Alan, P. | bioRxiv Science

bioRxiv2022

Automatic sub-precision membrane contact site detection identifies convoluted tubular riboMERCs

Identification and morphological analysis of mitochondria-ER contacts (MERCs) by fluorescent microscopy is limited by sub-pixel resolution inter-organelle distances. Application of a Membrane Contact Site (MCS) detection algorithm, MCS-DETECT, to 3D STED super-resolution image volumes reconstructs sub-resolution MERCs. MCS-DETECT shows that elongated ribosome-studded riboMERCs, present in HT-1080 but not COS-7 cells, are morphologically distinct from smaller smooth contacts and larger contacts induced by mitochondria-ER linker expression in COS-7 cells. riboMERC expression is reduced in Gp78 knockout HT-1080 cells and induced by Gp78 ubiquitin ligase activity in COS-7 cells. Knockdown of the riboMERC tether RRBP1 eliminates riboMERCs in both wild-type and Gp78 knockout HT-1080 cells. By MCS-DETECT, Gp78-dependent riboMERCs present complex tubular shapes that intercalate between and contact multiple mitochondria, that are lost upon RRBP1 knockdown. MCS-DETECT of 3D whole cell super-resolution image volumes therefore identifies a novel dual regulatory mechanism for tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity. eTOC SummaryApplication of the sub-pixel resolution Membrane Contact Site (MCS) detection algorithm, MCS-DETECT, to 3D STED super-resolution image volumes identifies a novel dual regulatory mechanism for tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.

cell biology↗

bioRxiv2021

Gp78-mediated basal mitophagy promotes mitochondrial health and limits mitochondrial ROS production

Mitochondria are major sources of cytotoxic reactive oxygen species (ROS) that contribute to cancer progression. Mitophagy, the selective elimination of mitochondria by autophagy, monitors and maintains mitochondrial health and integrity, eliminating ROS-producing mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis under basal conditions remain poorly understood. Gp78 E3 ubiquitin ligase is an endoplasmic reticulum membrane protein that induces mitochondrial fission and mitophagy of depolarized mitochondria. Here, we report that CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells increased mitochondrial volume and rendered cells resistant to carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitophagy. These effects were phenocopied by knockdown of the essential autophagy protein ATG5 in wild-type HT-1080 cells. Use of the mito-Keima mitophagy probe confirmed that Gp78 promoted both basal and damage-induced mitophagy. Application of a spot detection algorithm (SPECHT) to GFP-mRFP tandem fluorescent-tagged LC3 (tfLC3)-positive autophagosomes reported elevated autophagosomal maturation in wild-type HT-1080 cells relative to Gp78 knockout cells, predominantly in proximity to mitochondria. Mitophagy inhibition by either Gp78 knockout or ATG5 knockdown reduced mitochondrial potential and increased mitochondrial ROS. Live cell analysis of tfLC3 in HT-1080 cells showed the preferential association of autophagosomes with mitochondria of reduced potential. Basal Gp78-dependent mitophagic flux is therefore selectively associated with reduced potential mitochondria promoting maintenance of a healthy mitochondrial population and limiting ROS production.

cell biology↗