KINETOCHORES IN THE FISSION YEAST, SCHIZOSACCHAROMYCES POMBE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. By tandem-affinity-purification (TAP), the He lab has purified three major kinetochore protein complexes from fission yeast (Liu, 2005). Among them, the NMS and Sim4 complexes are constitutive elements of a core kinetochore structure. By metal shadowing EM, purified NMS and Sim4 complexes are both seen as fibrils (He X., unpublished). Fibrils are commonly observed by EM at several types of kinetochores (e.g. McEwen et al., 1998 and McIntosh J.R., unpublished results). While the protein sequence conservation of kinetochore components is not strong, it is possible that the common structural features (fibrils in this case), rather than sequence similarity in individual proteins, provides the basis for a ubiquitous mechanism that is shared by all types of kinetochores. One tool now available for the studying the functional significance of the NMS complex is a temperature sensitive allele of one of its subunits, nuf2-1. Kinetochores in nuf2-1 cells grown at their restrictive temperature fail to attach to the spindle, (Nabetani et al., 2001). However, under the same condition, the NMS complex remains intact and is properly localized on the kinetochores (He, X. unpublished). We will determine whether the NMS fibrils are still found at kinetochores and whether there is a change in their structure or position relative to the kMTs. The third kinetochore complex, Dam1, has been purified only from mitotic extracts (Liu, 2005). In vivo, the Dam1 complex associates with kinetochores and spindle microtubules specifically in mitosis. A recombinant form of budding yeast Dam1 complex was shown to assemble a ring around MTs in vitro (Miranda et al., 2005; Westermann et al., 2005). However, no such ring structure around kMTs has yet been seen in vivo. We will use a combination of the immuno-gold labeling and EM tomography to determine the localization of the Dam1 complex relative to the kMTs and provide the most powerful evidence about the presence or the absence of a Dam1 ring. Using Immuno-EM and EM tomography of cells with GFP tags on 3 of the Dam1 subunits, the Dam1 complex can be localized in thin (70nm) sections, and any possible fine structural features, such as rings, that are associated with the Dam1 complex will be sought within the section.