Figure 3. Myosin II enriches at mitochondrial constriction sites.
(A) U2OS cell transfected with mito-BFP and ER-green, then fixed and stained with anti-P-MRLC (myosin regulatory light chain, phosphoserine 19). Arrowhead shows mitochondrial constriction site. Scale bar, 10 µm.
(B) U2OS cells labeled with mitotracker (red), then fixed, stained with anti-PMRLC (green), and imaged by spinning disc confocal microscopy with 5–7 z-sections taken (200 nm z steps). Arrowheads denote constriction sites. Two examples shown. For each, left is maximum intensity image and right is 3D reconstruction. Scale bar, 2 µm.
(C) U2OS cell transfected with mito-dsRed and GFP-MIIA, then imaged live by confocal microscopy (single Z-plane). Arrow points to MIIA accumulation at fission site. Scale bar, 2 µm. See also Movie S1.
(D) Single confocal slices of “apical” mitochondria, located adjacent to the nucleus at > 1.4 µm from the ventral surface of the cell, avoiding interference from abundant ventral stress fibers. U2OS cells labeled with mitotracker (red), then fixed and stained with anti-P-MRLC (green). Cells either untreated (left), transfected with siRNA oligos against INF2 for 72 hrs (middle), or treated with 0.5 µM Latrunculin B for 60 min (right). Scale bar, 20 µm. See Figure S3 for quantification of mitochondria-associated P-MRLC puncta upon Myosin II and mitochondrial fission these treatments, as well as close-up image of apical mitochondria, P-MRLC, and actin filaments.