Myosin filament assembly

The question of why dephosphorylated smooth muscle myosin filaments exhibit greater stability in vivo than in vitro remains unanswered. One possibility is that the intracellular concentration of myosin in vivo exceeds the critical concentration necessary for filament assembly even for 

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Rho Controls organization of actin cytoskeleton and gene expression (F-actin bundling, myosin filament assembly) Rho (A-E), Cdc42, Rac (1-3) Anchored to plasma membrane by lipids, and translocates to cytosol... 

Smith RC, Cande WZ, Craig R, Tooth PJ, Scholey JM, Kendrick-Jones J (1983) Regulation of myosin filament assembly by light-chain phosphorylation. Philos Trans R Soc Lond B Biol Sci 302 7382... 

In a previous section we mentioned the significance of myosin filament structure. In nematodes two forms of myosin-II, myosin A and B, are required for proper filament stmcture (Epstein, 1988). The two forms of myosin are expressed at the proper time to allow for correct filament assembly. An accessory protein called paramyosin is also required for correct filament assembly. In vertebrate cardiac muscle, there are also two isoforms of myosin-II a-myosin and p-myosin. The proper ratio of these two proteins is of utmost importance for proper muscle activity. The incorrect synthesis of a- and P-myosins results in a severe cardiac disorder known as hypertrophic cardiomyopathy. Genetic transmission of the disease occurs in about 55% of families. The inherited condition is called familial hypertrophic cardiomyopathy (FHC), and this condition is a leading cause of sudden death in young athletes. 

Organization into macromolecular structures. There are no apparent templates necessary for the assembly of muscle filaments. The association of the component proteins in vitro is spontaneous, stable, and relatively quick. Filaments will form in vitro from the myosins or actins from all three kinds of muscle. Yet in vitro smooth muscle myosin filaments are found to be stable only in solutions somewhat different from in vivo conditions. The organizing principles which govern the assembly of myosin filaments in smooth muscle are not well understood. It is clear, however, a filament is a sturdy structure and that individual myosin molecules go in and out of filaments whose structure remains in a functional steady-state. As described above, the crossbridges sticking out of one side of a smooth muscle myosin filament are all oriented and presumably all pull on the actin filament in one direction along the filament axis, while on the other side the crossbridges all point and pull in the opposite direction. The complement of minor proteins involved in the structure of the smooth muscle myosin filament is unknown, albeit not the same as that of skeletal muscle since C-protein and M-protein are absent. 

The superstructure of smooth muscle actin filaments is differentiated from those of striated muscle by the absence of the troponins and the lateral organization by association of the filaments with dense bodies instead of with the Z-line. How these differences are encoded is again not at all clear. However, the myofibrillar structure and the alignment of the alternating actin and myosin filaments is apparently due primarily to dense bodies and the actin-actinin macrostructures. As the bent dumbbell shaped actins assemble into filaments they are all oriented in the same direction. The S-1 fragments of myosin will bind to actin filaments in vitro and in... 

Myosin may be extracted via high-ionic-strength buffers and purified. Synthetic thick filaments of myosin spontaneously assemble upon lowering the ionic strength of its solution, exhibiting the morphological characteristics of native thick filaments. This process initiates with myosin monomers assembled into parallel dimers. The dimers assemble into antiparallel tetramers, the tetramers into octamers, and the octamers into minifilaments... 

Fibrous proteins represent a substantial subset of the human proteome. They include the filamentous structures found in animal hair that act as a protective and thermoregulatory outer material. They are responsible for specifying much of an animal s skeleton, and connective tissues such as tendon, skin, bone, cornea and cartilage all play an important role in this regard. Fibrous proteins are frequently crucial in locomotion and are epitomised by the muscle proteins myosin and tropomyosin and by elastic structures like titin. Yet again the fibrous proteins include filamentous assemblies, such as actin filaments and microtubules, where these provide supporting structures and tracks for the action of a variety of molecular motors. 

In thinking about X-ray diffraction from this assembly, a number of the sarcomere components contribute to the observed patterns in ways that have been the subject of detailed analysis. In the A-band, these include the myosin filament backbone, where the coiled-coil a-helical myosin rods pack together, the myosin head arrays in the bridge regions of the myosin filaments, the non-myosin A-band proteins titin and C-protein (MyBP-C), and the A-band parts of the actin filaments. Very little has been seen in X-ray patterns so far that appears to be related to the M-band, probably... 

Liang W, Warrick HM, Spudich JA. 1999. A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly. J Cell Biol 147 1039-1047. 

In telophase, nuclei for each daughter cell form at the two poles, and the mitotic spindle apparatus disappears. Furthermore, nuclear membranes, nuclear lamina, nuclear pores, and nucleoli are reformed. The cell is now ready for cytokinesis, which is physical division of the cytoplasm. The cytoplasm divides as actin/myosin filaments contract and pinch off the plasma membrane, which results in two daughter cells that enter into Go or Gi for another round of division. The main checkpoint that exists during M phase in mammalian cells is the spindle checkpoint it is in place to ensure proper microtubule assembly, proper cell division, and that each daughter cell receives one copy of DNA. 

Filament assembly depends on characteristics of the myosin tail, especially the LMM, in which much of the amino acid sequence exhibits a heptapeptide repeat. If the repeat is represented as ABCDEFG, residues A and D are hydrophobic and lie at points where the a-helices... 

LC20 phosphorylation produces two pronounced effects it promotes the ability of myosin monomers to assemble into filaments, and it markedly increases (100-fold) the ATPase activity of myosin compared to un-phosphorylated filamentous myosin. How unphosphory-lated myosin filaments can remain stable is not certain, but may be related to binding to myosin of independently expressed MLCK fragments containing the myosin binding domain of MLCK. How LC20 phosphorylation increases the ATPase activity so dramatically is still unknown. 

Kinetic studies have shown that filament initiation is more difficult than subsequent elongation (Cross et al., 1991). In a system where assembly-disassembly might play a large role, for example, in nonmuscle vertebrate cells, this property predicts that the rate at which monomers become available for polymerization could alter both the number and length of myosin filaments that are formed. Thus control of kinase activity, which controls the number of assembly competent extended monomers, could be a factor in determining subsequent polymerization. 

In vitro, it is clear that smooth muscle myosin filaments can assemble into side-polar filaments that are never seen with skeletal muscle myosin (Craig and Megerman, 1977) (Fig. 4). The key feature of a side-polar filament is that all myosin heads have the same polarity along one edge of the filament, and the opposite polarity on the other edge, with bare zones on either end of the filament (Fig. 5). The heads of myosin in a bipolar filament, in contrast, reverse polarity at the filament center, thus producing a central bare zone. 

An additional property of CD in thin filaments is to cross-link thick and thin filaments owing to its ability to bind to actin and myosin. This suggests an additional role of CD in directing and stabilizing filament assembly and possibly in latch (see Section IX). 

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