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Endoplasmic reticulum : nanodomain organization and communication with mitochondria Gao, Guang

Abstract

Communications between the endoplasmic reticulum (ER) and mitochondria occur at specialized nanodomains, ER-mitochondria contact sites, which are hubs for calcium signaling, lipid transport, mitochondrial biogenesis, and mitochondrial DNA distribution. Thus, the regulation of ER-mitochondria contacts is vital for the cell. In this dissertation, I elucidate the regulation of ER-mitochondria contacts and the nanodomain organization of the ER and present a 3D whole-cell ER-mitochondria contact map. Firstly, Autocrine Motility Factor Receptor (Gp78) is localized throughout the ER with a subpopulation of mitochondria-associated ER labeled by anti-Gp78 3F3A. The epitope that 3FA binds is localized to an 8 amino acid motif that contains Ser-538, a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limits Gp78 ability to enhance ER-mitochondria contacts, induce mitochondrial fission and degrade mitofusin1/2 but does not impact Gp78 in vitro E3 ubiquitin ligase activity. p38 MAPK phosphorylation of Gp78 S538, therefore, regulates Gp78-dependent ER-mitochondria contacts and mitochondria dynamics. Secondly, diffraction-limited microscopy has hindered efforts to study nanodomains of ER and ER-mitochondria contacts. Here, STimulated emission depletion (STED) super-resolution microscopy reveals nanodomain/periodicity (maxima and minima) of ER tubules. Lumenal (ERmoxGFP) and membrane (Sec61βGFP) ER reporters are enriched at distinct nanodomains of peripheral ER tubules. Derlin-1 and calnexin are significantly enriched only in the periodic ER lumenal minima, which is disrupted by CLIMP-63 knockdown. Overexpression of CILMP-63 or reticulon alters ER tubule nanodomain organization and the preferential distribution of calnexin to ER nanodomains. This suggests that CLIMP-63 and reticulon define, segregate, and organize lumenal ER nanodomains from ER membrane protein complexes. Lastly, the ER presents tubules and sheets. However, the studies of ER-mitochondria mostly focus on ER tubule-mitochondria contacts. In this work, EM and live cell imaging show distinct types of ER-mitochondria contacts. 3D STED shows that the ER contains peripheral tubules, peripheral fenestrated sheets, and central sheet-like structures and further that all these ER structures contact with mitochondria. Thus, the application of super-resolution microscopy to ER-mitochondria contacts has identified multiple types of ER-mitochondria contacts in 3D, which will open up new avenues on ER-mitochondria contact studies.

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