The overall objective of this research program is to determine the molecular and structural mechanisms which regulate microtubule (MT) and spindle assembly dynamics and the mechanisms that move chromosomes. A major strength of our application is the development and application of new techniques in quantitative optical microscopy, fluorescent analog cytochemistry, caged compounds, laser optical traps and scissors, video- and digital imaging to measure the dynamics of individual MTs and associated processes in living cells and reconstituted preparations in vitro. The major specific aims are: (1) develop new light microscopy methods for tracking the motion of objects like kinetochores and microtubule ends, optical traps for measuring forces and manipulating objects relative to MTs, digital imaging methods for photon limited analysis of molecular and structural dynamics for spindles in tissue cells, extracts and in genetic organisms like yeast and Drosophila; and photoactivation techniques for GFP and other caged fluorophores and compounds for analysis of spindle and MT dynamics: (2) use high resolution video and digital microscopy assays of purified and cell extract systems to determine the molecular basis of MT dynamic instability, identify cell cycle dependent regulatory factors (non motor and MT motors) and obtain tubulin peptide ligands from phage-displayed random peptide libraries; (3) employ laser optical traps, laser scissors and calibrated glass microneedles to measure MT mechanical properties, force generation by MT polymerization/depolymerization, the strength of MT tip attachment molecules (TACs or couplers), kinetochore forces, pole-complex forces and polar ejection forces on chromosome arms and MT motor activity; (4) use fluorescence photoactivation approaches to determine the functions and dynamics of the molecular components of spindle fibers, kinetochores and spindle pole-complexes during congression and anaphase in vertebrate tissue cells, plant cells and cytoplasmic extracts; (5) analyze the motility of minus kinesins and their function in spindle pole organization and poleward force generation; (6) use high resolution digital imaging of genetically manipulated Drosophila and yeast cells to determine the functions of different proteins in mitotic spindle dynamics, chromosome segregation and cell cycle regulation using Drosophila and yeast cells.
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