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 µm/sec. (B) Keratocyte seen by scanning electronmicroscopy, showing its broad, flat lamellipodium and small cell body, including the nucleus, carried up abovethe substratum at the rear. (C) Distribution of cytoskeletal filaments in this cell. Actin filaments (red) fill the largelamellipodium and are responsible for the cells rapid movement. Microtubules (green) and intermediatefilaments (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 culturedfibroblast. At the front of the cell, filopodia,lamellipodia, and ruffles project from the cellmembrane. At the rear of the cell, the tail is firmlyattached to the surface. The arrow indicates thedirection of movement. (b) Fluorescence micrographof a fan-shaped fibroblast, stained with rhodaminephalloidin. Visible are numerous actin bundles in thelamellipodia 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 stablecomplexes 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 thirdphase, the ends of actin filaments are in a steady state with monomeric ATP G-actin. After their incorporation into a filament, subunits slowly hydrolyzeATP 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 thestart 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 abundle. (b) Long cross-linking proteins such as filamin are flexible and thus cancross-link pairs of filaments lying at various angles.
Figure 22-6. Cross-linkage of actin filamentnetworks to the plasma membrane in platelets,muscle cells, and epithelial cells. (a) In platelets athree-dimensional network of actin filaments isattached to the integral membrane glycoproteincomplex Gp1b-IX by filamin. Gp1b-IX also bindsto proteins in a blood clot outside the platelet.Platelets also possess a two-dimensional corticalnetwork of actin and spectrin similar to thatunderlying the erythrocyte membrane. (b) In musclecells dystrophin attaches actin filaments to anintegral membrane glycoprotein complex. Thiscomplex binds to laminin and agrin in theextracellular matrix (ECM). (c) In epithelial cells,the ERM protein, ezrin, and EBP50 crosslink anactin filament to the cystic fibrosis transmembraneconductance receptor. After activation, ezrinunfolds and oligomerizes to form head-to-taildimers. The head domain binds EBP50, while thetail domain binds actin.
Figure 18-34. The circumferentialbelt is located near the apical surfaceof epithelial cells. In epithelial tissue,a belt of actin and myosin filamentsrings the inner surface of the celladjacent to the adherens junctions,where cell-cell contacts aremaintained. The circumferential beltis attached by linker proteins to cell-adhesion molecules in the plasmamembrane (Chapter 22).
Figure 22-38. Experimental demonstration thatmyosin II is required for cytokinesis. The activity ofmyosin II was inhibited either by deleting its gene orby microinjecting anti-myosin II antibodies into acell. A cell that lacked myosin II was able to replicateits DNA and nucleus, but it failed to divide; thisdefect caused the cell to become large andmultinucleate. In comparison, an untreated cellduring the same period continued to divide andformed a multicellular ball of cells in which each cellcontained 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 outdifferent functions. The head domain binds actin and has ATPase activity. The light chains, bound to the neck domain, regulate the head domain. The taildomain 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 rigidcoiled-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 togenerate a paired-head and neck fragment, called heavy meromyosin (HMM), and a rodlike tail fragment, called light meromyosin (LMM). Furtherdigestion 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 aglass slide to form a chamber through which solutions can flow. A solution of actin filaments, made visible by staining with rhodamine-labeled phalloidin, isallowed to flow into the chamber, and individual filaments are observed under a fluorescence light microscope. (The coverslip in the diagram is showninverted from its orientation on the flow chamber to make it easier to see the positions of the molecules.) (b) Sliding movements of fluorescent actin filamentsgenerated by the myosin head can be quantified by video microscopy. These photographs show the positions of three actin filaments (numbered 1, 2, 3) at30-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 assayconditions (ionic strength, temperature, ATP concentration, calcium concentration, etc.). [Part (b) courtesy of M. Footer and S. Kron.]
Figure 22-28. The titin-nebulinfilament system stabilizes thealignment of thick and thinfilaments in skeletal muscle. (a) Atitin filament attaches at one end tothe Z disk and spans the distance tothe middle of the thick filament.Thick filaments are thus connectedat both ends to Z disks through titin.Nebulin is associated with a thinfilament from its (+) end at the Zdisk to its ( ) end. The large titinand nebulin filaments remainconnected to thick and thinfilaments during muscle contractionand generate a passive tension whenmuscle is stretched. (b) To visualizethe titin filaments in a sarcomere,muscle is treated with the actin-severing protein gelsolin, whichremoves the thin filaments. Withouta supporting thin filament, nebulincondenses at the Z disk, leaving titinstill attached to the Z disk and thickfilament.
Figure 22-26. Schematic diagram showing location of cappingproteins that stabilize the ends of actin thin filaments. CapZ (green)caps the (+), or barbed, ends of filaments at the Z disk, andtropomodulin (yellow) caps the ( - ), or pointed, ends of thinfilaments. The presence of these two proteins at opposite ends of athin filament prevents actin subunits from dissociating duringmuscle contraction.
Figure 22-27. The sliding-filamentmodel of contraction in striatedmuscle. The arrangement of thickmyosin and thin actin filaments inthe relaxed state is shown in thetop diagram. In the presence ofATP and Ca2+, the myosin headsextending from the thick filamentspivot, pulling the actin thinfilaments toward the center of thesarcomere. Because the thinfilaments are anchored at the Zdisks (purple), this movementshortens the sarcomere length inthe contracted state (bottom).
Figure 22-22. The coupling of ATP hydrolysis tomovement of myosin along an actin filament. In theabsence of bound nucleotide, a myosin head binds actintightly in a "rigor" state. When ATP binds (step 1 ), itopens the cleft in the head, disrupting the actin-bindingsite and weakening the interaction with actin. Freed ofactin, the myosin head hydrolyzes ATP (step 2 ), causing aconformational change in the head that moves it to a newposition, closer to the (+) end of the actin filament, whereit rebinds to the filament. As phosphate (Pi) dissociatesfrom the ATP-binding pocket (step 3 ), the myosin headundergoes a second conformational change the powerstroke which restores myosin to its rigor conformation.Because myosin is bound to actin, this conformationalchange exerts a force that causes myosin to move the actinfilament. The diagram shows the cycle for a myosin IIhead that is part of a thick filament, but other myosinsattached to a membrane are thought to operate accordingto 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) Ininvertebrate 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 Ca 2+concentrations <10 6 M, the myosin LCkinase is inactive, and a myosin LC phosphatase, which is not dependent on Ca 2+ for activity,dephosphorylates the myosin LC, causing muscle relaxation. (c) Activation of Rho kinase also leads tophosphorylation of the myosin regulatory LC at ser 19.
Figure 18-42. Steps in keratinocyte movement. In a fast-movingcell such as a fish epidermal cell, movement begins withextension of one or more lamellipodia from the leading edge ofthe cell (step 1); some lamellipodia adhere to the substratum viafocal adhesions (step 2). Then the bulk of the cytoplasm in thecell body flows forward (step 3). The trailing edge of the cellremains attached to the substratum until the tail eventuallydetaches and retracts into the cell body (step 4). See the text formore discussion.
igure 22-43. A model of the molecular events at theleading edge of moving cells. The polymerization of actinfilaments at the (+) end, stimulated by profilin located atthe leading-edge membrane, pushes the membraneoutward. Other proteins like Vasp and Arp2/3 mayparticipate in directing assembly. Simultaneously, cofilininduces the loss of subunits from the ( ) ends offilaments. Arp2/3 and actin cross-linking proteinsstabilize the actin filaments into networks and bundles. Inaddition, myosin I is thought to link actin filaments to theleading-edge plasma membrane.
Figure 22-12 Model of the complementary roles of profilin and thymosin β4 in regulatingpolymerization of G-actin. (a) At the cell membrane, profilin is bound to PIP 2, a membranelipid, while most of the G-actin is complexed with thymosin β4 and thus cannot polymerize. (b)In response to an extracellular signal, such as chemotactic molecules that stimulate actinassembly, profilin is released from the membrane by hydrolysis of PIP 2. The released profilindisplaces thymosin β4, forming profilin G-actin complexes that can assemble into filaments.(c) The profilin-actin complexes interact with proline-rich proteins in the membrane, whereprofilin adds actin monomers to the (+) end of actin filaments. Eventually, the incorporation ofmonomers into filaments depletes the pools of profilin-actin and thymosin β4 actincomplexes. (d) ADP G-actin subunits that have dissociated from a filament are converted intoATP G-actin by profilin, thus helping to replenish the cytoplasmic pool of ATP G-actin.
Figure 22-39. Cytoplasmic streaming in cylindrical giantalgae. (a) The center of a Nitella cell is filled with a singlelarge water-filled vacuole, which is surrounded by a layerof moving cytoplasm (indicated by blue arrows). Anonmoving layer of cortical cytoplasm filled withchloroplasts lies just under the plasma membrane(enlarged lower figure). On the inner side of this layer arebundles of stationary actin filaments (red), all orientedwith the same polarity. A myosinlike motor protein (bluedots) carries portions of the endoplasmic reticulum (ER)along the actin filaments. The movement of the ERnetwork propels the entire viscous cytoplasm, includingorganelles that are enmeshed in the ER network. (b) Anelectron micrograph of the cortical cytoplasm shows alarge vesicle connected to an underlying bundle of actinfilaments. This vesicle, which is part of the endoplasmicreticulum (ER) network, contacts the stationary actinfilaments and moves along them by a myosinlike motor.[Part (b) from B. Kachar.]