin filaments. The movement of the ER network propels the entire viscous cytoplasm, including organelles that are enmeshed in the ER network. (b) An electron micrograph of the cortical cytoplasm shows a large vesicle connected to an underlying bundle of actin filaments. This vesicle, which is part of the endoplasmic reticulum (ER) network, contacts the stationary actin filaments and moves along them by a myosinlike motor. [Part (b) from B. Kachar.]

6

THE CYTOSKELETON