Ca(2+) effects on myosin-II-mediated contraction of pseudo-contractile rings and transport of vesicles in extracts of clam oocytes.

Abstract

Ca2 + is a key regulator of cytokinesis, the process by which the contractile ring constricts the cell to form the cleavage furrow. BAPTA (1,2-bis (aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid) has been used to investigate the role of Ca2+ in this process. Several studies have shown that buffering cytosolic Ca2+ with BAPTA blocks cytokinesis in a variety of cultured cells and fertilized eggs (1, 2). These results support a model of cytokinesis in which a local rise in free Ca2+ is part of the signaling pathway that regulates assembly of the cytokinetic ring of actin filaments. In addition, Ca2+ is required for Ca2+-calmodulin (CaM)-stimulated phosphorylation of the regulatory light chain of myosin-II by myosin light chain kinase (MLCK). Therefore, Ca2+ serves both as a signal for cytokinesis and as a regulator of myosin-II activity. We studied cytokinesis in vitro by reconstituting pseudo-contractile rings in M-phase extracts made from clam oocytes. In these extracts, actin filaments spontaneously organized into a cooperative, 3-D network of interconnected filaments. We refer to this self-organized network of actin filaments as a pseudo-contractile ring because it shares the following two fundamental properties with the contractile ring: (i) it exhibits myosin-II-mediated, antiparallel sliding of actin filaments; and (ii) it assembles during the M-phase of the cell cycle. Fortuitously, actin filaments within the pseudo-contractile ring co-aligned to form bundles (10-20 parallel filaments) that were thick enough to be visualized by Allen Video Enhanced Contrast (AVEC) Differential Interference Contrast (DIC) microscopy. Therefore, sliding of actin filaments could be monitored and studied in networks reconstituted in vitro. The term contraction refers to the myosin-II-mediated sliding of actin filaments in the network; the network itself does not shorten. In a previous study, we used a function-blocking myosin-IIspecific antibody to establish that the sliding of actin bundles observed in these pseudo-contractile rings is mediated by myosin-II (3). Using the same antibody, we showed that vesicle transport on actin filaments observed in these extracts is also mediated by myosin-II. In this report, we determined the effects of Ca2+ l to i esis, the proce s by which the i s t e cell to for the cleavage furrow. i )-et a e, , ',N'-tetr acetic i sti ate t e role of Ca2+ in this proce s. t at uffering cytosolic Ca2+ with