Mabuchi I, Hamaguchi Y, Fujimoto H, Morii N, Mishima M, Narumiya S
Department of Biology, College of Arts and Sciences, University of Tokyo, Japan.
Zygote. 1993 Nov;1(4):325-31. doi: 10.1017/s0967199400001659.
Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22,000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.
将海胆卵用肉毒杆菌C3外毒素进行显微注射，C3外毒素是一种来自肉毒杆菌的ADP核糖基转移酶，它能特异性地使rho蛋白进行ADP核糖基化并使其失活。在核分裂期间显微注射C3外毒素会干扰随后的卵裂沟形成。用罗丹明-鬼笔环肽染色在这些卵的赤道皮质层中未检测到肌动蛋白丝。当将C3外毒素显微注射到正在进行卵裂的卵中时，它会迅速破坏收缩环肌动蛋白丝并导致卵裂沟消退。然而，C3外毒素对核分裂没有明显影响，并且从显微注射的卵中发育出多核胚胎。相比之下，C3外毒素在受精后立即对皮质肌动蛋白丝的组织没有影响。在分离的卵裂沟中只有一种蛋白质（分子量22,000）被C3外毒素进行了ADP核糖基化。当通过二维凝胶电泳分析时，这种蛋白质与源自人血小板的ADP核糖基化的rhoA迁移率相同。这些结果有力地表明，一种类rho的小GTP结合蛋白选择性地参与了收缩环的组织和维持。
