Calculation of a Gap restoration in the membrane skeleton of the red blood cell: possible role for myosin II in local repair.

Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network.

[The locomotor-contractile system of thrombocytes--an optimal model for studying the physiological motor and contractile processes in the effector systems of the non-muscular cells]. / Oporno-sokratitel'naia sistema trombotsitov--optimal'naia model' dlia izucheniia fiziologicheskikh dvigatel'nykh i sokratitel'nykh protsessov v éffektornykh sistemakh nemyshechnykh kletok organizma.

The authors suggest a concept according to which the cytoskeleton and contractile proteins of platelets are the locomotor-contractile system playing a role of the effector apparatus of hemostatic function of these cells. Demonstrate the role of the contractile structures in leukocyte phagocytosis.

Gelsolin is Ca2+-sensitive regulator of actomyosin system in platelet.

Effects of gelsolin on the actomyosin system in platelet have been studied. MgATPase activity of platelet actomyosin is enhanced up to two folds by 200 nM of platelet gelsolin in the presence, but not in the absence of Ca ion. The half maximum enhancement is observed at the concentration of Ca2+ around 10(-5) M. The effect of gelsolin to enhance the ATPase activity of actomyosin is potentiated by tropomyosin, which is a Ca2+-insensitive actomyosin enhancer. The results indicate that gelsolin may control the activity of actomyosin system in platelets.

Calpain abolishes the effect of filamin on the actomyosin system in platelets.

Platelet filamin was shown to cross-link F-actin and inhibit actomyosin ATPase activity. Filamin was also shown to be degraded by calpain (calcium-activated neutral proteinase; CANP) when the platelet was activated. The consequences of the proteolysis of filamin on the actomyosin system have been investigated. When degraded by calpain in the presence of Ca2+, filamin loses its ability to cross-link F-actin. Under the same conditions, its inhibitory effects on the superprecipitation and ATPase activity of actomyosin are abolished. The result suggests that the degradation of filamin is favorable for contraction of the activated platelets.

Caldesmon specifically inhibits the effect of tropomyosin on actomyosin system in platelet.

Caldesmon, a calmodulin and actin binding protein, has been shown to exist in platelet. In this report, it is shown that caldesmon specifically inhibits the effect of tropomyosin to enhance the actomyosin ATPase activity in platelet. Platelet tropomyosin enhances the MgATPase activity of platelet actomyosin. This effect is abolished by platelet caldesmon. In the absence of tropomyosin, however, caldesmon has no effect on the ATPase activity. The inhibition is not due to displacement of the binding of tropomyosin to F-actin by caldesmon. The result indicates that caldesmon is the specific inhibitor of tropomyosin in resting platelet.

Mersalyl, a sulfhydryl reagent, alters the solubility of myosin and cytoskeletal proteins of human platelets.

We have examined the effect of a mercurial sulfhydryl reagent, mersalyl, on the protein composition of cytoskeletons by SDS-polyacrylamide gel electrophoresis after treatment of human platelets with Triton X-100 (Triton) containing mersalyl and Ca2+, and have found that mersalyl alters the protein composition of cytoskeletons in a Ca2+-dependent manner. At 1 X 10(-7) M Ca2+, 0.2 mM mersalyl, which represents approximately the equivalent amount of sulfhydryl of platelet suspensions that we used, specifically made myosin insoluble. The amount of myosin in Triton-mersalyl residues was increased by increasing the Ca2+ concentration of Triton lysis buffer. Actin-binding protein, 235 kDa polypeptide and alpha-actinin-like protein were decreased in Triton residues by mersalyl at Ca2+ concentrations less than 1 X 10(-7) M, while these polypeptides in Triton residues were increased by mersalyl in the presence of more than 2 X 10(-7) M Ca2+. Electron microscopic study revealed the presence of thick filaments with an appearance similar to that of the thick filaments of platelet myosin. Thus, the modification with mersalyl of sulfhydryls of platelet polypeptides along with changes in Ca2+ concentrations within a physiological range leads to changes in solubility of, and filament formation of, myosin, actin and other cytoskeletal proteins.

An actomyosin contractile mechanism for erythrocyte shape transformations.

The membrane skeleton of the human erythrocyte consists of many short actin filaments that are multiply cross-linked by long, flexible spectrin molecules into a continuous network in the plane of the membrane. The mechanical properties expected for this spectrin-actin network can account for the tensile strength of the erythrocyte membrane and for the remarkable deformability of the cells, yet not for their characteristic biconcave shape. Recently, an authentic vertebrate myosin as well as a non-muscle form of tropomyosin have been identified and purified from erythrocytes. The myosin is present with respect to the actin in an amount comparable to actin-myosin ratios in other non-muscle cells, and there is enough tropomyosin to almost completely coat all of the short actin filaments in the membrane skeleton. The implications of these unexpected discoveries for the molecular organization of the cytoskeleton are discussed, and a mechanism is proposed by which myosin could interact with the membrane-associated actin filaments to influence erythrocyte shape and membrane properties.

Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity.

In an actomyosin fraction isolated from human platelets, phosphorylation of the 20,000-dalton light chain of myosin is stimulated by calcium and the calcium-binding protein calmodulin. The enzyme catalyzing this phosphorylation has been isolated by using calmodulin-affinity chromatography. Platelet myosin light chain kinase activity was monitored throughout the isolation procedures by using the 20,000-dalton smooth muscle myosin light chain purified from turkey gizzards as substrate. The partially purified myosin kinase requires both calcium and calmodulin for activity and has a specific activity of 3.1 mumol of phosphate transferred to the 20,000-dalton light chain per mg of kinase per min under optimal assay conditions. Km values determined for ATP and myosin light chains are 121 microM and 18 microM, respectively. Of several substrates surveyed as phosphate acceptors (alpha-casein, histone II-A, phosphorylase b, protamine, histone V-S, and phosvitin), only the 20,000-dalton myosin light chain is phosphorylated at a significant rate. These results suggest that platelet myosin light chain kinase is a calcium-dependent enzyme and that the requirement for calcium is mediated by the calcium-binding protein calmodulin.

Contractile properties of actomyosin from human blood platelets.

Actomyosin was purified from human blood platelets and used to form threads via extrusion. A sensitive tensiometer was employed to measure isometric tension and velocity of isotonic shortening of the threads in the presence of MgATP. Using fully phosphorylated myosin, we obtained values for maximum isometric tension (Po) and maximum velocity of contraction (V max) that were similar to those reported for threads composed of skeletal muscle actomyosin. Po was found to be directly proportional to the level of phosphorylation of the 20,000-dalton myosin light chain. We also studied the effect of phosphorylation on superprecipitation of platelet actomyosin. Fully phosphorylated myosin produced rapid clearing and superprecipitation, while myosin with a low level of bound phosphate underwent rapid clearing but did not superprecipitate. We have concluded from these results that: 1) the interaction between platelet actin and myosin produces tension and motion that is similar to that produced by skeletal muscle actin and myosin and 2) phosphorylation of the 20,000-dalton myosin light chain in important in controlling the production of force by platelet actin and myosin.