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Back to 2005 Research Tim Mitchison Mitotic Spindle assembly and dynamics In this project the students prepared extracts from Xenopus eggs, and used them to assemble meiosis II spindles. One group examined the role of the Ran pathway in spindle assembly using mutant Ran proteins that were constitutively active or inactive. They were able to confirm the important of ran for spindle assembly, and obtained some new information on the kinetics of spindle disassembly when Ran is inhibited. Another group assayed microtubule polymerization at kinetochores, using a new reagent combination (nocodazole + vanadate) to remove non-kinetochore microtubules. They pulsed green tubulin into spindles assembled with red tubulin, and were able to measure polymerization at kinetochores. They also investigated the effects of nocodazole and vanadate alone. This project led to a post-course collaboration. Rebecca Heald (UC Berkeley) is using the new reagent combination to assay kinetochore fiber assembly in her lab Biochemistry of cytokinesis This project was initiated in response to a challenge from one of our ’05 scholars, Randy Shekman. He pointed out the lack of an inn vitro system to dissect the mechanism of cytokinesis, and we responded by trying to set up such a system in Xenopus egg extracts. We found that extracts prepared in the absence of actin depolymerizing drugs would undergo rapid gelation-contraction if they were in M phase, but not in I phase. This process is actin and myosin dependent, and may recapitulate assembly of the mitotic cortex and/or cleavage furrow. We developed a useful quantitative assay for gelation-contraction in which 1 ul drops of extract containing rhodamine-actin were spread on a coverslip under oil (figure 1). Gelation-contraction was observed by fluorescence imaging at low mag (2x objective) and quantified using a MATLAB algorithm written by the students. They used these tools to probe the role of actin polymerization and the Rho pathway. This system may provide a useful way of reconstituting one aspect of cytokinesis. It lead to a post course project. Christine Field (Harvard Med School) is developing the geleation contraction assay in her lab. Towards a Scaling Relationships for Nucleii. In the 04 course, Martin Raff, one of our scholars, challenged us with the question of how the physical dimensions of cells are established. This year we tried to determine scaling relationships for nuclei, that is, how nuclear volume and surface area change if the amount of DNA changes. We treated cells with low concentrations of different poisons of the mitotic spindle (taxol, nocodazole, monastrol). We found that after 18rs in low concentrations of drug, many cells had multiple micronuclei, because they went through mitotis without correctly segregating chromosomes (see figure 2). We stained these micronuclei for nuclear pores and nuclear lamins, and wrote image analysis software to measure amounts of DNA, volume and surface area. Our preliminary results suggest that smaller nuclei with less DNA tend to have a lower density of DNA and a lower density of nuclear pores (figure 2, note nuclear pore density in insets). This suggests that swelling of nuclei is driven by surface area expansion, rather than volume expansion. Figures.  Figure 1. Gelation-contraction of Xenopus egg extracts as a possible in vitro model of cleavage furrow assembly. Rhodamine-actin (1uM) was added to extracts from unfertilized eggs. Part of the extract was treated with Ca++ to change the cell cycle state for mitosis to interphase. Droplets of extracts were spread under oil, and imaged with a 2x objective. The images show a few stills from the time lapse record. Note that mitotic extract undergoes rapid gelation-contraction. Interphase extracts take much longer, and contraction tends to be uneven.  Figure 2. DNA and nuclear pore staining of a normal BSC1 cell (monkey) compared to a cell that has gone through mitosis in the presence of taxol, resulting in micronucleaus formation. Note that the DNA density tends to be lower in the smaller nuclei. Note also that the nuclear pore density is also lower in some cases (eg insets). |
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