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 subpixel resolution interorganelle distances. Here, the membrane contact site (MCS) detection algorithm, MCS-DETECT, reconstructs subpixel resolution MERCs from 3D super-resolution image volumes. 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 formation is associated with increased mitochondrial potential, reduced in Gp78 knockout HT-1080 cells and induced by Gp78 ubiquitin ligase activity in COS-7 and HeLa cells. Knockdown of 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. MCS-DETECT of 3D whole-cell super-resolution image volumes, therefore, identifies novel dual control of tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.