We are using cryofixed, freeze-substituted material to study the kinetochores and their associated microtubule (MT) ends in mitotic mammalian cells. PtK cells are grown on chips of Thermanox cover slips, high-pressure frozen, freeze-substituted, and sectioned at 250-300 nm. 3-D reconstructions of kinetochore regions are obtained using dual-axis HVEM tomography. The walls of these MTs are much straighter than in chemically fixed material and the shapes of MT ends can be identified more reliably. In cryofixed material, the walls of most kinetochore MTs flare outward at their plus ends, a morphology characteristic of disassembling MTs in vitro (Mandelkow et al., JCB 114:977, 1991). Portions of 3 kinetochores have been reconstructed from a cell in prometaphase, 2 from metaphase, 4 from early anaphase, and 2 from mid-anaphase, with ~15 MTs found on each kinetochore. The degree of flaring has been quantified and found to be similarly distributed at all mitotic stages, with some MTs dramatically flared, most modestly flared, and some negligibly flared. In particular, such a distribution was found for two sister kinetochores from a metaphase cell, and for two sister kinetochores from a prometaphase cell. These findings suggest that the flaring is not simply indicative of disassembly. We propose that it is a morphology characteristic of MTs interacting with the kinetochore machinery. ?
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