February 4 , 2009
3 pm in Howey Physics Room N110

 

Ekaterina L. Grishchuk
University of Colorado at Boulder

"Biological nanomachines for moving chromosomes:

motors and polymers that generate force"

 

Poleward chromosome movement during mitosis is dependent upon the activities of minus-end directed, microtubule-dependent motors, and it requires the depolymerization of microtubules (MTs). 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. Although deletion of all three such motors leads to elevated chromosome loss, the maximum rate of poleward kinetochore movement is unaffected. 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.

To analyze the mechanism of chromosome movement via MT disassembly we directly measured the depolymerization force in vitro. Our results suggest that a single disassembling MT can generate about ten times the force that is developed by a motor enzyme. To capture this energy, the kinetochore must have a specialized molecular “coupler”. The Dam1/DASH protein complex from budding yeast kinetochore can form rings around MTs and track their shortening ends in vitro, so they may be the relevant coupler. We have quantified the MT-dependent diffusion, sliding, and cargo moving properties of Dam1 complexes in vitro, using fluorescence microscopy, laser trapping and mathematical modeling. We propose that rings follow MT depolymerization as a result of protofilament power strokes that force the relocations of strong Dam1-tubulin bonds. Modeling suggests that this “forced walk” mechanism enables the ring to keep a firm grip on shortening MT ends while yielding significant efficiency in energy transduction. Such features appear well suited for budding yeast, where each kinetochore is stably attached to only one MT. Chromosome couplers in higher eukaryotes, however, are likely to be based on fibrillar structures. Electron tomography of kinetochore MTs in PtK cells reveals fibrils that appear to transmit tension from bending tubulin protofilaments to the centromeric chromatin. Our biophysical studies of the conserved NDC80 kinetochore complex interacting with depolymerizing MTs in vitro suggest that it can provide processive coupling to MT depolymerization, so these proteins are likely to be an integral part of kinetochore motility in all eukaryotic cells.