Katya Grishchuk - Research Associate

Poleward chromosome movement during mitosis is dependent upon the activities of minus-end directed, microtubule-dependent motors, and it requires the depolymerization of microtubules. To learn the respective roles of these factors we have taken two approaches. In the first we are characterizing the roles of minus-end directed motor enzymes in vivo using fission yeast. This organism has three such motors: dynein and two kinesin-14s, Pkl1p and Klp2p. The maximum rate of poleward kinetochore movement was unaffected by the deletion of any or all of these motors, suggesting that microtubule depolymerization itself can cause such movements in vivo. However, Klp2p, which localizes to kinetochores, contributed to the processivity of poleward movement by promoting the shortening of kinetochore fibers. Pklp1 and dynein were required for efficient and accurate chromosome bi-orientation. In pkl1Δ, whose product normally localizes to the spindle and poles, the checkpoint that monitors chromosome bi-orientation was defective, leading to premature anaphase. Electron microscopy suggests that Pkl1p contributes to error-free bi-orientation by promoting the normal organization of spindle poles, while dynein helps to anchor microtubule minus ends. Thus, motor activities help to ensure the expediency and accuracy of chromosome segregation, but chromosome motility in vivo may occur in their absence and is likely to be driven by MT depolymerization.
 
Given the importance of MT depolymerization for mitosis, we are also studying this phenomenon in vitro, using a novel experimental system that allows the direct measurement of the depolymerization force. By conjugating glass microbeads to microtubules through strong inert linkages, and by using laser tweezers we have shown that depolymerizing microtubules can exert a brief tug on an attached bead. Analysis of these interactions with a molecular-mechanical model of microtubule structure and function suggests that a single disassembling microtubule can generate about ten times the force that is developed by a motor enzyme. This mechanism may therefore be the primary machinery for chromosome motion. We have recently extended this approach to demonstrate that a ten-subunit protein complex from budding yeast kinetochores, the Dam1/DASH ring, is an effective coupler for chromosome motility, because it can achieve a good transduction of energy from MT disassembly while maintaining a firm grip on the shortening MT tip. These two experimental systems, one in vivo and one in vitro, have great potential for analyzing the mechanisms of chromosome movement and the ways in which novel chemicals can specifically block mitotic progression while leaving other biological processes untouched.