Fig. 2.

Localizing ER-mitochondrial-associated membranes (MAMs) and verification of the IMM-dynamics in correlation to the proximity/distance to MAMs. a HeLa cells were transfected with ER-RFP, stained with MTG and imaged with live N-SIM. Both channels were auto Otzu thresholded. Overlap regions of ER and mitochondria were determined as MAMs and the areas of co-localization were incrementally dilated using 5, 10, or 20 iterations. The IMM-dynamics measured according to Fig. 1b were cropped to the dilated areas and subsequently quantified. b HeLa cells transfected with ER-RFP and control siRNA or OPA1-specific siRNA, stained with MTG, were imaged with live N-SIM. CM-dynamics over the entire mitochondrion and in MAM-close regions (five iterations) were quantified. Images and analyses were obtained from at least five cells in each of eight experiments. c HeLa cells stained with MTG and transfected with ER-RFP were imaged with N-SIM. Cells were incubated for 45 min in 2Ca-buffer or 0Ca-buffer with 50 μM BAPTA-AM. CM-dynamics of the complete mitochondria and that of MAM-related areas (five iterations) were quantified. Images and analyses were obtained from at least five cells in each of six experiments. Bars represent mean ± SEM