| Research Abstract |
We have established CHO mutants defective in the late endocytic pathway (LEX mutants). We have found that one of the mutants, LEX2, was defective in late endosomal sorting due to the lack of an enzyme involved in the later stage of cholesterol biosynthesis, and showed that (1) cholesterol is required for the sorting from MVBs to the Golgi and that (2) MVB reorganization is important for this late endosomal sorting. To study the dynamics of GFP-tagged organelles or transports/targeting of GFP-tagged proteins in single cell under a microscope, we have developed a novel microphotometric assay system by using GFP-visualization techniques coupled with semi-intact cell system. Using the assay system, (1) we have visualized and investigated the morphological changes of the ER-network in CHO cells during mitosis using the GFP-tagged HSP47. Time-lapse images and photobleaching experiments revealed that the ER-network remains almost continuous throughout the cell cycle, but is partially severed
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during mitosis. The cell cycle-dependent behavior of the ER-network, disruption followed by reformation, was reconstituted in SLO-permeabilized CHO cells using mitotic and interphase cytosol. Remarkably, the reformation of the disrupted ER-network was achieved through two sequential fusion reactions. The first process was mediated by NSF/α-SNAPs, and typical membranous intermediate structures were generated to connect the disrupted ER-tubules. The second fusion was mediated by p97/p47/VRF135, the same minimal components required for the homotypic fusion events in Golgi cisternae regrowth after mitosis. (2) We have visualized and reconstituted the vesicular transports between the Golgi and ER in semi-intact CHO cells. Based on the kinetic analysis, we found that, in the presence of mitotic cytosol, anterograde transport from the ER to Golgi was inhibited depending on cdc2 kinase but the retrograde transport was not. The transitional-ER (tER), which is a platform of ER-derived transport vesicles, was found to be disrupted in the presence of mitotic cytosol. Such perturbation of ER-Golgi transports might result in the relocation of the Golgi components to the ER during mitosis. Less
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