Dynamics and Mechanics of Mitosis in Drosophila: Mechanisms of Anaphase B

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DESCRIPTION (provided by applicant): During mitosis, the mitotic spindle uses microtubules (MT) plus mitotic motors to coordinate chromatid-to-pole motility (anaphase A) and spindle elongation (anaphase B). The aim of the work described here is to provide a comprehensive molecular and quantitative explanation of anaphase B. The conceptual framework underlying the proposal is that spindle elongation depends on an interpolar (ip) MT sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an "on-off" switch. We will explore a model in which the pre-anaphase B spindle is maintained at a steady state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. In response to cyclin B degradation at the end of anaphase A; (i) a MT catastrophe gradient causes ipMT plus ends to invade the overlap zone where outward ipMT sliding occurs; and (ii) ipMT minus end depolymerization ceases so flux is turned "off", tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. The specific aims are: 1. To continue our biochemical and structural analysis of the interactions between purified kinesin-5 and MTs, in order to improve our understanding of the sliding filament mechanism underlying anaphase B and its regulation; 2. To determine how the network of MT polymerases, depolymerases, crosslinkers and sliding motors cooperate to create the MT catastrophe gradient and turn off poleward flux in response to cyclin B degradation; And 3. To analyze the dynamics and structural reorganization of spindle MTs, motors and MAPs associated with the transition from pre-anaphase B to anaphase B. This multidisciplinary project will utilize protein biochemistry and motility assays, in vivo imaging and electron microscopy, the genetic and biochemical manipulation of living cells, together with quantitative modeling. We aim to learn how the anaphase spindle functions as a macromolecular machine to elongate itself and pull apart sister chromosomes, and thus to provide insights into how defects in its function can give rise to genomic instability, birth defects and cancer. PUBLIC HEALTH RELEVANCE: This basic science research project is aimed at understanding the mechanism by which the mitotic spindle coordinates the accurate segregation of the genetic material, a fundamental process that underlies the propagation of all life on Earth. An improved understanding of the normal mechanisms of mitosis may illuminate defects in this process that lead to genomic instability, birth defects and cancer; and also may help us understand the changes in this process that occur in the asymmetric mitoses that underlie stem cell divisions. This, in turn, could lead to improvements in the treatment of mitosis-related diseases, e.g. through the use of inhibitors that target specific mitotic proteins as potential anti-cancer agents.