Self-organization of microtubules in interphase cells

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DESCRIPTION (provided by applicant) The broad objective of the parent proposal for this international collaborative supplement is to understand the mechanisms of the formation and centering in vivo of the radial array of cytoplasmicn microtubules (MTs). 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 organize MTs into a radial array. Specific aims focus on analysis of the roles of MT dynamics and MT motors in the attainment of the radial MT organization. Experimental system involves cytoplasmic fragments of pigment cells, melanophores, lacking the centrosome. The supplemental proposal is directly related to the principal aims of the parent grant. The supplemental proposal is motivated by the observations made by the foreign collaborator during the visits to the Pl's laboratory, which indicate an apparent paradox in MT organization field. Although the self-organization mechanisms, which depend on the MT dynamics and the activity of MT motors are responsible for the focusing of MTs in the absence of the centrosome, they are likewise essential for the establishment and centering of the centrosomal radial MT array. A combination of experimental techniques that include digital fluorescence microscopy, laser microsurgery, photoactivation and expression of dominant-negative subunits will be used to elucidate the mechanisms of self-organization of and cenetering of the centrosomal radial MT array. Specifically, we will determine the role of a minus-end MT motor, cytoplasmic dynein, in the focusing of MTs at the centrosome and test the mechanisms of centering force production.