The cytoskeletal microfilament protein, actin, and its molecular motor, myosin, play critical roles in fundamental eukaryotic cellular functions that involve movement, whether whole cell locomotion or intracellular movements such as cytokinesis (cell division). The overall goal of this project is to gain a more thorough understanding of the actin-myosin mediated contractile mechanisms that govern cell shape, movement and division by characterizing the organization of actin and myosin in intact cells and by identifying the molecular interactions involved in regulating cytoskeletal assembly and function. The apparent interplay between force production and assembly of the contractile apparatus has important implications in mechanically regulating cell division. To address this problem, it is critical to obtain more complete information on the molecular organization of actin and myosin during active cell division. As with muscle contraction, knowledge of both the global orientation and polarity of actin filaments is crucial to determining how forces are applied to the membrane, while information on the molecular organization of myosin is essential to understanding how and where force production is generated during cell division. The experiments will focus on understanding the structural assembly and regulation of actin-myosin in actively motile, dividing cells using a combination of imaging, biochemical, biophysical, and molecular biological approaches. Specifically, experiments will focus on mapping the global organization, orientation, polarity, and interaction of actin and myosin filaments followed in live dividing NRK cells, with subsequent analysis of molecular structure using high-resolution ultrastructural methods. Methodology will include high-resolution, single-cell analysis on actively dividing cells and single cell correlative light and electron microscopy methods, combined with the development and application of a variety of molecular probes for actin and myosin function (including conventional fluorescent reagents, GFP-analogs, and photoactivatable caged probes) to define the molecular structure, filament polarity, and axial organization of actin and myosin within actively cleaving cells. This work is expected to produce the first structural-dynamic analysis of actin-myosin contractility in dividing cells, and will directly test specific aspects of the "contractile ring" hypothesis originally put forward in the literature nearly 30 years ago. The results of these studies will provide a comprehensive integrated understanding of the biomechanical basis for cell cleavage, and will provide a foundation of knowledge for subsequent experiments addressing the role force plays in regulating actin-myosin based contractility in cytokinesis.
