CHARACTERIZATION OF MIA1P MUTANTS IN 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. Mia1p is a previously unknown coiled-coil protein whose localization and function during mitosis are being investigated. This protein localizes to both the known microtubule organizing centers in S. pombe: the spindle pole body (SPB) and the post-anaphase array. Analysis of serial sections from rapidly frozen, freeze-substituted cells embedded in either HM20 or Epon/ Araldite has enabled us to characterize the phenotype of the Mia1p overexpression mutant. Cells of the Mia1p overexpression strain exhibit nuclear envelope invaginations and protrusions (i.e., panhandles) during mitosis. The invaginations of the nuclear envelope extend into the nucleus and form a sac-like structure within the nucleus-proper, with a narrow mouth opening to the cytoplasm at the nuclear periphery. The invaginations contain material from the cytoplasm. Serial section analysis suggests that the invaginations may be the result of SPB insertion that normally occurs in early mitosis (i.e., prophase.) Electron tomography of semi-thick sections ought to resolve the question concerning SPB insertion and formation of the invaginations in the Mia1p overexpression strain. The nuclear envelope also forms long panhandle-like structures that contain MTs. Generally, it would seem to be the case that a double-panhandle condition exists in most of these nuclei. SPBs occur either at the distal end of the panhandles or at the junction of the panhandle with the nucleus proper. Serial section analysis suggests that at least one of the duplicated SPBs may lack an outer plaque component, possibly the result of either a defect in SPB duplication and/ or SPB insertion. It is currently unknown whether the MTs of a single SPB are responsible for the dual panhandle condition or if both SPBs are involved in this process. Electron tomography of semi-thick sections ought to resolve the questions concerning SPB morphology and the precise interactions of MTs as they pertain to panhandle formation.