CHARACTERIZATION OF MIA1P MUTANTS IN SCHIZOSACCHAROMYCES POMBE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Mia1p是一种以前未知的螺旋状蛋白，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pombe中已知的微管组织中心：纺锤杆体（SPB）和后期阵列。对快速冷冻、冷冻替代的细胞在HM20或Epon/ Araldite中包埋的连续切片分析，使我们能够表征Mia1p过表达突变体的表型。Mia1p过表达菌株的细胞在有丝分裂过程中表现出核膜内陷和突出（即panhandles）。内陷的核膜延伸到细胞核内，在核内形成囊状结构，在核周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。连续切片分析提示内陷可能是SPB插入的结果，通常发生在有丝分裂早期（即前期）。半厚切片的电子断层扫描应该能解决在Mia1p过表达菌株中SPB插入和内陷形成的问题。核膜也形成长盘柄状结构，其中包含mt。一般来说，在大多数核中似乎存在双盘柄条件。spb要么发生在长柄的远端，要么发生在长柄与核的交界处。序列切片分析表明，至少有一个复制的SPB可能缺乏外部空斑成分，可能是SPB复制和/或SPB插入缺陷的结果。目前尚不清楚是否单个SPB的mt负责双长柄条件，或者是否两个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形态和mt的精确相互作用的问题，因为它们与狭长状地层有关。

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.