3D STRUCTURE OF ER IN MITOTIC AND INTERPHASE CELLS

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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. 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.