CHARACTERIZATION OF MIA1P MUTANTS IN SCHIZOSACCHAROMYCES POMBE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。Mia1p是一种未知的卷曲卷曲蛋白，其在有丝分裂过程中的定位和功能正在研究中。该蛋白定位于金黄色葡萄球菌中已知的微管组织中心：纺锤体极体(SPB)和后期阵列。对HM20或EPON/Araldite中嵌入的快速冷冻、冷冻替代细胞的连续切片的分析使我们能够表征Mia1p过表达突变体的表型。Mia1p高表达株的细胞在有丝分裂过程中表现出核膜内陷和突起(即长柄)。核膜的内陷延伸到核内，在核内形成囊状结构，在核的外围有一个狭窄的开口通向细胞质。内陷含有来自细胞质的物质。连续切片分析表明，内陷可能是SPB插入的结果，这种插入通常发生在有丝分裂的早期(即早期)。半厚切片的电子断层扫描应该能解决Mia1p过表达菌株中SPB插入和内陷形成的问题。核包膜还形成含有MTS的长柄状结构。一般说来，这些原子核中的大多数似乎都存在双狭长的情况。SPB发生在盘柄的远端或盘柄与固有核的交界处。连续切片分析表明，至少一个复制的SPB可能缺少外部斑块成分，可能是SPB复制和/或SPB插入缺陷的结果。目前尚不清楚是单个SPB的MTS导致了双重狭长柄情况，还是两个SPB都参与了这一过程。半厚切片的电子断层扫描应该解决有关SPB的形态和MTS的精确相互作用的问题，因为它们与狭节的形成有关。

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.