Actin filaments have very important roles in the cell. In a half decade their detailed structure was revealed by EM. However their interaction with their accessary proteins in the cell is unclear, so it is important to know how to interact with each other in biological conditions. Here we combined cryo-ET and image processing with computer. Tilt series from EM have very low S/N ratio, which restricts resolution, so we overcome the problem with that. We obtained high resolution structure of "in vivo" actin filaments in filopodia by cryo-ET. Moreover, the interactions between actin and fascin were revealed. Hence our techniques have potential to reveal interaction between actin and various interaction proteins such as motor protein at subnanometer resolution. Contractile actin stress fibers play important roles in various physiological processes, such as migration, survival, and differentiation. However, their biophysical characteristics and structural components are largely unknown.
1P195 非筋細胞から単離したアクチンストレスファイバーの成分に ついてTo identify the critical constituents required for the contraction, we have tried to develop an in-vitro contractile system of actin stress fibers isolated from nonmuscle cells. We demonstrate that the phosphorylation level of myosin regulatory light chains is maintained in our isolation procedure, and the stress fibers are highly contractile even without Rho kinases. This system enables us to measure the contractile force of single stress fibers, which has never been reported. Myosin mutant G680V from Dictyostelium discoideum cannot move actin filament in vitro. Previously, we have reported that the sliding velocity of an actin filament was remarkably accelerated in a intermittent manner by mixing of the slight amount of the mutant into skeletal HMM. In order to examine the capability of the mutant in solution, G680V-HMM was added to assay in solution. The actin filament in the condition also demonstrated two-times faster velocities than those without mutants. Furthermore, the movement was not intermittent but continuous. Neither intact nor NEMtreated HMM demonstrate any acceleration. These results suggest that the attaching of G680V myosin molecules to an actin filament might alter the sliding capacity of the filament. Eukaryotic flagellar movements are based on the sliding between microtubules (MTs) powered by dynein. Dynein is thought to be regulated by a central pair and radial spokes, but the mechanism is not understood. Chlamydomonas mutants lacking the flagellar central structures are nonmotile, but the mutant axonemes display beating in the presence of ATP and various kinds of salts. Here we performed X-ray fiber diffraction studies to examine the structural base for the motility. We found that the diameters in mutants were smaller than that in wild type (~185 nm) by 2-3 nm, and that the diameters increased by 1-2 nm under the motility recovery conditions. This suggests that the central structures regulate the axoneme diameter to induce the effective dynein-MT interactions. During cytokine...
b{nding ef D-cofi[in was also observed for F-R-actin, suggesting that the cooperative binding ofcofi[in was not origin-dependent phenomenon We are now examining the cooperative binding of D-HMM to F-D-actin. We will pfesent detailed data of the cooperative interactions containing mutually exclusive binding at this meeting.
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