Chapter 3 Cytoskeleton © 2020 Elsevier Inc. All rights reserved. Figure 3–1 . Organization of a skeletal muscle. A skeletal muscle consists of bundles of fibers called fasciculi. Each fasciculus consists of a bundle of long, multinucleated muscle fibers that are the cells of the muscle tissue. Within the muscle cells are the myofibrils, which are composed of highly organized arrangements of myosin II (thick) and actin (thin) filaments. The extreme structural organization of the myofilaments is the basis for the striated appearance of skeletal muscle. The myofilaments are organized into the functional units of skeletal muscle, the sarcomere, which extends from one Z disk to the next. Actin thin filaments extend from the Z disk (light-staining I band) toward the center of the sarcomere, where they interdigitate with the myosin thick filaments (dark-staining A band). Cross sections through the sarcomere near the Z disk (1) show the ~ 8-nm actin thin filaments, whereas sections in the regions of the A band (4) demonstrate that each − 15-nm-thick filament is surrounded by a hexagonal array of six actin thin filaments. Sections through the sarcomere near the center in the segment of the A band referred to as the H band show the organization of the myosin thick filaments (2), whereas cross sections through the center of the H band demonstrate a network of filaments that participate in the assembly of the thick filaments to form the M line (3). (Modified from Bloom W, Fawcett DW. A Textbook of Histology, 10th ed. Philadelphia: WB Saunders, 1975.) © 2020 Elsevier Inc. All rights reserved. 2 Figure 3–2. Electron microscopy of skeletal muscle. A longitudinal section through a skeletal muscle cell demonstrates the regular pattern of cross-striations derived from the myofibrils. As shown in this low-magnification electron micrograph, the skeletal muscle cell has many myofibrils aligned in parallel. In this repeating structure, one can easily discern the Z disk. Scale bar = 0.3 μm for A, I, and H bands and M line of the sarcomere. (inset) Terminal cisternae of the sarcoplasmic reticulum (SR) and associated transverse tubule (T). (Courtesy Dr. Phillip Fields.) © 2020 Elsevier Inc. All rights reserved. 3 Figure 3–3. Structure of globular (G) and filamentous (F) actin. (A) G-actin is a 43-kDa monomer with four structural domains. ATP and ADP bind to G-actin within the groove separating domains 1 and 3. (B) F-actin is a helical filament composed of polymerized G-actin monomers (spheres). The filament undergoes a complete turn of the helix every 14 G-actin monomers, or 37 nm. © 2020 Elsevier Inc. All rights reserved. 4 Figure 3–4. Polymerization of actin. The polymerization of actin occurs in three stages: (1) a lag phase in which an actin trimer nucleation site is formed; (2) a polymerization phase, during which G-actin monomers are added preferentially at the plus end of the actin filament; and (3) a steady state, at which actin monomers are being added at the plus end at the same rate they are being removed at the minus end. © 2020 Elsevier Inc. All rights reserved. 5 Figure 3–5. Formation of actin thin filaments and their arrangement in the sarcomere. Globular actin monomers polymerize through head-to-tail association to form the helical filamentous (F-actin) form of actin. Thin filaments are built from the specific association of the F-actin filaments with the rodlike tropomyosin molecule, which lines the grooves of the actin filament, and the troponin polypeptide complex. In the sarcomere the thin filaments are anchored at the Z disk through their interactions with binding proteins, principally cap Z and α-actinin. The exact structure of the Z disk is unknown; the protein interactions shown in this diagram are based on the in vitro capabilities of the isolated cap Z and α-actinin proteins. As illustrated the specific protein interactions of the Z-disk proteins immobilize the thin filaments at their plus (+) ends, thereby maintaining the polarity of the actin thin filaments in the sarcomere. The minus (−) ends are capped by tropomodulin. © 2020 Elsevier Inc. All rights reserved. 6 Figure 3–6. Structure of myosin II and its cleavage by papain. Myosin II is a 150-nm-long fibrous protein, with two globular heads. Treatment of myosin II with the proteolytic enzyme papain releases the two myosin heads, or SF1 fragments, from the myosin rod. © 2020 Elsevier Inc. All rights reserved. 7 Figure 3–7. Actin filaments have a polarity. The polarity of actin filaments can be visualized by labeling with myosin SF1 fragments. (A) This is an electron micrograph of in vitro formed actin filaments that have bound myosin SF1 fragments. The myosin fragments bind to the actin filaments, demonstrating their polarity. The myosin heads look like arrowheads that all point to the minus (−) ends of the actin filament, the barbed ends facing the plus (+) ends of the filaments. (B) In a sarcomere the barbed or plus (+) ends are attached to the Z disk. When actin filaments are bound to the cytoplasmic surface of the plasma membrane, it is the plus end that is associated with the membrane. The example shown here is the attachment of actin filaments to the tip of the microvillus. ([A] Courtesy Dr. Roger Craig, University of Massachusetts.) © 2020 Elsevier Inc. All rights reserved. 8 Figure 3–8. Formation of myosin thick filaments. (A) Thick filament formation is initiated by the end-to-end association of the rodlike tail domains of myosin II molecules. (B) This results in the formation of the bipolar thick filament, with globular heads at either end separated by a 160-nm central bare zone consisting of myosin II tail domains. At the filament ends the myosin globular head domains protrude from a 10.7-nm-diameter central core at intervals of 14 nm. The successive myosin heads rotate around the fiber, which forms a filament containing six rows of myosin head domains to contact the adjacent thin filaments of the sarcomere. © 2020 Elsevier Inc. All rights reserved. 9 Figure 3–9. Titin and nebulin: accessory proteins of the skeletal muscle sarcomere. The location of the proteins titin and nebulin within the sarcomere is shown. Titin, a large protein that has elastic properties and links the myosin thick filaments to the Z disks, helps maintain their location in the sarcomere. Nebulin, a large filamentous protein anchored at the Z disk, is in close apposition to the actin thin filaments. Their close association with the thin filaments suggests that the nebulin fibers serve to organize the actin filaments of the sarcomere. © 2020 Elsevier Inc. All rights reserved. 10 Figure 3–10. Sliding filament model of muscle contraction. Muscle contraction occurs by the sliding of the myofilaments relative to each other in the sarcomere. (A) In relaxed muscle the thin filaments do not completely overlap the myosin thick filaments, and a prominent I band exists. (B) With contraction, movement of the thin filaments toward the center of the sarcomere occurs, and because the thin filaments are anchored to the Z disks, their movement causes shortening of the sarcomere. The sliding of thin filaments is facilitated by contacts with the globular head domains of the bipolar myosin thick filaments. © 2020 Elsevier Inc. All rights reserved. 11 Figure 3–11. Illustration of the ATP-driven myosin-actin interactions during contraction. The binding of ATP to a myosin head group causes release from the actin filament (step 1). The hydrolysis of ATP to ADP + Pi readies the myosin head to contact an actin filament (step 2). The initial contact of the myosin with an actin filament causes the release of Pi and a tight binding of the actin filament (step 3). This tight binding induces a change in conformation of the myosin head, such that it pulls against the actin filament, the power stroke (step 4). This change in conformation is accompanied with the release of ADP. The binding of an additional ATP causes a release of the actin filament and a return of the myosin head to a position ready for another cycle. © 2020 Elsevier Inc. All rights reserved. 12 Figure 3–12. Diagram of Ca2+-mediated movements of troponin and tropomyosin filaments during muscle contraction. (A) In the relaxed muscle the tropomyosin filament is bound to the outer domains of seven actin monomers along the actin filament. The troponin complex is bound to the tropomyosin by the rod-shaped troponin T (TnT) polypeptide. (B) In the presence of Ca2+, troponin C (TnC) binds the calcium, causing the globular domain of troponin [TnC and troponin I (TnI)] to move away from the tropomyosin filament. (C) This movement permits the tropomyosin to shift to a position that is farther inside the groove of the helical actin filament, allowing the myosin heads to make contact with the released sites of the actin monomers. © 2020 Elsevier Inc. All rights reserved. 13 Figure 3–13. Diagram of part of a skeletal muscle fiber, illustrating the organization of the sarcoplasmic reticulum (SR) and transverse tubule (t-tubule) networks. The SR is a specialized smooth endoplasmic reticulum (ER) that in muscle serves as a store for Ca2+ ions. The SR forms a membranous tubule network that surrounds the myofibrils. At the A-I band junctions of the sarcomere, the SR forms a more regular channel, referred to as the terminal cisternae. Two terminal cisternae are separated by a second tubule system, the t-tubules, which are special invaginations of the sarcolemma. These three membrane-bound tubules form a structure known as the triad: a t-tubule flanked on either side by a terminal cisternae of the SR, at the region of the A-I junction of the sarcomere.