Analysis of ER-mitochondria contacts by correlative fluorescence microscopy and soft X-ray tomography of mammalian cells

Abstract

Mitochondrial fission is important for organelle transport, quality control and apoptosis. Changes to the fission process can result in a wide variety of neurological diseases. In mammals, mitochondrial fission is executed by the GTPase dynamin-related protein 1 (Drp1; encoded by DNM1L), which oligomerizes around mitochondria and constricts the organelle. The mitochondrial outer membrane proteins Mff, MiD49 (encoded by MIEF2) and MiD51 (encoded by MIEF1) are involved in mitochondrial fission by recruiting Drp1 from the cytosol to the organelle surface. In addition, endoplasmic reticulum (ER) tubules have been shown to wrap around and constrict mitochondria before a fission event. Up to now, the presence of MiD49 and MiD51 at ER-mitochondrial division foci has not been established. Here, we combine confocal live-cell imaging with correlative cryogenic fluorescence microscopy and soft x-ray tomography to link MiD49 and MiD51 to the involvement of the ER in mitochondrial fission. We gain further insight into this complex process and characterize the 3D structure of ER-mitochondria contact sites.

Cooperative and independent roles of Drp1 adaptors Mff and MiD49/51 in mitochondrial fission

“…4B). This result supports other findings showing that MiD51 and Mff might assemble with Drp1 at the same fission foci (Elgass et al, 2015). Interestingly, no ER proteins were found to be enriched in this analysis.…”

“…In addition to Drp1 and adaptors, mitochondrial fission also involves the endoplasmic reticulum (ER) and actin, which aid in the formation of constriction sites for Drp1 assembly with Mff (Friedman et al, 2011) and MiD proteins (Elgass et al, 2015). To date, it has not been clear whether these adaptors interact and/or perform separable roles in Drp1-mediated mitochondrial fission.…”

Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling

“…2, E and G); (5) HA-Mff and MiD51-GFP failed to assemble into foci to distribute smoothly through the extended mitochondrial tubules in Drp1-KO cells (Fig. 2, F and G); (6) consistent with previous studies (Friedman et al, 2011;Elgass et al, 2015), 91% of HAMff (n cells = 4) and 94% of MiD51-GFP (n cells = 4) localized as foci on the MAM (Fig. 2, H and I); and (7) Mff functioned as a Drp1 recruiter on the MOM and mediated mitochondrial fission in the absence of MiD proteins (Fig.…”

The coming of age of the mitochondria–ER contact: a matter of thickness

“…81 In mammals, correlative cryogenic fluorescence microscopy and soft X-ray tomography (CFM-SXT) revealed the presence of ER extensions 168 nm long and 80 nm in diameter that contact mitochondria at vision sites enriched in the mitochondrial dynamics proteins Mid49 and Mid51 (mitochondrial dynamics proteins of 49 and 51 kDa). 82 By implication, a MERC with a length longer than 100 nm should not be a fission-MERC.…”