Microtubule Dynamics and Chromosome Movements During Meiosis

This research project is aimed at resolving the mechanisms of chromosome congression and chromosome segregation during meiosis. Dr. LaFountain recently completed a very successful sabbatical stay in Conly Rieder's lab at the Wadsworth Center in Albany, where he discovered that there are a variety of forces acting on chromosomes during meiosis in insect spermatocytes. That work provided indirect evidence that spermatocyte spindles are engaged in microtubule flux -- a process in which tubulin subunits are constantly added to microtubule plus ends and removed from minus ends. Findings from laser microsurgery implicate flux in mediating the poleward transport properties of spermatocyte spindles. The main objective of the research is to analyze microtubule flux directly in living spermatocytes in relation to the movement of chromosomes attached to microtubules. Fluorescent speckle microscopy will be used to fluorescently label discrete domains within unlabeled microtubules. From the comparison of velocities of moving kinetochores to the velocities of speckles on kinetochore fibers attached to those kinetochores, the relative contributions made by flux and kinetochore motors to the mechanism underlying movement will be revealed. This approach not only will test the "flux machine" model for chromosome segregation during anaphase, it is expected to provide important data regarding the mechanism underlying the congression of chromosomes to the spindle equator. The research aiming to speckle-label spindle microtubules is built on findings obtained from recently completed feasibility studies done in collaboration with Christopher Cohan in the Department of Anatomy at Buffalo. These studies have revealed the conditions for the successful microinjection of fluorescently conjugated tubulin subunits into spermatocytes, ideal material for the study of meiosis in vitro but heretofore regarded to be unsuitable for microinjection methods. During congression, the positioning process that occurs during prometaphase and results in the alignment of chromosomes on the metaphase plate prior to their segregation to the spindle poles, chromosome velocities usually exceed those at anaphase, suggesting the involvement of kinetochore-based motors. To test that hypothesis, the laser workstation in the Rieder lab will be used to bisect congressing bivalents (conjoined homologous chromosomes) into two "univalents," each of which will contain the kinetochores of one of the homologues and will be analyzed for its capability to move poleward immediately after the cutting operation. Data from such operations performed at successive time intervals during the course of prometaphase will test two competing models for congression. The traction fiber model predicts the two univalents will move in opposite directions toward the pole that each faces. By contrast, the motile kinetochore model predicts only the leading univalent will continue moving poleward; the trailing univalent would be expected to be in a "neutral" state and not move at all. Broader impact: it is expected that Dr. LaFountain will continue integrating his research into educational activities, including teaching formal undergraduate courses that include a laboratory component and training graduate students in the research laboratory. He also gives presentations in non-academic settings such as community groups.