STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria

Abstract

The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle.

Keywords: MICOS; MitOS; membrane architecture; nanoscopy.

Fig. 1.

Mitofilin is localized in individual clusters in the mitochondria of primary adult human fibroblasts. (A) Cell overview. The mitochondrial network of a primary fibroblast is labeled with an antiserum against mitofilin (green). The microtubule cytoskeleton (red), the nucleus (blue) and the actin cytoskeleton (gray) are labeled as well. (B and C) STED super-resolution microscopy (C) reveals that mitofilin is localized in discrete clusters, which are blurred and not resolvable using diffraction-limited confocal microscopy (B) of the same region. (Insets) Magnification of the boxed areas. (Scale bars: 20 μm in A; 1 μm in B and C.)

Fig. 2.

Mitofilin cluster distribution is denser in the perinuclear mitochondria. (A) Overview image of a fibroblast labeled with an antiserum against mitofilin and imaged by STED microscopy. (B and C) Magnification of the boxed areas in A. (D and E) Averaged intensity profiles across the indicated mitochondrial tubule sections within the respective boxed areas in B and C. (Scale bars: 20 μm in A; 1 μm in B and C.)

Fig. 3.

Mitofilin clusters are localized at the horizontal sides of mitochondria. (A and B) Two-color STED microscopy of fibroblast mitochondria labeled with antisera against mitofilin and the beta-subunit of the mitochondrial F₁F_o-ATPase (ATPS) (A) or with antisera against the outer membrane protein Tom20 and ATPS (B). In the overlay images, ATPS is shown in red, and mitofilin and Tom20 are in green. (C and D) Averaged intensity profiles across the indicated mitochondrial tubule sections within the boxed areas in A and B. The averaged ATPS intensity profile is shown in red, and the profiles of mitofilin and Tom20 are in green. (Scale bars: 1 µm.)

Fig. 4.

STED microscopy of MINOS. (A) Distribution of MINOS components in mitochondria of primary adult human fibroblasts. The cells were labeled with antibodies against the indicated proteins. (B) Localization of mitofilin in different mammalian cell lines [HeLa cells, neonatal human fibroblasts (Neo), Vero cells, and U2OS cells]. In each case, representative STED images of mitochondria located in the cell periphery are shown. (Scale bars: 1 µm.)

Fig. 5.

Submitochondrial localization of mitofilin using quantitative immunoelectron microscopy. (A) Immunogold labeling of mitofilin in HeLa cells. A representative mitochondrion is shown. The arrow points to the position of a gold particle. (B) Localizations of the gold particles (red) as determined by immunogold labeling of mitofilin plotted on a scheme representing a part of a mitochondrion. The histogram shows the fraction of gold particles within the indicated distance to the crista junction. The histogram and the graphical representation are based on the same measured gold particle localizations. OM, outer membrane; IM, inner membrane. (Scale bar: 100 nm.)

Fig. 6.

Cristae junction orientation and cristae morphology analyzed by dual-axis electron tomography. Primary adult human fibroblasts were grown on Aclar discs. (A and B) Single slices of the tomogram (grayscale) overlaid with reconstructions of segmented cristae membranes (green). Cristae junctions are denoted by orange spheres. Shown are a top view of a mitochondrion (A) (growth surface in plane with the tomogram) and a side view of the same mitochondrion (B). Note the lamellar cristae and the preferential orientation of the cristae junctions at the sides of the organelle, so that they are in parallel with the growth surface. (C) Magnification of a single slice of the tomogram (top view). The arrows point to the same cristae junctions as marked by the arrows in A. (Scale bar: 100 nm in A and C; 50 nm in B).