Organization and dynamics of actin and myosin during cytokinesis in mammalian epithelial cells

Cytokinesis, the process of physically separating cells for division, requires the precise orchestration of numerous physical, mechanical, chemical and biological processes. For these processes to function well, complex coordination of various proteins, with crosstalk between them, either as signaling molecules or as just plain structural components that contribute to the physical separation must exist. Actin, a structural polymer and myosin, a motor are two proteins that contribute to this process significantly. Both proteins are assembled in the contractile ring and together are responsible for the process of constriction. A thorough understanding of the behavior of these proteins, in the contractile ring as well as outside in a cell undergoing cytokinesis is therefore important to prevent possible defects that might lead to deleterious diseases. In this dissertation research, a combination of techniques are made use of, that involve live imaging of fluorescently labeled proteins in cells undergoing cytokinesis along with the use of drugs that either disrupt the structure (integrity) or function of cytokinetic proteins. I generated two LLCPK1 (pig epithelial cell lines); one that stably expresses GFP-actin and the other that stably expresses Tandemn Dimer RFP-myosin regulatory light chain (TDRFP-MRLC). Live imaging and analysis of cells expressing GFP-actin shows that actin in the contractile is highly dynamic and need to be dynamic. Evidence is presented for new roles of Myosin II, in addition to generating the force for cytokinesis. Myosin not only contributes to disassembly of actin in the contractile ring but is also required to maintain actin in the equatorial region. Live imaging of the cell lines that expresses TDRFP-MRLC or GFP-actin helped in the better understanding of the role of microtubules in simultaneously regulating actin and myosin dynamics, not only in the contractile ring to allow ingression, but also in preventing contractile activity outside in the contractile ring. Cytokinesis involves other proteins besides actin and myosin, which help in their recruitment, assembly, ingression and subsequent disassembly. Decreasing the accumulation of actin in the contractile ring, by treatment with Latrunculin B facilitated the examination of spatial and temporal events involved in building the ring. Actin, myosin and other proteins organized as nodes that coalesce during ingression, similar to the fission yeast. We conclude that this mode of cytokinesis a highly conserved feature of cytokinesis