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控中的分子动力学和参与其中，我们研究了微管破坏对肌动蛋白应激纤维和焦点黏附组装的影响以及细胞形态。我们发现，在饥饿的Balb/C3T3细胞中，所有的微管干扰药物包括秋水仙胺和长春花碱都能快速和可逆地诱导含有纽蛋白的肌动蛋白应激纤维和局灶性连接的形成，并伴随着激活的细胞流动性。相比之下，紫杉醇，一种稳定微管的药物，完全抑制了破坏微管的药物的这些作用。微量注射Rho GTP酶的特异性抑制剂C3ADP-核糖基转移酶可阻断微管破坏所诱导的应力纤维和焦点粘连组装。这些结果表明，微管含有信号分子，通过激活Rho信号级联来调节应力纤维和焦点附着的形成。我们推测，微管释放和应力纤维诱导因子将内在可变和不规则的Ar肌动蛋白细丝动力学与细胞运动过程中的协调和定向运动联系在一起。为了确定影响Rho活性的因素，我们进一步分析了胞质和膜组分中的Rho激活活性。我们发现微管结合蛋白部分具有抑制RhoGDI的活性，表明微管部分含有RhoGDI抑制因子。进一步的研究表明，微管部分的主要成分微管蛋白直接结合并抑制RhoGDI的活性。这些结果清楚地表明，微管蛋白抑制RhoGDI，导致RhoG-蛋白从RhoGDI释放，甚至激活Rho G-蛋白。被激活的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.