Successful mitosis requires the equal segregation of the genome from mother to daughter cells. This occurs on the mitotic spindle, an intracellular machine that uses microtubules (MTs) and MT-based motor proteins to coordinate chromosome movements with cell division. The goal of our proposed studies is to elucidate the motor-mechanisms that position chromosomes on the spindle. Our central hypothesis is that this involves the cooperative activity of multiple motors functioning simultaneously to generate a dynamic balance of complementary and antagonistic forces. Specifically, motors positioned on kinetochores generate forces directed toward the spindle poles while motors positioned on chromosome arms generate forces directed toward the metaphase plate. We posit that when these 'poleward' and 'plateward' forces precisely balance, a steady-state structure forms and chromosomes maintain a stable position, as during metaphase. Tipping this balance, via the up- or down-regulation of a subset of motors, results in specific chromosome movements such as chromatid-to-pole motion during anaphase A. To study this, we will carry out the following specific aims using Drosophila early embryos as our primary experimental system:
Aim 1) Characterize the rates and structural basis of chromosome motility with high spatial and temporal resolution.
Aim 2) Test the hypothesis that the kinetochore binding motors, dynein/dynactin and KinI kinesins, work cooperatively to generate poleward forces on chromosomes.
Aim 3) Test the hypothesis that the chromosome-arm binding motors, KLP38B and Nod, work cooperatively to generate plateward forces on chromosomes.
Aim 4) Examine the functional inter-relationships that exist between sets of poleward and plateward motors to determine whether chromosomes are subjected to counterbalancing motor-generated forces. Our overall experimental strategy is to utilize the results of analyses of individual motors to formulate and test broader hypotheses regarding how these motors work collectively to drive the coherent and tightly controlled reorganization of the genome that must occur during cell proliferation. Because defects in this process lead to numerous human maladies, including birth defects and cancer, our findings should provide insights into the causes of these diseases and suggest potential therapeutic approaches to their treatment.
