Molecular Dissection of the "Pacman-Flux" Machinery Used to Move Chromosomes

DESCRIPTION (provided by applicant): The ability of chromosomes to move towards opposite poles of the mitotic spindle is fundamental for proper cell division and defects in this process are believed to be an initial step in tumorigenesis. Poleward chromosome motility occurs by a "Pacman-flux" mechanism: Chromosomes induce the depolymerization of attached microtubule plus-ends, termed "Pacman", while being reeled into spindle poles by poleward tubulin flux" stimulated by depolymerization of microtubule minus-ends. The goal of the studies outlined in this proposal is to elucidate the molecular machinery that stimulates and controls "Pacman-flux"-based chromosome motility. The central hypothesis of this work is that chromosome motility is controlled by a mechanistically diverse array of proteins which employ distinct targeting and mechanisms-of-action to control the polymerization state of microtubule ends. There are two specific aims: Aim 1) Elucidate the molecular pathway that stimulates and controls the velocity of microtubule minus-end depolymerization and poleward flux. Studies in this aim evaluate the flux-related functions of microtubule severing proteins, multiple kinesin-13s and kinesin-13 phosphorylation. Aim 2) Elucidate the molecular pathway by which chromosomes induce the depolymerization of microtubule plus-ends. Studies in this aim evaluate the Pacman-related functions of CLIP-170/190, microtubule severing proteins, and multiple kinesin-13s. The fruit fly Drosophila melanogaster is the primary experimental system used in these studies. Live cell techniques for visualizing spindle and chromosome dynamics have been optimized in Drosophila S2 cells and embryos and thus these cells provide an ideal context within which to study how the manipulation of protein function impacts "Pacman-Flux". Live cell microscopic studies of mitosis will be complemented by biochemical and molecular approaches to provide an in-depth understanding of whether and how specific classes of proteins drive chromosome segregation. Additional studies are performed in human cells to determine whether aspects of our proposed pathways are evolutionary conserved. Defects in chromosome segregation lead to human maladies such as birth defects and cancer. An understanding of how this process occurs normally should provide insights into the molecular etiology of these diseases and suggest therapeutic strategies for their treatment.