Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología, IHEM (CONICET-UNCuyo), Facultad de Ciencias Médicas, Argentina.
Exp Cell Res. 2012 Mar 10;318(5):478-88. doi: 10.1016/j.yexcr.2012.01.002. Epub 2012 Jan 10.
Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking.
胞吐作用是一个高度调控的多阶段过程,由多个功能可定义的阶段组成,包括募集、靶向、连接、预激发和分泌囊泡与质膜的对接,随后是钙触发的膜融合。精子的顶体反应是一个复杂的、依赖钙的调节性胞吐作用。外顶体膜和细胞膜之间的多个部位融合导致顶体内容物的释放和顶体周围膜的丧失。对于介导这个特殊融合模型中的膜对接的分子,我们知之甚少。在神经元中,已经提出形成三元 RIM/Munc13/Rab3A 复合物作为突触囊泡对接的关键组成部分。以前,我们证明 Rab3A 定位于人精子的顶体区域,刺激顶体胞吐作用,并参与膜融合的早期阶段。在这里,我们报告 RIM 和 Munc13 也存在于人精子中,并定位于顶体区域。与 Rab3A 一样,RIM 和 Munc13 参与了内顶体钙流出之前的预融合步骤。通过使用抗体和重组蛋白的功能测定,我们表明 RIM、Munc13 和 Rab3A 在顶体胞吐作用过程中相互作用。最后,我们通过电子传输显微镜报告说,隔离 RIM 和 Rab3A 改变了钙激活顶体胞吐作用过程中顶体膜与质膜的对接。我们的结果表明,RIM/Munc13/Rab3A 复合物参与顶体胞吐作用,并且 RIM 和 Rab3A 在膜对接中起核心作用。