Fig. 1: Forces induce mitochondrial elongation.
a, Mitochondrial length analysis in MCF10A-RAS cells cultured for 24 h on stiff (E = ~15 kPa) versus soft (E = ~0.5 kPa) fibronectin-coated polyacrylamide hydrogels (n = 71 (soft) and 95 cells (stiff) from four experiments). Scale bars, 10 μm. b,c, Mitochondrial length analysis in D2.0R cells (b) and MEFs (c) cultured as in a (n = 69 (stiff) and 48 cells (soft) in b; n = 57 (stiff) and 76 cells (soft) in c; both from three experiments). d, Mitochondrial length analysis in MCF10A-RAS cells transfected to express dominant-negative RHOA T19N or treated for 3 h with inhibitors of ROCK and MLCK (Y27632 and ML7; YM) or with the SMIFH2 inhibitor of the formin actin-nucleating proteins (n = 25 (GFP), 30 (RHOA T19N), 71 (DMSO), 89 (YM) and 93 cells (SMIFH2); from two experiments for GFP and RHOA T19N and from four experiments for the remainder). Scale bar, 10 μm. e, Mitochondrial length analysis in MEFs treated as in d (n = 86 (DMSO) and 71 cells (YM) from four experiments). Scale bar, 10 μm. f, Mitochondrial length analysis in mouse lung endothelial cells (MLECs) treated for 18 h with 4-OHT to trigger CreERT2-mediated Talin1 inactivation (Tln1 KO) (n = 26 (WT) and 25 cells (Tln1 KO) from two experiments). Scale bar, 10 μm. g, Mitochondrial length analysis in MCF10A-RAS cells transfected with control (siCO) or PIEZO1/2 siRNAs (n = 29 (siCO), 64 (siPIEZO1/2a) and 71 cells (siPIEZO1/2b) from three experiments). h, Mitochondrial length analysis in 3T3L1 cells cultured on large (10,000 μm2), medium (1,024 μm2) or small (300 μm2) micropatterned fibronectin islands for 24 h (n = 61 (large), 51 (medium) and 96 cells (small) from four experiments). Scale bars, 10 μm. i,j, Mitochondrial length analysis in 3T3L1 (i) and MCF10A-RAS cells (j) cultured under sparse (low-density (LD)) or dense (high-density (HD)) conditions for 72 h. Where indicated, cells were locally released from crowding by scratching the monolayer for 8 h (scratch) (n = 89 (LD) and 193 cells (HD) from four experiments in i; n = 69 (LD), 192 (HD without scratching) and 143 cells (HD with scratching) from three experiments in j). Scale bar, 10 μm. k, HUVECs forming mature monolayers were left untreated (static) or conditioned for 18 h with a flow yielding a nominal WSS of 1.4 or 4.0 Pa. Top left, analysis of cell orientation in response to flow (0° is the flow direction). Bottom left, representative TOMM20 immunofluorescence. Right, mitochondrial length analysis (n = 161 (static), 175 (1.4 Pa) and 202 cells (4.0 Pa) from three experiments). Scale bar, 30 μm. l, Mitochondrial length analysis in HaCaT cells cultured under dense conditions on a fibronectin-coated silicon membrane for 72 h then subjected to tonic uniaxial stretching for 3 h (stretch) (n = 128 (no stretch) and 153 cells (stretch) from three experiments). Bidirectional arrow represents the direction of stretching. Scale bar, 20 μm. m, Mitochondrial area analysis (right) from transmission electron microscopy images (left) of basal keratinocytes from normal mouse skin (CO) versus skin subjected to expansion for 6 d (EXP)26 (n = 88 (CO a), 78 (CO b), 68 (EXP c), 76 (EXP d) and 62 mitochondria (EXP e) in ≥15 cells per mouse). Scale bars, 2 μm (top images) and 500 nm (magnified images). The data are presented as means ± s.d. (a–l) or means and single points (m), with representative pictures. Statistical significance was determined by two-tailed Student’s t-test. In a–e and h–l, P values were calculated on punctate mitochondria. Source numerical data are available.
