3D STRUCTURE OF ER IN MITOTIC AND INTERPHASE CELLS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们正在使用电子断层扫描的快速冷冻，冷冻替代非洲绿色猴肾（BSC 1）细胞，以评估变化的内质网（ER）的精细结构在细胞分裂的不同阶段。 整个有丝分裂细胞的三维荧光显微镜研究表明，ER网络在有丝分裂的每个阶段都经历了形态学的变化，所有这些都不同于间期ER模式。 电子断层扫描正被用来补充LM的工作，并将研究结果扩展到精细结构水平。 到目前为止，我们已经准备了9个间期细胞的断层数据集，19组中期细胞，3组后期细胞和5组末期细胞。 初步结果证实了间期BSC 1细胞中存在池状和网状ER结构域。 虽然这些结构域是连续的，但它们主要位于细胞质的不同部分;池状结构域更靠近细胞核，而网状区域更靠近细胞周边。 在有丝分裂过程中，最明显的是在中期和后期，ER组分被发现在细胞的边缘，有丝分裂纺锤体区之外。 在这些阶段，网状结构域是不存在的，ER只出现作为短，与核糖体结合到外表面的池。 我们目前正在研究有丝分裂最早期（前期和前中期）和最晚期（后期和末期）的ER特征。 晚期细胞的研究也可能揭示核膜重组的早期步骤。 免疫荧光研究表明，某些ER标志物的不均匀分布，我们预计，免疫电子显微镜将被用来确定细胞质ER域的分子，以及结构，水平上的特点。

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. We are using electron tomography of rapidly-frozen, freeze-substituted African green monkey kidney (BSC1) cells to evaluate changes to the fine structure of the endoplasmic reticulum (ER) during the different stages of cell division. Three dimensional fluorescence microscope studies of whole mitotic cells suggest that the ER network undergoes morphological changes in each phase of mitosis, all of which differ from the interphase ER pattern. Electron tomography is being used to complement the LM work and extend the findings to the fine structural level. We have so far prepared 9 tomographic datasets of interphase cells, 19 sets of cells in metaphase, 3 sets of anaphase and 5 sets of telophase cells. Initial results confirm the presence of cisternal and reticular ER domains in interphase BSC1 cells. Although these domains are continuous, they are largely situated in different parts of the cytoplasm; Cisternal domains are nearer the nucleus while the reticular areas are nearer the cell periphery. During mitosis, most notably in metaphase and anaphase, ER components are found at the edges of the cell, outside of the mitotic spindle zone. In these phases, reticular domains are absent and ER appears only as short, cisternae with ribosomes bound to the outer surfaces. We are currently working to characterize ER in the earliest (prophase and prometaphase) and latest (late anaphase and telophase) stages of mitosis. Studies of late telophase cells may also shed light on the early steps of nuclear envelope reformation. Immunofluorescence studies suggest an uneven distribution of certain ER markers and we anticipate that immunoelectron microscopy will be used to determine if cytoplasmic ER domains can be characterized on the molecular, as well as the structural, level.