Organization of cytoplasm is crucially important to all the processes that occur in living cells, including compartmentalization, cell metabolism, and cell division. It is believed that the system of microtubules (MTs) plays a key role in organizing the cytoplasm by forming a radial array that supports membrane traffic and determines positions of organelles. Formation of the radial array is generally assumed to be a result of MT nucleation at the centrosome, which often locates in the center of a cell. Recent studies indicate, however, that cells have a remarkable self-organizing capacity and that MT motors, which normally use MTs as 'rails', may in turn arrange MTs into a radial array. The mechanisms of self-organization remain largely unknown. The overall goal of the proposed research is to elucidate the self-organization mechanisms that define formation and centering of the radial MT array in vivo. Our recent observations established experimental system, which allows addressing these questions. The system involves cytoplasmic fragments of pigment cells, melanophores. In melanophores, thousands of pigment granules rapidly aggregate to the center or redisperse throughout the cytoplasm. The granules move by means of MT motors. Cytoplasmic fragments of melanophores lacking the centrosome rapidly form and position to the center the radial array of cytoplasmic MTs. Formation of such an array requires the presence of pigment granules, MT dynamics and the activity of a MT motor, cytoplasmic dynein. A combination of experimental techniques that include digital fluorescence microscopy, photobleaching, photoactivation, laser microsurgery and microinjection of motor-specific probes will be used to elucidate mechanisms of self-organization and self-centering in melanophore fragments. Specifically, we will unravel the mechanism of MT nucleation on the pigment aggregates that controls spatial organization of MTs. We will locate the sites of production of the centering force and determine the role for MT motors in positioning of the radial MT array. Finally, we will determine the minimal complement of molecular components essential for the self-organization and self-centering by reconstituting these phenomena in vitro. These studies are important for understanding the general mechanisms of cellular organization but also for the design of new therapeutic strategies aimed at prevention and treatment of disease.
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