The longterm goal of this project is to determine the mechanisms that cells use for assembling and regulating the interactions of the actin and myosin filaments that form the cleavage furrow. A number of microscopic imaging methods will be used with living cells that have been microinjected with various probes, to analyze the distribution of the major component molecules of the cleavage ring. The first aim will be to determine how actin filaments are assembled into the region of the cortical membrane where the future contractile ring will form. The source of the actin that composes the contractile ring is uncertain. Considering the importance of this division process for the cells, there may be two independent methods for directing actin to the cleavage furrow. Experiments are planned to test this hypothesis by determining if and when the filaments move along the cortical surface to the cleavage furrow and if there is elongation of the pre-existing actin filaments in the furrow region, and to determine if actin monomers add to one or both ends of the existing actin filaments in the furrow regions. Experiments are also planned that will utilize aequorin to measure fluctuations in calcium levels during the assembly and contraction of the cleavage ring in these dividing tissue culture cells. The calcium levels inside cells are thought to be regulated by the endoplasmic reticulum. Confocal microscopy of living cells is planned to determine if there is any co-distribution of the endoplasmic reticulum and actin filaments in the assembling and contracting cleavage ring. All of the proposed experiments are designed to determine the mechanisms responsible for a basic process present in all animal cells: cytokinesis. %%% The ability to reproduce is the most fundamental, defining property of life. Cell division is an intrinsic part of this phenomenon for all cellular organisms. For single-celled organisms, reproduction is, simply, cell division. For multicellular organisms, cell division is key to reproduction, by generating germ-line cells (gametes, spores) and for regenerating whole organisms from spores or zygotes (development). The division of one cell into two is a remarkably controlled process; the precise geometry of cleavage is critical to the survival of the daughter cells and/or the organism. The cleavage mechanism in animal cells generally involves an actomyosin based contractile apparatus, the contractile ring, which girdles the cell at the inside of the cleavage furrow. This project will use experimental manipulations and state-of-the-art microscopic observations directly on living cells to better understand the cleavage process.
