Comparative Analysis of SPLICS and MCS-DETECT for Detecting Mitochondria-ER Contact Sites (MERCs).

Abstract

Detection of mitochondria-ER contacts (MERCs) from diffraction limited confocal images commonly uses fluorescence colocalization analysis of mitochondria and endoplasmic reticulum (ER) as well as split fluorescent probes, such as the split-GFP-based contact site sensor (SPLICS). However, inter-organelle distances (∼10-60 nm) for MERCs are lower than the 200-250 nm diffraction limited resolution obtained by standard confocal microscopy. Super-resolution microscopy of 3D volume analysis provides a two-fold resolution improvement (∼120 nm XY; 250 nm Z), which remains unable to resolve MERCs. MCS-DETECT, a membrane contact site (MCS) detection algorithm faithfully detects elongated ribosome-studded riboMERCs when applied to 3D STED super-resolution image volumes. Here, we expressed the SPLICS_L reporter in HeLa cells co-transfected with the ER reporter RFP-KDEL and label fixed cells with antibodies to RFP and the mitochondrial protein TOM20. MCS-DETECT analysis of 3D STED volumes was compared to contacts determined by co-occurrence colocalization analysis of mitochondria and ER or the SPLICS_L probe. Percent mitochondria coverage by MCS-DETECT derived contacts was significantly smaller than those obtained for colocalization analysis or SPLICS_L, and more closely matched contact site metrics obtained by 3D electron microscopy. Further, STED analysis localized a subset of the SPLICS_L label to mitochondria with some SPLICS_L puncta observed to be completely enveloped by mitochondria in 3D views. These data suggest that MCS-DETECT reports on a limited set of MERCs that more closely corresponds to those observed by EM.