Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria-ER contacts

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.