3D STRUCTURE OF ER IN MITOTIC AND INTERPHASE CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们正在使用快速冷冻、冷冻替代的非洲绿猴肾(BSC1)细胞的电子断层扫描来评估细胞分裂不同阶段内质网(ER)精细结构的变化。对整个有丝分裂细胞的三维荧光显微镜研究表明，ER网络在有丝分裂的各个阶段都经历了形态变化，所有这些都不同于间期ER模式。电子断层扫描被用来补充LM的工作，并将发现扩展到精细结构水平。到目前为止，我们已经准备了9组间期细胞、19组中期细胞、3组后期细胞和5组末期细胞的断层数据。初步结果证实在间期BSC1细胞中存在脑池和网状ER结构域。虽然这些区域是连续的，但它们大多位于细胞质的不同部分；池区域更靠近细胞核，而网状区更靠近细胞外围。在有丝分裂过程中，最明显的是中期和后期，内质网成分位于细胞边缘，在有丝分裂纺锤体区外。在这些阶段，网状结构域不存在，内质网仅表现为短小的脑池，核糖体与外表面结合。我们目前正在研究有丝分裂的早期(前期和中期)和后期(后期和末期)的内质网特征。对晚期末期细胞的研究也可能有助于揭示核膜重塑的早期步骤。免疫荧光研究表明，某些ER标记的分布不均匀，我们预计免疫电子显微镜将被用于确定细胞质ER结构域是否可以在分子水平和结构水平上进行表征。

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.