Cell division in animal cells culminates in cytokinesis, the separation of two daughter cells. The force generating apparatus for this cleavage is the contractile ring, a transient actin-myosin organelle that is attached to and exerts tension on the plasma membrane. Many facets of cytokinesis remain to be elucidated. These include the source, composition, and interaction of contractile ring components, the regulation of contraction, and the spatial positioning of the contractile ring. The proposed experiments address several of these aspects. Monoclonal antibodies, which were prepared against actin-associated proteins, have allowed identification of several protein components of the contractile ring in sea urchin eggs. These proteins will be purified and characterized in vitro, particularly with regard to the proteins with which they interact. The functions of the proteins in vivo will be investigated in microinjection experiments with the antibodies prepared against them. The regulation of contraction will be examined by investigating whether changes in myosin phosphorylation occur at cytokinesis when the amount of myosin in the cytoskeleton increases. The results of the proposed experiments will allow a molecular description of the functions of several components of the contractile ring and thus increase our understanding of this dynamic structure. %%% Reproduction is the most fundamental aspect of all living things. Cell division is an intrinsic part of this phenomenon for all cellular organisms, both prokaryotic and eukaryotic. For Protistans (single-celled organisms), reproduction is, simply, cell division. For multicellular organisms, cell division is key to reproduction, not only for generating germ-line cells (gametes, spores) but also for regenerating whole organisms from spores or zygotes (development). In multicellular organisms, tight regulation of the mechanism whereby daughter cell cytoplasms are partitioned from each other during cell division, including the precise position of the cleavage furrow, is invariably crucial for subsequent development of the organism. Thus, this project addresses one of the most fundamental aspects of animal life on earth. The cleavage mechanism is known to involve an actomyosin based contractile apparatus, the contractile ring, which girdles the cell at the cleavage furrow at the cytoplasmic face of the plasma membrane. The goals of this project are to discover what other proteins, besides actin and myosin, are in the ring, how the ring components function to cause the constriction of the membrane at the cleavage furrow, and how all this is regulated. The approaches to be used will combine rigorous in vitro biochemical analysis with experimental manipulations and observations in vivo.
