Studying cytokinesis in vitro.

Cytokinesis is the final stage of the cell division, through which the cellular constituents of mother cells are partitioned into two daughter cells resulting in increase in cell number.  Cytokinesis in many eukaryotes involves the closure of an actomyosin based contractile ring.  The relative contributions of actin polymerization, disassembly and myosin II function in ring closure are not fully understood. Here we show the establishment of an in vitro system for the study of actomyosin ring assembly and closure in fission yeast Schizaosaccharomyces pombe.  We have purified the actomyosin rings, which constrict upon ATP addition.  The constriction and disassembly of these rings were inhibited upon addition of the type II myosin inhibitor Blebbistatin and can be slowed down by the F-actin stabilizer phallacidin.  Surprisingly, unlike in intact cells, neither actin polymerization nor Cofilin-based severing was required for closure of actomyosin rings.  These studies establish that actin disassembly and myosin II function are sufficient for the mechanics of actomyosin ring closure.  Unlike eukaryotes bacterial cell division is orchestrated by a tubulin homologue, FtsZ, which polymerizes to form a ring like structure at the cell division site.  The mechanisms regulating the assembly and organization of FtsZ molecules into rings are not fully understood.  We show that FtaZ filaments assemble into cytoplasmic rings of a diameter of 0.5mm when expressed in fission yeast.  These rings were formed by process of closure and spooling of linear bundles.  We conclude that the FtsZ can self assemble into a ring structure and this process may involve the hydrolysis of FtsZ bound GTP and lateral associations between FtsZ filaments.  In future these in vitro system will provide mechanistic insights into diverse sets of cytokinetic apparatus.