Actin mechanics

Allison, S.D. et al.. Effects of drying methods and additives on structure and function of actin mechanisms of dehydration-induced damage and its inhibition. Arch. Biochem. Biophys., 358,171, 1998. 

Ishijima A, Kojima H, Funatsu T, Tokunaga M, Higuchi H, Tanaka H and Yanagida T 1998 Simultaneous observation of individual ATPase and mechanical events by a single myosin molcule during interaction with actin Ce//92 161-71... 

Zigmond, 1988). The ATP-hydrolysis that accompanies actin polymerization, ATP —> ADP + Pj, and the subsequent release of the cleaved phosphate (Pj) are believed to act as a clock (Pollard et ah, 1992 Allen et ah, 1996), altering in a time-dependent manner the mechanical properties of the filament and its propensity to depolymerize. Molecular dynamics simulations suggested a so-called back door mechanism for the hydrolysis reaction ATP ADP - - Pj in which ATP enters the actin from one side, ADP leaves from the same side, but Pj leaves from the opposite side, the back door (Wriggers and Schulten, 1997b). This hypothesis can explain the effect of the toxin phalloidin which blocks the exit of the putative back door pathway and, thereby, delays Pi release as observed experimentally (Dancker and Hess, 1990). 

Pollard et al., 1992] Pollard, T. D., Goldberg, I., and Schwarz, W. H. Nucleotide exchange, structure, and mechanical properties of filaments assembled from ATP-actin and ADP-actin. J. Biol. Chem. 267 (1992) 20339-20345... 

Wriggers and Schulten, 1998] Wriggers, W., and Schulten, K. Investigating a back door mechanism of actin phosphate release by steered molecular dynamics. Biophys. J. Submitted. 

Microfilaments and Microtubules. There are two important classes of fibers found in the cytoplasm of many plant and animal ceUs that are characterized by nematic-like organization. These are the microfilaments and microtubules which play a central role in the determination of ceU shape, either as the dynamic element in the contractile mechanism or as the basic cytoskeleton. Microfilaments are proteinaceous bundles having diameters of 6—10 nm that are chemically similar to actin and myosin muscle ceUs. Microtubules also are formed from globular elements, but consist of hoUow tubes that are about 30 nm in diameter, uniform, and highly rigid. Both of these assemblages are found beneath the ceU membrane in a linear organization that is similar to the nematic Hquid crystal stmcture. 

Contraction of muscle follows an increase of Ca " in the muscle cell as a result of nerve stimulation. This initiates processes which cause the proteins myosin and actin to be drawn together making the cell shorter and thicker. The return of the Ca " to its storage site, the sarcoplasmic reticulum, by an active pump mechanism allows the contracted muscle to relax (27). Calcium ion, also a factor in the release of acetylcholine on stimulation of nerve cells, influences the permeabiUty of cell membranes activates enzymes, such as adenosine triphosphatase (ATPase), Hpase, and some proteolytic enzymes and facihtates intestinal absorption of vitamin B 2 [68-19-9] (28). 

Thickness of the laminar layer is deterrnined both by the need to reproduce fine detail in the object and by the penetration depth of the actinic laser light into the monomer bath (21,76). There is thus a trade-off between precision of detail in the model and time required for stereohthography, ie, the number of layers that have to be written, and an optimum Light-absorbing initiator concentration in the monomer bath corresponding to the chosen layer thickness. Titanocene-based initiators, eg, bis-perfluorophenyltitanocene has been recommended for this apphcation (77). Mechanistic aspects of the photochemistry of titanocenes and mechanisms of photoinitiation have been reviewed (76). 

In the presence of calcium, the primary contractile protein, myosin, is phosphorylated by the myosin light-chain kinase initiating the subsequent actin-activation of the myosin adenosine triphosphate activity and resulting in muscle contraction. Removal of calcium inactivates the kinase and allows the myosin light chain to dephosphorylate myosin which results in muscle relaxation. Therefore the general biochemical mechanism for the muscle contractile process is dependent on the avaUabUity of a sufficient intraceUular calcium concentration. 

FIGURE 17.23 The mechanism of skeletal muscle contraction. The free energy of ATP hydrolysis drives a conformational change in the myosin head, resulting in net movement of the myosin heads along the actin filament. Inset) A ribbon and space-filling representation of the actin—myosin interaction. (SI myosin image courtesy of Ivan Rayment and Hazel M. Holden, University of Wiseonsin, Madison.)... 

Stimulation of PKA and PKC inhibit NHE5 activity, as does hyperosmolarity, resembling the responsiveness of NHE3. NHE5 may also be regulated by the actin cytoskeleton, but further work is required to validate this notion and clarify the exact mechanism. 

Studies on muscle contraction carried out between 1930 and 1960 heralded the modem era of research on cytoskeletal stmctures. Actin and myosin were identified as the major contractile proteins of muscle, and detailed electron microscopic studies on sarcomeres by H.E. Huxley and associates in the 1950s produced the concept of the sliding filament model, which remains the keystone to an understanding of the molecular mechanisms responsible for cytoskeletal motility. 

More than 50 proteins have been discovered in the cytosol of nonmuscle cells that bind to actin and affect the assembly and disassembly of actin filaments or the cross-linking of actin filaments with each other, with other filamentous components of the cytoskeleton, or with the plasma membrane. Collectively, these are known as actin-binding proteins (ABPs). Their mechanisms of actions are complex and are subject to regulation by specific binding affinities to actin and other molecules, cooperation or competition with other ABPs, local changes in the concentrations of ions in the cytosol, and physical forces (Way and Weeds, 1990). Classifications of ABPs have been proposed that are based on their site of binding to actin and on their molecular structure and function (Pollard and Cooper, 1986 Herrmann, 1989 Pollard et al., 1994). These include the following ... 

The diversity of these subcellular actin structures is remarkable and appears to be determined by the interactions of many actin-binding proteins (ABPs) as well as by changes in the concentrations of intracellular signaling molecules such as Ca and cAMP, by small GTP-binding proteins, and by signals arising from mechanical stress. Approximately 50% of the actin molecules in most animal cells are unpolymerized subunits in the cytosolic pool and exist in a state of dynamic equilibrium with labile F-actin filamentous structures (i.e., new structures are formed while existing structures are renewed) (Hall, 1994). 

Stress fibers are parallel bundles of actin filaments that develop in the cytoplasm of fibroblasts from the cortical actin network in response to mechanical tension. These often bind to the plasma membrane at focal contacts and, through transmembrane linker glycoproteins, to the extracellular matrix. Thus, actin filaments of stress fibers indirectly Join to the inner face of the plasma membrane through molecular assemblies of attachment proteins, which include an actin-capping protein, a-actinin, vinculin, and talin (Small, 1988). 

Blood platelets are key players in the blood-clotting mechanism. These tiny fragments of cytoplasm are shed into the circulation from the surface of megakaryocytes located in the bone marrow. When the lining of a blood vessel is injured, activated platelets release clotting factors, adhere to each other and to damaged surfaces, and send out numerous filopodia. The shape changes that occur in activated platelets are the result of actin polymerization. Before activation, there are no microfilaments because profilin binds to G-actin and prevents its polymerization. After activation, profilin dissociates from G-actin, and bundles and networks of F-actin filaments rapidly appear within the platelet. 

Pollard, T.D. Cooper, J.R. (1986). Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Ann. Rev. Biochem. 55, 987-1035. 

The myosin head has long been shown to induce, even in low ionic strength buffers, polymerization of G-actin into decorated F-actin-S i filaments that exhibit the classical arrowhead structure (Miller et al., 1988 and older references therein). However, to date, the molecular mechanism of this polymerization process remains unknown. 

The kinetics of F-actin-Si assembly from G-actin and Si via nucleation of actin filaments, followed by Si binding are not observed in a low ionic strength medium. Instead, the mechanism involves condensation of high affinity (G-actin)2 S complexes rapidly preformed in solution. Assembly of F-actin-Si in the presence of Si > G-actin is a quasi-irreversible process. This mechanism is therefore different from that involving the assembly of F-actin filaments, which is characterized by the initial, energetically unfavorable formation of a small number of nuclei representing a minute fraction of the population of actin molecules, followed by endwise elongation from G-actin subunits. 

Combeau, C. Carlier, M.-F. (1988). Probing the mechanism of ATP hydrolysis on F-actin using vaitadate and the structural analogs of phosphate BeF j and AIF J. J. Biol. Chem. 263,17429-17434. 

The myosins are a superfamily of proteins that have the ability to convert energy released by ATP is hydrolysis into mechanical work. There are many forms of myosin, all of which have ATPase activity and an actin-binding site that is located... 

The regulation of smooth muscle and nonmuscle myosin-II is substantially different from the mechanism described above for two important reasons. First, there is no troponin in smooth muscle and nonmuscle cells. Second, although the rate of hydrolysis of ATP by these myosins is low in the presence of physiological concentrations of Mg % the addition of actin does not necessarily result in the stimulation of ATP hydrolysis by smooth muscle or nonmuscle myosin-II. These observations suggest the presence of a unique mechanism for Ca " regulation in smooth and nonmuscle cells, and that these myosins require an activation process before actin can stimulate ATP hydrolysis. 

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