QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 10 August 2004
doi: 10.1242/jcs.01299

This Article Summary Full Text Full Text (PDF) Supplemental Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Varadi, A. Articles by Rutter, G. A. Search for Related Content PubMed PubMed Citation Articles by Varadi, A. Articles by Rutter, G. A.

Cytoplasmic dynein regulates the subcellular distribution of mitochondria by controlling the recruitment of the fission factor dynamin-related protein-1

Aniko Varadi^1,*, Linda I. Johnson-Cadwell¹, Vincenzo Cirulli², Yisang Yoon³, Victoria J. Allan⁴ and Guy A. Rutter^1,

¹ Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK
² The Whittier Institute for Diabetes, Laboratory of Developmental Biology, University of California San Diego, La Jolla, CA 92037, USA
³ Center for Basic Research in Digestive Diseases and Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA
⁴ School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK

View larger version (51K): [in a new window]   Fig. 1. Mitochondria are partially localised along microtubules in live HeLa cells. (A) Cells were co-transfected with 0.25 µg mito.DsRed and 1 µg -tubulin.EGFP. Forty-eight hours after transfection cells were imaged on an UltraVIEWTM Live Cell Confocal Imaging system in KRH buffer (see Materials and Methods) at 22°C. The boxed region in a is shown on an expanded scale in b); arrows in b indicate regions of colocalisation. (B) Microtubules were depolymerised with 10 µM nocodazole. The boxed region in a is shown on an expanded scale in b. Bars, 1.0 µm. (C) Dynactin subunits (p50 and p150Glued) and dynein are associated with a mitochondrial fraction. Heavy mitochondrial pellet (HMP) was obtained after spinning the postnuclear supernatant (PNSN) at 3000 g for 10 minutes (see Materials and Methods). An equal amount (3 µg) of protein was loaded each lane. The intensity of the protein bands was measured using NIH ImageJ software (http://rsb.info.nih.gov/ij/). PHMSN, post-heavy mitochondrial supernatant; DIC, dynein intermediate chain.

View larger version (99K): [in a new window]   Fig. 2. Overexpression of dynamitin (p50) induces mitochondrial collapse around the nucleus and MTOC. (A) HeLa cells were co-transfected with 0.5 µg mito.DsRed (a,b,e,f) and 1 µg pAdTrack-CMV (empty vector/control) (a,b) or p50.EGFP (e,f). Mitochondria were visualised with MitoTrackerRedTM (c,d,g,h) in cells transfected with either pcDNA3 (empty vector/control) (c,d) or p50.pcDNA3 (g,h). Hatched boundaries in c and d indicate the cell periphery. Twelve hours after transfection, cells were either imaged on a Leica TCS-NT confocal microscope (a,b,e,f) or fixed (c,d,g,h) and were then immunostained with a mouse monoclonal anti-p50 antibody (1:250) and visualised with an Alexa Fluor 488 goat anti-mouse secondary antibody (1:500) before confocal imaging. p50-overexpressing cells were identified by exciting either EGFP or the secondary antibody at 488 nm. DsRed or MitoTrackerRedTM fluorescence was visualised in the same cells by exciting at 568 nm. Typical confocal images of DsRed-labelled mitochondria in control or p50-expressing cells are shown in a and e, MitoTrackerRedTM fluorescence in c and g, composite images b,d,f and h. Note the re-localisation of mitochondria close to the nucleus in p50-expressing cells (e versus a, g versus c). Bars, 10 µm. (B) Reconstruction of adjacent transmission electron microscopic fields showing a (B) control-HeLa cell with randomly distributed mitochondria throughout the cytosol and a (C) p50-transfected HeLa cell with mitochondria redistributed to the perinuclear region within 2-3 µm of the nuclear membrane. Mitochondria are largely absent at the cell periphery. (D) Same section as shown in C at higher magnification. Tight association of mitochondria with the ER is evident. ER, endoplasmic reticulum; m, mitochondria; nu, nucleus; pm, plasma membrane.

View larger version (137K): [in a new window]   Fig. 3. Overexpression of p50 alters mitochondrial morphology and ultrastructure. Transmission electron micrographs of p50-expressing HeLa cell cross-sections at high magnification show unusually branched (A) or elongated (B) morphology of the mitochondria. Arrowhead in A indicates the branching of mitochondria; Arrowheads in B indicate a single mitochondrion of 10 µm in length. Notice the dramatic loss of cisternae in the mitochondria marked with an asterisk in A.

View larger version (65K): [in a new window]   Fig. 4. The effect of p50-overexpression on mitochondrial distribution is mimicked by anti-dynein antibody. (A) Cells were transfected with 0.5 µg mito.DsRed for 16 hours then microinjected together with either 1 mg ml–1 control IgG (a,b) or a mouse monoclonal anti-dynein intermediate chain (DIC) antibody (c,d) and 1 mg ml–1 Oregon Green BAPTA dextran. Two to four hours after injection, antibody-treated cells were identified by exciting Oregon Green at 488 nm and using fluorescein isothiocyanate filters for fluorescence emission on the Leica confocal microscope. Typical 568 nm in vivo confocal images of mitochondria in control IgG (a) and anti-DIC antibody (c) injected cells; b and d composite images. Bars, 10 µm. (B) The collapsed mitochondria phenotype was scored in cells transfected with p50 or microinjected with the anti-DIC antibody. Data are presented as the number of cells exhibiting this phenotype as a fraction of the total number of cells transfected or microinjected. Two-hundred p50-expressing and ten microinjected cells were analysed in five independent experiments. (C) Effects of p50 overexpression on endocytic organelles. HeLa cells were transiently transfected with p50.EGFP (as described above), and fixed and stained 12 hours later. Immunocytochemistry was performed using a (a,b) monoclonal anti-TGN38 antibody (Lee and Banting, 2002) and a (c,d) monoclonal anti-lysosome-associated membrane protein LAMP-1 antibody (a marker of late endosomes and lysosomes (Ihrke et al., 1998). Fluorescence of an Alexa 568 goat anti-mouse secondary antibody was then visualised in a and c at 568 nm. Intrinsic EGFP fluorescence was visualized at 488 nm in b and d, which are composite images also showing TGN38 and LAMP1 immunostaining, respectively .Arrowheads in a) indicate the position of p50-expressing cells; arrowheads in c) indicate the extreme shift of LAMP-positive late endosomes and lysosomes into the periphery after p50 overexpression. Asterisks indicate control cells. Bars, 10 µm. (D) Overexpression of p50 has no effect on mitochondrial membrane potential (mit). Cells were incubated with 1 µM TMRE for 30 minutes and confocal images were subsequently acquired. Using identical confocal settings, TMRE fluorescence was 28±3.5 (n=6) and 30±3.1 (n=14) arbitrary units in control (a) and p50-expressing (b) cells, respectively. Bars=10 µm (A-E). Hatched boundaries in A, C and D, obtained from an overlay with the transmitted image of the cell, indicate the cell periphery.

View larger version (40K): [in a new window]   Fig. 5. Kinesin I heavy chain and dynactin complex interact in HeLa cell extract. (A) p150Glued was precipitated from HeLa cell extracts by a monoclonal antibody and blots were probed with a monoclonal anti-kinesin-heavy-chain antibody (SUK4) right panel) or a monoclonal anti-p50 antibody (left panel). Control samples (-Cont.) were precipitated by an unrelated monoclonal antibody. Five µg of protein were loaded from the cell homogenate; the equivalent to 35 µg protein was used as starting material for immunoprecipitation and an equal amount of protein was loaded from the immunoprecipitated samples (lanes p150Glued and -Cont.). (B) Depolymerisation of actin filaments partially restores the normal distribution of mitochondria in p50-expressing cells. Cells were co-transfected with 0.5 µg mito.DsRed and 1 µg p50.EGFP. Twenty hours after transfection cells were incubated with latrunculin B (25 µg ml–1) in KRH buffer at 37°C and then imaged on an Ultra VIEWTM Live Cell Confocal Imaging system. Mitochondria of the same cell (a) before the addition of latrunculin B, (b) after a 30-minute incubation or (c) after a 60 minute-incubation with the drug.

View larger version (50K): [in a new window]   Fig. 6. Mitochondrial movements in live HeLa cells. Cells were co-transfected with 0.25 µg mito.DsRed and (A) 1 µg padTrack-CMV or (B,C) p50.EGFP. Twelve hours after transfection, cells were imaged on an UltraVIEWTM Live Cell Confocal Imaging system in KRH buffer at 37°C; images were acquired at 2 Hz. Boxed regions on the control cell (A) are enlarged in panels a and b. Indicated by the white arrowheads is a single moving mitochondrion whose original starting position is labeled with a red asterisk. Direction of movement is indicated by yellow arrows. (B,C) Mitochondrial distribution in HeLa cells expressing moderate (B) and high (C) levels of p50.EGFP. Note the long interconnected mitochondria in bottom panels Ba and b, and the collapsed mitochondrial structure in bottom panel Cc. (D) Twenty-five mitochondrial regions were selected in ten control cells (black bars), and in cells with moderate (grey bars) and high (white bars) p50 expression and movements of mitochondria were analysed (see Materials and Methods, and Results). Movements of mitochondria are most apparent on dynamic images (see supplementary material Movies 1-5).

View larger version (59K): [in a new window]   Fig. 7. Subcellular localisation of endogenous Drp1 in HeLa cells. HeLa cells were first fixed with cold methanol-acetone (1:1) and endogenous Drp1 and tubulin were detected by immunofluorescence with an anti-Drp1 (1:250) and an anti--tubulin (1:1000) antidody. (a) mito.DsRed; (b,f) Drp1; and (e) -tubulin-immunofluorescence; (c,g) overlay of a and b, and e and f, respectively. Boxed areas in c and g are shown on an expanded scale in I, and II and III respectively. the enlarged image (III) was rotated 90° clockwise. Arrows indicate Drp1 assoc iation with punctate vesicular structures that are aligned along mitochondria (I) and microtubules (II,III).

View larger version (57K): [in a new window]   Fig. 8. Overexpression of p50 alters Drp1 localisation to mitochondrial membranes. (A) Endogenous Drp1 was stained in (a) empty vector or (b) p50.EGFP-transfected cells (n=15 cells in three independent experiments). Boxed areas in are shown on an expanded scale below (c,d); image in right upper corner shows the distribution of mitochondria in the same cells. Notice the filamentous and dispersed staining of Drp1 in the images of control and in p50 cells. (B) Overexpression of wild-type Drp1 restores normal distribution of mitochondria in p50 cells. Mitochondria were visualised with MitoTrackerRedTM in cells transfected with (a) empty vector, (b) p50.EGFP, (c) p50.EGFP+Drp1wt and (d) Drp1K38A (n=8 cells in three independent experiments). Hatched boundaries, obtained from an overlay with the transmitted image of the cell, indicate the cell periphery. Bars, 10 µm (C) Overexpression of p50 reduces the amount of Drp1 on mitochondrial membranes. Cells transfected with p50.EGFP (p50) or empty vector (Cont.) were fractionated as described in Materials and Methods. The post-nuclear supernatant (PNSN), post-mitochondrial supernatant (PMSN), mitochondrial pellet (Mito.P) and microsomal pellet (MP) were probed with a rabbit polyclonal anti-human Drp1 antibody. An equal amount of protein (26 µg/lane) was loaded from each fraction. The antibody recognised Drp1 at the expected size (80 kDa). (D) Overexpression of wild-type Drp1 restores the normal amount of Drp1 on mitochondrial membranes in p50-expressing cells. Post-nuclear supernatant (PNSN) and the mitochondrial pellet (Mito.P) were prepared from control cells (Cont.) and from cells overexpressing Drp1 (Drp1) and Drp1+p50 (Drp1+p50). An equal amount of protein (16 µg) was loaded in each lane. The blots were scanned and quantified with NIH ImageJ software (http://rsb.info.nih.gov/ij/).

View larger version (28K): [in a new window]   Fig. 9. The Drp1-dynactin complex interacts in HeLa cell extracts. p50, p150Glued and Drp1 were immunoprecipitated (IP) from HeLa cell extracts using the corresponding antibodies. Immunoprecipitations were performed in the presence (+NP40) and absence (-NP40) of detergent. Blots were probed with a monoclonal anti-p150Glued antibody (A,B) or a monoclonal anti-p50 antibody (C). Control samples (-Cont.), precipitated by an unrelated monoclonal antibody or rabbit pre-immune serum. Five µg of protein were loaded from the cell homogenate, and 500 µl were used as starting material for immunoprecipitation. Within each panel, an equal amount of protein was loaded from the immunoprecipitated samples (lanes p150Glued and -Cont.). Drp1*, cells expressing p50; p50*, cells expressing Drp1K38A.

View larger version (27K): [in a new window]   Fig. 10. Possible interaction of Drp1 (red) and dynein-dynactin complexes (green) with microtubules and mitochondria. Efficient fission of mitochondria (Control) facilitate transport of these organelles by anterograde motors including kinesin (yellow). Disruption of dynein-dynactin (p50) leads to the loss of Drp1 from mitochondria, which become more interconnected and consequently a poorer substrate for anterograde transport.