(Funded partly by NIH GMS 40198 to C. Rieder). We are exploring the idea of using IVEM or HVEM tomography to reconstruct the ultrastructure that underlies events recorded in vivo by video-enhanced LM prior to fixation. This year we showed, for the first time, that tomography could be effectively used in same-cell correlative LM/3D EM projects. Using our laser microsurgery system we can produce chromosomes containing a single kinetochore that appear by LM to become attached via spindle microtubules to both poles and then move onto the spindle equator (i.e., congress). If this is true it reveals that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochore during spindle formation and chromosome motion, and it implies that vertebrate kinetochores consist of multiple domains, that the motility states of each domain is regulated independently of one another, and that each domain contains the complete molecular machinery for kineto chore function. To evaluate this possibility we used IVEM tomography and Sterecon to precisely determine the distribution of microtubule ends around the single kinetochore region on chromosome that congressed with just one associated kinetochore. The results of this study are discussed under the Highlights section, and clearly demonstrate the utility of tomography to same cell correlative LM/EM studies.. Khodjakov, A., R.W. Cole, B.F. McEwen, K. F. Buttle and C.L. Rieder. 1997. Chromosome fragments possessing only one kinetochore can congress to the spindle equator in PtK1 cells. J. Cell Biol., 136:229-241 (with cover photograph).
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