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First published online 6 March 2007
doi: 10.1242/jcs.03418

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The SUMO protease SENP5 is required to maintain mitochondrial morphology and function

¹ University of Ottawa Heart Institute, Rm H445A, 40 Ruskin Street, Ottawa, Ontario, K1Y 4W7, Canada
² Ontario Genomics Innovation Centre, Ottawa Health Research Institute, 501 Smyth, Ottawa, Ontario K1H 8L6, Canada

View larger version (31K): [in this window] [in a new window]   Fig. 1. SENP5 regulates mitochondrial fission. (A) COS-7 cells transfected as indicated were fixed and examined for mitochondrial morphology by examining the OCT:CFP signal. Values represent the mean ± s.d. of three coverslips, examining 50-100 cells from each. Fragmented, mitochondria with a stokes radius of 1. (B) Representative images of the OCT-CFP matrix marker shown as an example of the rescue phenotype. (C) Lysates of COS-7 cells (transfected as indicated) examined by SDS-PAGE, and then probed with anti-His6, anti-FP, anti-DRP1, anti-Mfn2 and anti-Tom20 antibodies. (D) Quantification of DRP1 protein levels obtained from the blots in C was expressed as a percentage of the intensity found in cells transfected with SUMO1:His alone. The values were normalized to the levels of Tom20 in each lane to ensure equal loading (n=3). UTF, untreated cell fraction.

View larger version (72K): [in this window] [in a new window]   Fig. 2. SENP5 is broadly distributed and deSUMOylates multiple mitochondrial substrates. (A) COS-7 cells transfected with SENP5:YFP were fixed; YFP fluorescence indicates that SENP5 localizes mainly in nucleolus with clear cytosolic and nuclear staining (bar, 10 µm). (B) COS-7 cells were harvested and fractionated to obtain mitochondria and cytosol. In addition to the total extracts, 50 µg of each fraction were loaded in a gel to examine endogenous SENP5 distribution. Markers of different fractions (HSP70 for cytosol, HSP60 and Tom20 for mitochondria) are indicated. (C) Untransfected COS-7 cells or those expressing SENP5:YFP were harvested and mitochondria were isolated. Of total cell lysate (Tot) or mitochondriaenriched fractions (Mit) 100 µg were separated by SDS-PAGE to examine the endogenous mitochondrial SUMO1 conjugates. Shown are short and long exposures of the anti-SUMO1 western blots (top and middle panel), together with anti-DRP1 and anti-Tom20 detection (bottom panel). SUMO1 conjugates in the mitochondrial fraction are indicated by circles, arrows indicate DRP1 high molecular mass conjugates. UTF, untreated cell fraction.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Silencing of SENP5 results in increased DRP1 SUMOylation. (A) SENP5 silencing increased DRP1 conjugates. COS-7 cells transfected with either shRNA vector alone (vector) or with SENP5-specific shRNA (SENP5 RNAi1), were selected in the presence of 5ug/ml puromycin for 12 days before collection; 100 µg of the total lysates were separated by SDS-PAGE and western blots with the indicated antibodies are shown. (B) Quantification of the SENP5 and DRP1 protein levels in control (Vector) or SENP5-silenced [shRNA (SENP5)] cells from eight different experiments. In each case, 100 µg of total cell extract was loaded and signals from the western blots were quantified using the Alpha Innotech HD Gel documentation system. (C) Immunoprecipitation of DRP1 from control (Vector) or SENP5-silenced (RNAi1) cells reveals an increase in mono-SUMOylated DRP1. COS-7 lysates from cells stably expressing vector alone or SENP5 shRNA were immunoprecipitated with either anti-DRP1 antibody or mouse IgG. Immunoprecipitates were run in duplicates on a 4-20% gradient gel; one blot was probed for endogenous DRP1 and the other for SUMO1. (D) Immunoprecipitation of DRP1 from SUMO1:FLAG-transfected COS-7 cells stably expressing vector alone or SENP5 shRNA. COS-7 cells stably expressing vector alone or SENP5 shRNA were transfected with SUMO:Flag for 24 hours prior to immunoprecipitation with either anti-DRP1 antibody or mouse IgG. Immunoprecipitates were run on duplicate gels; one blot was probed with a monoclonal anti-Flag antibody, the second with the anti-SUMO antibody. Following this, both gels were probed with anti-DRP1 antibody. The increased total level of DRP1 precipitated within the SENP5-silenced cells appears as a doublet. Only the top band of this doublet overlaps directly with the anti-Flag and anti-SUMO1 antibody signal (arrow).

View larger version (65K): [in this window] [in a new window]   Fig. 4. Silencing of SENP5 alters mitochondrial morphology, and increases DRP1 and SUMO1 recruitment. (A) COS-7 cells treated or not with shRNA targeting SENP5 were infected with DRP1:YFP, fixed and stained with anti-Tom20 antibody. Images were taken with the Poloychrome IV monochrometer on the Olympus IX70 microscope; Tom20 staining (white), DRP:YFP (green). Bars, 15 µm. (B) Amplified images of mitochondria from different cells treated with shRNA targeting SENP5 stained with anti-Tom20 antibody. Confocal images show enlarged mitochondria (*) and mitochondrial fragments (arrows), typical of cells that express shRNA targeting SENP5. Bars, 5 µm. (C) Cristae are not significantly altered in SENP5-silenced cells. COS-7 cells stably expressing vector (left panels) or SENP5 shRNA (right panels) were processed for EM. Two representative images are shown for each condition. Many smaller mitochondria were observed in the SENP5-silenced cells (arrows), larger mitochondria lost their thin, tubular morphology and appeared more spherical. Asterisks represent cristae sectioned longitudinally through the lamella, which were more readily observed within those cells expressing SENP5 shRNA. Bars, 500 nm. (D) Enlarged boxed areas shown in A showing recruitment of DRP1:YFP to mitochondria in control cells (top panels) and SENP5-silenced (bottom panels). Bars, 2 µm. Note the relatively even distribution of DRP1 puncta along mitochondria within control cells. Mitochondria that lack SENP5 show many enrichments of DRP1:YFP along the ridges of the enlarged mitochondria (arrows). (E) Images shown represent enlarged regions of confocal sections taken from live cells expressing either control shRNA (top panels) or SENP5 shRNA (bottom panels) that were transfected with SUMO1:YFP (green) and the matrix marker pOCT:CFP (white). In control cells, SUMO1:YFP is occasionally observed at active sites of fission as shown previously (Harder et al., 2004). In SENP5-silenced cells, similar SUMO1:YFP puncta are also observed at sites of fission; however, there are an increased number of SUMO1:YFP puncta on the mitochondria that did not appear to be at active fission sites (at least during the time of imaging). These still images are taken from a live confocal video which shows SUMO1:YFP puncta with respect to the dynamic fission activity of mitochondria (supplementary material Movie 3A,B). Bars, 2 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 5. Interference with DRP1 rescues the SENP5-silenced phenotype. (A) COS-7 cells stably expressing vector only (panels i,ii,iii) or SENP5-specific shRNA (panels iv,v,vi) were infected with the dominant-negative DRP1-K38E-CFP-HA (infection controls are shown in insets of ii and v), or treated with dsRNA to knock-down expression of DRP1 (panels iii,vi; knock-down controls are shown in supplementary material Fig. S1) were fixed in 4% PFA for staining with monoclonal antibody against cytochrome c. Bars, 10 µm (i,ii,iv,v) and 5 µm (iii,vi). (B) Quantification of the mitochondrial phenotype observed in A. Each bar represents the mean ± s.d. of cells from three coverslips, with 50 cells examined per coverslip.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Mitochondrial fusion is inhibited upon silencing of SENP5. (A) COS-7 cells stably transfected with shRNA vector alone (top panels) or to shRNA targeting SENP5 (bottom panels) were transfected with pOCT:PAGFP and the regions of interest indicated were activated at 405 nm. Stacks were obtained by imaging cells at 488 nm every 10 minutes for 40 minutes, after which all of the mitochondria containing PAGFP were activated. Masks were obtained where each voxel having a fluorescent signal above the threshold was scored as white and single slices taken from the stacks are shown at 1 minute post activation, at 40 minutes and after complete activation. Bars, 10 µm. (B) Quantification of the data obtained from control cells (n=10) and from SENP5-silenced cells (n=13). The percent increase in the spread of the activated signal is shown for each cell. The circled data points represent the cell whose corresponding images are shown in A. The data show that there is an average of 21% of mitochondrial fusion within 40 minutes in COS-7 cells, whereas SENP5-silenced cells have significantly reduced rates of fusion (P<0.001).

View larger version (60K): [in this window] [in a new window]   Fig. 7. Downregulation of SENP5 leads to increased reactive oxygen species (ROS) production. (A) COS-7 cells stably expressing either vector (top panels) or silenced for SENP5 shRNA (middle and bottom panels) were incubated at 37°C with 10 µM hET; oxidized hET (red) and MitofluorRed (green) were observed at the times indicated. Note the increased intensity of red fluorescence at 60 minutes in cells that contain shRNA targeting SENP5. The bottom panel shows a higher magnification of the aberrant mitochondria within these cells. (B) Average fluorescence intensity per cell of oxidized hET and MitofluorRed from 244 SENP5-silenced cells and 270 cells containing the vector. Data were collected (using identical laser intensities and scanning parameters) from live cells over a 90-minute period, imaging 10-20 cells per field. (C) Average fluorescence of oxidized hET per cell was plotted for each field as a function of imaging time. Each point represents the average hET fluorescence of 10-20 cells. Control cells did not significantly increase their ROS production over time, whereas the rate of ROS production in SENP5-silenced cells is increased. (D) COS-7 cells stably expressing either vector or shRNA targeting SENP5 were harvested and duplicate samples of 105 cells were incubated at 37°C with 10 µM hET for 40 minutes in culture medium. Then, fluorescence emission scans were collected to examine the reduced (emission peak, 420 nm; excitation, 355 nm) and oxidized hET (emission peak, 620 nm; excitation, 518 nm). Each value represents the mean ± s.d. of two independent aliquots (*P<0.01). (E) Same as D, but cells had been previously infected or not with DRP1-K38E-CFP-HA. Each value represents the mean ± s.d. of two independent aliquots (*P<0.01, **P<0.01).