The overall objective of this project is to determine the molecular and structural mechanisms which regulate microtubule (Mt) assembly and move chromosomes. Because chromosome movement is tightly coupled to the dynamics of Mt assembly, our approach continues to be focused on establishing the pathways and regulation of Mt assembly. A major strength of our research effort is the development and application of new techniques in quantitative optical microscopy, fluorescene analog cytochemistry and digital image processing and analysis to measure Mt associated processes in living cells and in purified preparations in vitro. The overall objective is to reconstitute life-like spindle assembly and chromosome movements from purified centrosomes, chromosomes, tubulin, regulatory proteins, molecular motors such as kinesin and other supporting components. The major specific aims are: 1) to determine the pathways of tubulin assembly-disassembly for kinetochore Mts (kMts) and non- kMts during chromosome-to-pole movements in living cells by using a fluorescent tubulin analog, fluorescence redistribution after photobleaching (FRAP), digital image processing and immunocytochemistry; 2) to test the """"""""dynamic instability"""""""" model and provide functional assays for the action of microtubule associated proteins (MAPs) by measuring directly the dynamics of Mts assembled in vitro from purified tubulin and potential regulatory MAPs; 3) to identify MAPs which regulate microtubule assembly; 4) to reactivate life-like Mt dynamics in spindles isolated from sea urchin embryos; 5) to determine the structure and composition of the spindle matrix and centrosomes; 6) to reconstitute chromosome movements in vitro from purified components of the mitotic apparatus; and 8) to study the mechanisms of chromosomes ejection and submicroscopic transport in the half-spindle.
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