Mitochondrial fission involves the preconstriction of an organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and non-muscle myosin II through a mechanism that remains unclear, largely due to the unknown cytoskeletal ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded in an interstitial cytoskeletal network that contains abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise part of this interstitial network. Non-muscle myosin II is found adjacent to mitochondria but is not specifically enriched at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas dynamic myosin II clouds are present in the vicinity of constrictions. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure on the mitochondrial surface and thus initiates curvature-sensing mechanisms that complete mitochondrial constriction.
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We thank H. N. Higgs for helpful discussions and reagents, A. York and M. Schell for reagents and the members of the Svitkina lab, M. Shutova and N. Efimova, for comments on the manuscript. This work was supported by NIH grant nos. R01 GM 095977 and R01 GM 106000 to T.M.S.
Integrated supplementary information
Supplementary Fig. 1 Additional examples and 3D views of the ultrastructure of the cell interior revealed by PREM in glial cells.
Exposure of the cell interior by saponin-mediated extraction (a-d) or by nitrocellulose-mediated unroofing (e,f). (a, e) Broad windows into the cell interior formed as a result of saponin-mediated extraction (a) or nitrocellulose-mediated unroofing (e) expose membrane organelles associated with the cytoskeleton. Boxed regions in a and e are enlarged in b and f, respectively. (b, f) Enlarged boxed region from a and e, respectively, shown as anaglyph images generated from stereo pairs taken at ±11º. To view 3D organization, use red-cyan anaglyph glasses with red filter over the left eye. These image show unconstructed mitochondria (purple) that interact with putative ER structures (yellow). Cytoskeletal filaments are present in the cytoplasm. They often interact with the ER and sometimes intersect mitochondria. (c, d) PREM anaglyph images corresponding to panels b and c of main Fig. 1. Scale bars: 2 µm (a and e), and 200 nm (b, c-f). The images are representative of n = 6 independent experiments, which gave similar results.
Supplementary Fig. 2 Control experiments for the procedures associated with nitrocellulose-mediated unroofing.
(a) Time lapse imaging of HeLa cells expressing GFP-Ftractin and stained with MitoTracker Deep Red. At t = 0 sec, cell culture medium was replaced with PEM buffer containing 2 µM phalloidin and 10 µM taxol and maintained at room temperature (~22ºC). Imaging was also performed at room temperature. Arrows point to individual mitochondria or F-actin structures to indicate that no apparent changes was induced by the PEM buffer during live cell imaging. The images are representative of n = 2 independent experiments. (b) Confocal images of COS-7 cells expressing Mito-BFP (green) and mCherry-Sec61β (red). Control: cells were directly fixed and imaged. NC-treated: cells were subjected to nitrocellulose-mediated unroofing, fixed, imaged by confocal microscopy, processed for PREM and imaged by electron microscopy. In this sample, some cells were unroofed (bottom row), whereas others remained intact (middle row). The overall organization of Mito-BFP and mCherry-Sec61β is similar in all three cases. Scale bars: 5 µm. The images are representative of n = 3 independent experiments, which gave similar results.
Supplementary Fig. 3 Constitutively active INF2-A149D-CAAX induces abundant actin filaments at the ER surface.
(a-c) Confocal microscopy of a COS-7 cell expressing GFP-INF2-A149D-CAAX (AD-INF2), the ER marker mCherry-Sec61β (Sec61β) and mitochondrial marker Mito-BFP (Mito), and stained with Alexa Fluor 680 phalloidin (F-actin). (a, b) Individual fluorescence channels showing the nearly entire cell (a) and an enlarged boxed region (b). (c) Dual-channel overlays in different combinations of the same region as in b reveal partial colocalization between GFP-INF2-A149D-CAAX, mCherry-Sec61β and F-actin. (d-h) CLEM of mCherry-INF2-A149D-CAAX-expressing COS-7 cell. (d) Fluorescence microscopy of mCherry-INF2-A149D-CAAX in an expressing cell. (e, f) PREM (e) and PREM/fluorescence overlay (f) images of the boxed region in d. (g, h) The INF-A149D-CAAX-positive ER regions are coated with dense actin filament arrays. Both panels show enlargements of the same region outlined by a purple box in f either as PREM/fluorescence overlay (g) or by PREM only (h). Mito, mitochondria. Arrowheads mark actin filaments originating from INF-A149D-CAAX-positive ER and running toward the mitochondrion. See 3D version of panel H in Supplementary Fig. 4 (panel s3). Scale bars: 5 µm (a, d-f), 2 µm (b, c) and 500 nm (g, h). The images are representative of n = 2 independent experiments, which gave similar results.
Supplementary Fig. 4 3D anaglyph versions of the images shown in the main-text figures and Supplementary Fig. 3, as indicated by panel labels.
(a) Dynamics of mitochondria, the ER and F-actin in a HeLa cell expressing Mito-BFP (white), GFP-Ftractin (green) and mCherry-NMIIA (red) after addition of ionomycin at time 0:00 (min:sec). Ionomycin treatment leads to accumulation of F-actin that largely overlaps with the ER, whereas mitochondria are mostly located in the voids of the network. However, sites of mitochondrial constriction and fission (arrows and arrowheads) overlap with both F-actin and ER signals. (b) Dynamics of mitochondria, F-actin and NMIIA in a HeLa cell expressing Mito-BFP (white), GFP-Ftractin (green) and mCherry-NMIIA (red) after addition of ionomycin at time 0:00 (min:sec). Initiation of mitochondrial constriction correlates with the appearance of F-actin and, occasionally, NMIIA clouds intersecting the constriction (arrows and arrowheads). Subsequently, F-actin remains at the constriction sites until mitochondrial fission, whereas NMIIA mostly fluctuates in the mitochondrion vicinity. (c) A 3D PREM image of a constricted mitochondrion (purple) in an unroofed HeLa cell treated with ionomycin for 1.5 min. Membranous structures likely representing the ER are shaded in yellow. Actin filaments apparently originating from the ER intersect the mitochondrion close to the constriction site. Scale bars: 1 µm (a, b) and 200 nm (c). The images are representative of n = 2 independent experiments, which gave similar results.
(a) F-actin assembly in COS-7 cells after ionomycin treatment. COS-7 cell expressing mCherry-Ftractin is shown before (top) and after (middle) ionomycin treatment. Merged image (bottom) of these two time points shows reddish color in the cell center indicating F-actin assembly in the cytoplasm after ionomycin treatment. (b) Time-lapse sequence of a mitochondrial fission event in COS-7 cell expressing Mito-BFP (white), mCherry-Ftractin (red) and GFP-NMIIB (green) after addition of ionomycin at time 0:00 (min:sec). Initiation of mitochondrial constriction (arrow, t=0:21) correlates with the appearance of F-actin and NMIIB clouds intersecting the constriction (arrows and arrowheads). Subsequently, F-actin remains at the constriction sites until mitochondrial fission, whereas NMIIA mostly fluctuates in the mitochondrion vicinity. (c) Immunogold PREM of NMIIB localization at mitochondrial constriction in an unroofed COS-7 cell treated with ionomycin for 5 min. Immunogold particles (18 nm) marked by yellow dots localize at comparable densities at the constrictions site and in the surrounding network. Mitochondrion is shaded in purple. Scale bars: 10 µm (a), 1 µm (b) and 200 nm (c). The images are representative of n = 2 independent experiments, which gave similar results.
Supplementary Figures 1–6, Supplementary Table title/legend, Supplementary Video titles/legends.
Three-dimensional organization of actin filaments at the mitochondrial constriction site in a COS-7 cell expressing GFP−K38A-Drp1.
Rotenone treatment induces actin assembly in the cytoplasm in association with mitochondria.
Ionomycin treatment leads to F-actin assembly in association with the ER.
Dynamics of mitochondria, F-actin and NMIIA in HeLa cells (example 1).
Dynamics of mitochondria, F-actin and NMIIA in HeLa cells (example 2).
Dynamics of mitochondria, F-actin and NMIIB in COS-7 cells.
3D organization of actin filaments and immunogold-labelled NMIIA at the mitochondrial constriction site in a HeLa cell treated with ionomycin for 2 min.
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