(Supported in part by GMS 40198 to C. Rieder and NSF MCB 9420772 to B. McEwen) The strategy of the proposed research is to use electron microscopic tomography (EMT), video light microscopy and 3-D immuno-electron microscopy to evaluate several current hypothesis concerning chromosome motion and kinetochore/centromere complex (KCC) organization. The latter include models of the interaction between kinetochore microtubules (kMT's) and the KCC during movement toward and away from the spindle pole; how the direction of motion is controlled; if the morphological changes associated with the dynamic instability of MTs in vitro can be detected in kMTs; the repeat subunit model for the kinetochore outer plate organization; and the possible role of specific DNA sequences in KCC function. The results of these investigations will significantly improve our understanding of how mitosis works, and lay the foundation for future studies that identify the chemical mechanisms responsible for many of the various chromosome motions during mitosis. This part of the study evaluates polar ejection forces. These forces push objects away from the spindle poles and produce chromosome motion away from the pole. It is generally assumed that polar ejection forces arise from elongating spindle microtubules (Mts) pushing against objects they encounter. To test this hypothesis we are tracing Mts that impinge upon chromosome fragments that have been cut from chromosome arms by laser surgery. To avoid confusion from interactions with Mts from both spindle poles, fragments are cut from monooriented newt lung chromosomes located in anaphase-like prometaphase cells where the spindle poles are too far apart to interact with the same chromosomes. The prediction is that strongly ejecting fragments will have significantly more Mts than weakly or non-ejecting fragments. A second question is do the Mts end at the fragment's surface, as predicted by a Mt pushing theory, or do they track along or through the fragment as predicted by a theory that ejection is generated by Mt motor molecules in the chromosome arms. Cells are filmed prior to, during, and after laser surgery to determine the """"""""strength"""""""" (i.e., rate) of the ejection force, and then fixed for 3D-EM. Mts are traced either in tomographic reconstructions of serial sections 0.35-.050 5m thick, or in serial-section reconstructions of 90nm thick sections. Thus far fourteen cells have been filmed and fixed. Four of these were lost during the embedding procedures. Of the remainder three showed strong ejection. One of these was analyzed via tomographic reconstruction of 0.50 5m thick sections but no Mts were found. However the analysis was limited by a large rip in the formvar support that appeared when the specimen was first put into the HVEM. Currently we are analyzing a second serial thick-section series and have thus far found only one Mt interacting with the ejected fragment. This Mt runs tangential to one edge of the fragment consistent with the motor model for ejection. Tomographic reconstructions are currently being computed f or three other serial sections in the series to make sure we haven't over looked any Mts. However, since no Mts are visible in the untilted projections from these series, it seems likely that the observed ejection is cause by a single Mt interacting with the chromosome fragment. We have cut a serial thin section (90 nm thickness) series from the third cell exhibiting strong ejection and the reconstruction process is currently under way. Earlier we completed a serial thin section reconstruction of a fragment that showed weak or no ejection and found no Mts. From the work thus far it is clear that only a few Mts are involved in polar ejection from anaphase-like prometaphase spindles in newt lung cells. For this reason we are developing an immunolabling protocol for staining Mts so they can be more readily identified and traced in tomographic reconstructions. This development will allow use to analyze 1.0.5m thick sections. This both saves time and enhances our ability to trace Mt continuity.
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