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First published online 24 January 2006
doi: 10.1242/jcs.02762

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Regulation of mitochondria distribution by RhoA and formins

Alexander A. Minin^1,2,*,, Alexander V. Kulik^1,3,*, Fatima K. Gyoeva¹, Ying Li², Gohta Goshima⁴ and Vladimir I. Gelfand⁵

¹ Institute of Protein Research, Russian Academy of Sciences, Moscow 119988, Russia
² Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
³ Moscow Institute of Physics and Technology, Moscow 141700, Russia
⁴ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94107, USA
⁵ Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA

View larger version (36K): [in a new window]   Fig. 1. Distribution of fluorescently labeled mitochondria in CV-1 and BG2-C2 cells. Fluorescent images showing (A) a CV-1 cell transfected with the plasmid pEYFP-Mito and (B) Drosophila BG2-C2 cells transfected with pAc-EGFP-Mito plasmid. See Movies 1 and 2 in supplementary material. Bars, 10 µm.

View larger version (42K): [in a new window]   Fig. 2. LPA inhibits the motility of mitochondria in CV-1 cells through RhoA. (A,C,E) The first frame of the image sequence recorded, and (B,D,F) the plotted tracks of individual mitochondria in the same cells. (A,B) Control cells; (C,D) cells were treated with 5 µM LPA for 5 minutes; (E,F) cells were transfected with plasmid pcDNA3-EGFP-RhoA(Q63L). See also Movie 3 in supplementary material. Bar, 10 µm. (G) Quantification of mitochondrial motility in control cells, cells treated with 5 µM LPA or cells expressing EGFP-RhoA(Q63L) (RhoA-QL). Values are the mean percentage of movements exceeding 0.2 µm/second from all movements ± s.e.m.; P<0.05; n = number of cells and, in brackets, number of organelle movements.

View larger version (103K): [in a new window]   Fig. 3. Effects of a constitutively active mutant of mDia1 on the cytoskeleton of CV-1 cells. Cells were transfected with plasmids encoding EGFP-tagged mDia-N3. After fixation in 4% formaldehyde with 1% Triton X-100, cells were stained with TRITC-phalloidin to visualize F-actin (A) or with antibodies against -tubulin and TRITC-labeled secondary antibodies (C), and with antibodies against Glu-tubulin and FITC-labeled secondary antibodies to visualize stable microtubules (B,D). Transfected cells were found before fixation by EGFP fluorescence (shown by arrows). Bar, 10 µm.

View larger version (90K): [in a new window]   Fig. 4. A constitutively active mDia1 mutant, but not other proteins promoting actin polymerization, inhibits the motility of mitochondria. CV-1 cells were transfected with plasmids encoding EGFP-mDia-N3, EGFP-Rac1(Q61L), EGFP-Cdc42(Q61L) or EGFP-WA, and the movement of fluorescently labeled mitochondria was recorded by time-lapse video microscopy. The first frames of the image sequences, showing different mitochondria distribution in cells expressing EGFP-mDia-N3 (A), and EGFP-Rac1(Q61L) (B) (see Movies 4 and 5 in supplementary material). (D) The cell expressing EGFP-WA (shown with arrow) with increased level of F-actin visualized by TRITC-phalloidin staining. Bars, 10 µm. (C) Quantification of mitochondrial motility in cells expressing EGFP-mDia-N (mDia-N), EGFP-WA (WA), EGFP-Rac1(Q61L) (Rac1-QL) or EGFP-Cdc42(Q61L) (Cdc-QL). Values are the mean percentage of movements exceeding 0.2 µm/second from all movements ± s.e.m.; P<0.05; n = number of cells and, in brackets, number of organelle movements.

View larger version (116K): [in a new window]   Fig. 5. The constitutively active mutant of diaphanous induces mitochondrial anchoring in Drosophila BG2-C2 cells. BG2-C2 cells were transfected with a plasmid encoding EGFP-C-Dia. Cells were either fixed for cytoskeleton staining (A-F) or incubated with 100 nM Texas Red Mitotracker for the analysis of mitochondrial motility (G-I). (A,D,G) Fluorescent images that show the transfected cells. (C) Cells were stained with anti-tubulin antibody and TRITC-labeled secondary antibody. Microtubules in transfected cells form the characteristic rosette-like pattern. (F) Cells were stained with TRITC-phalloidin; the higher level of F-actin could be seen in transfected cells. (B,E,H) Phase-contrast images of cells shown in A,D,G, and (I) the first frame in the video sequence (Movie 6 in supplementary material) showing the distribution of elongated mitochondria in the transfected cell shown in G. Bars, 10 µm.

View larger version (38K): [in a new window]   Fig. 6. RNAi-mediated depletion of diaphanous increases the motility of mitochondria in BG2-C2 cells. Cell lines with fluorescently labeled mitochondria, peroxisomes and lysosomes were incubated in culture medium containing 10 µg per ml of dsRNA complementary to diaphanous mRNA. The content of target protein was estimated before treatment and after 3 and 7 days of incubation. The organelle motility in these cells was analyzed after 7 days of incubation. (A) Western blot analysis of diaphanous before and after incubation of cells with dsRNA for 3 and 7 days, respectively. Left panel shows results of serial dilutions of control cell extract used for calibration of the western blot. KHC, kinesin heavy chain. (B) Fluorescent image of cells with labeled mitochondria after 7 days of incubation with dsRNA. Bars, 10 µm. (C) Quantification of mitochondrial motility in cells after RNAi. The control cells were treated the same way as those exposed to RNAi with the only exception that dsRNA was omitted. Values are the mean percentage of movements exceeding 0.2 µm/second from all movements ± s.e.m.; P<0.05; n = number of cells and, in brackets, number of organelle movements. (D) The motility of lysosomes and peroxisomes labeled with EGFP-tagged probes (see Materials and Methods) was analyzed as described for mitochondria. Average travel distances and velocities were measured as described in the Materials and Methods, and fast movements were determined as in Fig. 2. Values are the mean percentage ± s.e.m.; P<0.05; n, number of cells and, in brackets, number of organelle movements.

View larger version (58K): [in a new window]   Fig. 7. Analysis of the role of ROCK in anchorage of mitochondria. CV-1 cells were transfected with plasmids encoding Myc-ROCK-RB/PH(TT) (ROCK-DN) (A,B) or Myc-ROCK-CAT (C,D). Cells were fixed; transfected cells were visualized by staining with anti-Myc antibody, and F-actin was visualized by TRITC-phalloidin. Bar, 10 µm. (E) Quantification of mitochondrial motility in cells expressing EYFP-Mito alone or EYFP-Mito and indicated mutant proteins in microinjected cells before or after treatment with 30 µM Y-27632 (Y27632) for 30 minutes or with 5.0 µM LPA for 5 minutes. Live cells expressing ROCK mutants were recognized by the presence of labeled mitochondria. Values are the mean percentage of fast movements ± s.e.m.; P<0.05; n, number of cells and, in brackets, number of organelle movements.

View larger version (18K): [in a new window]   Fig. 8. Disruption of F-actin by latrunculin B increases the motility of mitochondria. The movements of fluorescently labeled mitochondria in CV-1 (A) and BG2-C2 (B) cells were recorded using time-lapse video microscopy before or after incubation with either 0.2 µM latrunculin B (LB) for 20 minutes (A) or with 10 µM latrunculin B for 1 hour (B) and analyzed, as described in the Materials and Methods. For depletion of diaphanous by RNAi, BG2-C2 cells were incubated for 7 days with dsRNA, as in Fig. 7. For the analysis of mitochondrial motility in BG2-C2 cells transfected with EGFP-C-Dia (GFP-C-Dia), they were incubated with 100 nM Texas Red Mitotracker for 40 minutes, and mitochondrial movements were recorded by time-lapse video microscopy using red fluorescence filter set (see also Movie 7 in supplementary material). Values are the mean percentage of fast mitochondria movements ± s.e.m.; P<0.05; n, number of cells and, in brackets, number of organelle movements.

View larger version (49K): [in a new window]   Fig. 9. Expression of pEGFP-mDia-N3 in CV-1 cells affects the distribution of mitochondria caused by disruption of microtubules. Distribution of mitochondria (A) or F-actin (B) in cells that were either transfected with pEGFP-mDia-N3 (B) or co-transfected with pEYFP-Mito and pEGFP-mDia-N3 (A). After depolymerization of microtubules with nocodazole, cells were fixed in 4% formaldehyde. F-actin was stained with TRITC-phalloidin. Cell margins in A are indicated by dots. Transfected cells are shown by arrows. Bars, 10 µm.

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