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Интеллектуальная Система Тематического Исследования НАукометрических данных |
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Tyrosine kinases of myosin II phosphorylation pathway play a pivotal role in the focal adhesions’ (FA) formation, although their role in distribution of FAs is poorly elucidated. To describe the effects of ROCK and MLCK inhibitors on organization of FAs we cotransfected 3T3 fibroblasts with paxillinGFP and vinculinRFP plasmids and observed by timelapse fluorescence microscopy. At the active edge of the control cells we visualized FAs containing only paxillin as stretched structures in lamellipodia, FAs that contained both paxillin and vinculin (37% of total FA number) were located behind the active edge of lamellum, and no FAs containing vinculin alone were detected in this band. In the cell interior (more than 10 um from the cell margin) no paxillinonly FAs were visualized, 36% of adhesions in this area contained both paxillin and vinculin, while all others contained only vinculin. Both inhibitors did not prevent the formation of FAs, but induced their redistribution. Under both treatments paxillinonly FAs were visualized as numerous small spotlike structures near the cell margin. ROCK inhibitor Y27632 suppressed the membrane ruffling and led to disassembly of the stress fibers. Vinculinonly FAs appeared at the cell periphery, and the percentage of FAs containing both proteins decreased to 21%. FA organization in the cell interior remained intact. MLCK inhibitor ML7 induced the formation of long membrane protrusions and disorganization of peripheral actin, though stress fibers in the cell interior remained intact. MLCKinhibited cells assembled bright vinculincontaining FAs at the cell periphery and large vinculincontaining FA (median area 0.510.09 um compared to 0.340.09 um in control) were located in the cell interior. The percentage of FAs containing both proteins also decreased to 20%. Thus, MLCK and ROCK are essential regulators that independently contribute to the FA organization and functional heterogeneity in different cell parts.