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Vandevoorde, Kurt

University of British Columbia

Selective autophagic degradation of the mitochondria, or mitophagy, is a quality control mechanism through which mitochondria that are dysfunctional or no longer required are degraded. Dysregulation of mitophagy has been associated with oncogenesis and neurodegenerative diseases. Gp78 is an E3 ubiquitin ligase involved in ER-associated degradation and previously implicated in damage-induced mitophagy. Here we demonstrate that CRISPR Cas9 KO of Gp78 results in impaired basal and damage-induced mitophagy using both mtKeima and LC3-GFP-RFP assays which report on mitochondrial delivery to autolysosomes and autophagic flux, respectively. We demonstrate that impaired autophagic flux results in decreased mitochondrial potential and the accumulation of mitochondrial derived superoxide. Induction of mitochondrial fission through the overexpression of Drp1 fails to rescue decreased membrane potential in Gp78 KO cells suggesting that Gp78 regulation of mitophagy extends beyond the induction of mitochondrial fission. In our HT-1080 cell model we demonstrate that autophagosomes selectively localize to mitochondria exhibiting low membrane potential and that the regulation of membrane potential by Gp78 extends to additional cell lines other than our HT-1080 model. Mitochondria-endoplasmic reticulum (ER) contacts (MERCs) are regions of close apposition between the ER and mitochondria. These sites act as signaling hubs regulating lipid transfer, calcium signaling and mitochondrial dynamics. These structures are classified based on the type of ER (rough/smooth) associating with the mitochondria forming ribo-MERCs and smooth MERCs, respectively. Dysregulation of MERCs has been demonstrated in neurodegenerative diseases, cancer, and metabolic disorders. Here we demonstrate that COS7 and HT-1080 preferentially form smooth MERCs and rough MERCs, respectively. We have developed a novel Laplacian-based algorithm to detect MERCs in 3D super-resolution microscopy data capable of detecting endogenous and induced MERCs. We demonstrate that Gp78 and RRBP1 selectively regulate ribo-MERCs independent of one another. MERC regulation through the expression of Gp78 is dependent on GP78’s E3 ligase activity as RING-mut Gp78 failed to induce ribo-MERCs. MERCs generated through Gp78 expression exhibit convoluted tubular morphologies distinct from planar MERCs induced through artificial tether expression.

Text

2023-03-01

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/83873

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/