SLIDING FILAMENT MECHANISMS IN MITOTIC SPINDLES

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The overall aim of the project is to learn how multiple microtubule sliding motors contribute to the morphogenesis of the mitotic spindle in Drosophila syncytial blastoderm-stage embryos. These embryos are amenable to biochemical, genetic and cytological analysis of mitotic mechanisms, and previous work done in this laboratory showed how three mitotic motors, KLP61F, Ncd, and cytoplasmic dynein, cooperate fo position spindle poles during spindle assembly, maintenance and elongation. We want to further test the hypothesis that KLP61F and Ncd, located on interpolar microtubule bundles, and dynein localized on the cell cortex, cooperate to drive a sliding filament mechanism that precisely positions spindle poles. To do this we will use techniques already developed in the laboratory, including the purification and analysis of native motor proteins, the microinjection of function- blocking antibodies and mutant proteins, and the high resolution analysis of spindle pole positioning by time-lapse confocal microscopy of living embryos. We will augment these approaches with the use of Fluorescence Photobleaching, Fluorescence Photoactivation or Fluorescent Speckle Microscopy to directly visualize microtubule- microtubule and microtubule-cortical actin sliding in living embryos.