STUDY ON ROLE OF MICROTUBLE-BINDING AND RHO-ACTIVATING FACTORS IN CYTOKINESIS

为了深入了解微管和相关信号分子在细胞运动调节中的分子动力学和参与，我们研究了微管破坏对肌动蛋白应力纤维和粘着斑组装以及细胞形态的影响。我们发现，所有微管破坏药物包括秋水仙胺和长春碱都能迅速和可持续地诱导肌动蛋白应力纤维和含有黏着斑蛋白的局灶性粘附的形成，并伴有血清饥饿的Balb/c3 T3细胞中激活的细胞活力。与此相反，紫杉醇，一种微管稳定药物，完全抑制微管破坏药物的这些作用.显微注射rho GTP酶的特异性抑制剂C3 ADP-核糖基转移酶可阻断微管断裂诱导的应力纤维和粘着斑的组装。这些结果表明，微管含有信号分子，通过激活rho信号级联反应调节应力纤维的形成和局灶性粘附。我们假设微管释放和应力纤维诱导因子将内在可变和不规则的肌动蛋白丝动力学与细胞运动过程中的协调和定向运动联系起来。为了鉴定该因子，我们进一步分析了胞浆和膜组分中rho活化的活性。我们发现微管结合蛋白组分具有抑制rhoGDI的活性，表明微管组分含有rhoGDI抑制剂。进一步的研究表明微管蛋白（微管组分的主要成分）直接与rhoGDI结合并抑制rhoGDI的活性，这些结果清楚地表明微管蛋白抑制rhoGDI，导致rho G蛋白从RhoGDI中释放出来，并最终激活rho G蛋白。活化的rho促进肌动蛋白的聚合，这需要哺乳动物细胞的胞质分裂。

To obtain insight into the molecular dynamics and involvement of microtubles and the related signal molecules in the regulation of cell locomotion, we studied the influence of microtuble disruption on actin stress fibers and focal adhesion assembly in addition to cell morphology. We found that all microtuble- disrupting drugs including colcemid and vinblastine rapidly and rever sibly induce the for mation of actin stress fibers and focal adnesions containing vinculin, accompanied by activated cell molility ih ser um- starved Balb/c3T3 cells. In contrast, taxol, a microtuble- stabilizing drug, completely inhibited these effects of the microtuble- disrupting drugs. A microinjection of C3ADP-ribosyltransfer ase, a specific inhibitor of rho GTP ase, blocked the stress fiber and focal adnesion assembly induced by the microtuble disruption. These results suggested that microtubles contain signal molecules that regulate the for mation of stress fibers and focal adnesions by activating the rho signal cascade. We postulate that microtuble - releasing and stress fiber- inducing factors link the intrinsically variable and irregul ar actin filament dynamics to coor dinated and directional locomotion in the process of cell movement. To identify the factor, we further analyzed the activity of rho activation in cytoslic and membrane fractions. We found that microtuble- binding protein fractions had the activity for rhoGDI inhibition, indicating that microtuble fractions contain rhoGDI inhibitor. Further investigations showed that tubulin proteins, main component of microtuble fraction, directly bind to and inhibit the activity of rhoGDI.These results clearly show that tubulin proteins inhibit rhoGDI, resulting inreleasing of rho G-protein from RhoGDI and eventialy activating rho G- protein. The activated rho promotes polymerization of actin proteins which require cytokinesis of mammalian cells.