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

First published online 23 August 2005
doi: 10.1242/jcs.02537

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS 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 Yu, T. Articles by Yoon, Y. Search for Related Content PubMed PubMed Citation Articles by Yu, T. Articles by Yoon, Y.

Regulation of mitochondrial fission and apoptosis by the mitochondrial outer membrane protein hFis1

¹ Department of Anesthesiology and of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA
² Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA

View larger version (30K): [in a new window]   Fig. 1. Illustration of hFis1 constructs used in this study. All hFis1 constructs were tagged with the Myc-epitope at the N-terminus. The hFis1 transmembrane domain spanning the mitochondrial outer membrane is depicted in black near the C-terminal end. Wild-type hFis1 (hFis1-WT) contains six -helices (1-6) in its cytosolic domain. Helices 2-3 and 4-5 form the two TPR motifs, TPR1 and TPR2, respectively.

View larger version (51K): [in a new window]   Fig. 2. Deletion of 1-helix of hFis1 abolishes mitochondrial fragmentation and increases the DLP1-hFis1 interaction. (A) Deletions in the N-terminal cytosolic domain of hFis1 abolished mitochondrial fragmentation. Myc-tagged hFis1 full length (WT) and deletion constructs, hFis1[11-152], hFis1[21-152], hFis1[32-152], hFis1[61-152] and hFis1[92-152] were transfected into Clone 9 cells harboring GFP-labeled mitochondria and cells overexpressing the Myc-tagged proteins were detected by immunofluorescence using the anti-Myc antibody. Two-hundred to 300 untransfected cells and cells transfected with each construct were counted to score mitochondrial morphology. The upper three panels represent the three mitochondrial morphologies used for scoring the different mitochondrial phenotypes. Mitochondria were detected by GFP fluorescence and anti-Myc staining completely overlapped with the GFP signal. While untransfected cells contained normal tubular mitochondria (top left, bottom UnTr), mitochondria in cells overexpressing hFis1-WT were finely fragmented, showing a punctate mitochondrial phenotype (top middle, bottom WT). The number of cells containing fragmented mitochondria decreased as N-terminal amino acids were further deleted. The third phenotype that shows swollen, ball-shape mitochondria (top right) were prevalent in cells overexpressing Myc-hFis1[32-152]. Scale bar, 10 µm. (B) Deletion of the N-terminal region of hFis1 enhances the hFis1-DLP1 interaction. BHK-21 cells were transfected with full length (FL) or N-terminally truncated hFis1 constructs and whole cell extract (WCE) was subjected to immunoprecipitation by anti-DLP1 antibodies. Immunoprecipitated proteins were analyzed by immunoblotting with anti-DLP1 and anti-Myc antibodies. All Myc-tagged hFis1 constructs, FL and deletion mutants, were expressed in cells as detected in the WCE. Myc-hFis1[21-152], Myc-hFis1[32-152], and Myc-hFis1[61-152] were readily detected in immune complexes isolated by the anti-DLP1 antibody while little binding was obtained with cells expressing FL and Myc-hFis1[11-152]. No binding was detected with Myc-hFis1[92-152]. 1% of starting materials and 50% of immunoprecipitated materials were loaded onto the gel for immunoblot analyses.

View larger version (42K): [in a new window]   Fig. 3. Two TPR motifs are important for interaction with DLP1 or DLP1-containing complex. (A) Helix-breaking point mutations in 1 (L14P), 2 (L42P), and 3 (L58P), 4 (L77P), 5 (L91P), and 6 (L110P) were made in full-length hFis1 and overexpressed in Clone 9 cells to test their effects on mitochondrial fragmentation. The L42P, L58P, L77P, and L91P point mutations in helices forming TPR abolished the mitochondrial fragmentation effectively, showing that less than 15% of the point mutant-expressing cells contained fragmented mitochondria. L14P and L110P point mutations in non-TPR helices were less effective in abolishing mitochondrial fragmentation. (B) Gallery of cells expressing TPR mutations showing the dominant-negative mitochondrial phenotype. These mitochondrial morphologies were found throughout cells overexpressing four TPR mutants. Mitochondria are elongated and often severely entangled around the nucleus indicative of a fission-defective mitochondrial phenotype, suggesting that these point mutants exert a dominant-negative effect. N, nucleus; scale bar, 10 µm. (C,D) Morphometric analyses of mitochondrial elongation in cells expressing TPR mutants. Cell counting revealed that 25-60% of cells expressing TPR mutants displayed the dominant-negative mitochondrial phenotype whereas non-TPR mutants showed no mitochondrial elongation (C). For morphometric analysis, five mutant cells with less severely entangled mitochondria and five untransfected normal cells were selected, and pixel lengths were measured only in clearly discernible mitochondria. Pixel values representing the length of mitochondria were sorted from the longest to the shortest, and the five longest mitochondrial tubules from each cell are presented (D). Note that overall lengths of mitochondrial tubules are longer in mutant cells, showing approximately two-fold increase. (E) 4 and 5 participate in binding DLP1 or DLP1-containing complex. Three helix-breaking mutants L77P, L91P, and L110P in Myc-hFis1[32-152] were tested for DLP1 binding by co-immunoprecipitations. Both L77P and L91P mutations greatly reduced the binding to DLP1, whereas the L110P mutant showed the DLP1 interaction to the level similar to Myc-hFis1[32-152]. 1% of starting materials and 50% of immunoprecipitated materials were loaded onto the gel for immunoblot analyses.

View larger version (37K): [in a new window]   Fig. 4. The mitochondrial permeability transition contributes to mitochondrial swelling caused by overexpression of 1-deleted hFis1. (A) Clone 9 cells were transfected with Myc-hFis1[32-152] alone or co-transfected with Bcl-2 and Myc-hFis1[32-152], and scored for the swollen mitochondrial morphology. Co-expression of Bcl-2 with Myc-hFis1[32-152] reduced the number of cells containing swollen mitochondria by more than twofold, indicating that the mitochondrial permeability transition (MPT) played a role in mitochondrial swelling. Incubating cells transfected with Myc-hFis1[32-152] with MPT inhibitors bongkrekic acid (BA) or cyclosporin A (CsA) also decreased mitochondrial swelling. (B) Cobalt-quenched calcein measurement. Clone 9 cells transfected with Myc-hFis1[32-152] were loaded with calcein and CoCl2 and stained with MitoTracker Red. (Left panel: mito) Swollen mitochondria by overexpression of Myc-hFis1[32-152] were visible in cells with asterisks. (Right panel: calcein) Calcein fluorescence was greatly reduced in swollen mitochondria (cells with asterisks). Cobalt ions entered mitochondria and quenched calcein fluorescence, indicating that the MPT occurred in swollen mitochondria. (C) Quantitation of calcein fluorescence in mitochondria. Calcein fluorescence in the mitochondrial regions was quantified from digital images. Mitochondrial regions were identified based on MitoTracker staining. Because of cell-to-cell variations of calcein fluorescence, intensity values from each cell were plotted to show the distribution, and median values were indicated as arrows. Fluorescence intensity was measured in 43 cells each for untransfected (UT) and transfected ([32-152]) at 24 hours post transfection (black circle and triangle, respectively), and in 28 cells each for UT and [32-152] at 48 hours post transfection (gray symbols). Experiments were repeated three times and similar quantitative results were obtained in all three experiments.

View larger version (64K): [in a new window]   Fig. 5. Swollen mitochondria induced by overexpression of 1-deleted hFis1 contain increased DLP1. (A) Clone 9 cells transfected with Myc-hFis1-FL or Myc-hFis1[32-152] were immunostained with anti-Myc and anti-DLP1 antibodies. DLP1 puncta (green) distributed throughout the cytoplasm in cells expressing Myc-hFis1-FL (a) and associated with fragmented mitochondria stained with anti-Myc (red) shown in enlarged image (a'). However, in cells expressing Myc-hFis1[32-152] (b), the non-mitochondrial distribution of DLP1 appeared to be reduced while more DLP1 (green) associated with Myc-hFis1[32-152] (red) of swollen mitochondria, forming lager spots and patches of DLP1 (b'). Scale bar, 20 µm for a,b, and 10 µm for a',b'. (B) Cellular fractions isolated from untransfected cells and cells transfected with Myc-hFis1[32-152] were analyzed by immunoblotting. Each lane was loaded with 15-20 µg total protein and probed for DLP1 and cytochrome c for a mitochondrial marker. Note that no cytochrome c was detected in the cytosol from cells overexpressing Myc-hFis1[32-152], indicating that cytochrome c release did not occur at the time of fractionation (16 hours post-transfection). Most DLP1 was found in the cytosolic fraction and mitochondria contained a low level of DLP1 (left panel). PNS, post-nuclear supernatant; cyt, cytosolic fraction; mito, mitochondrial fraction. Densitometry was performed using ImageJ (NIH) software, and density of the DLP1 band in mitochondrial fraction was normalized against the cytochrome c band. DLP1 band density after normalization showed increased DLP1 in the mitochondrial fraction from the cells overexpressing Myc-hFis1[32-152] (right panel). (C) Mitochondrial fractions from untransfected cells, Myc-hFis1[32-152]-transfected cells, and cells co-transfected with Bcl-2 and Myc-hFis1[32-152] were immunoblotted for DLP1. Whereas more DLP1 was found in the mitochondrial fraction from cells transfected with Myc-hFis1[32-152], co-expression with Bcl-2 reduced the mitochondrial DLP1 to the level of untransfected cells (left panel). The right panel shows densitometric analysis.

View larger version (19K): [in a new window]   Fig. 6. DLP1 binding to hFis1, but not the DLP1-GTPase activity, is necessary for mitochondrial swelling induced by overexpression of 1-deleted hFis1. (A) Leucine-to-proline mutants (L42P, L58P, L77P, L91P, and L110P) in Myc-hFis1[32-152] were expressed in Clone 9 cells and mitochondrial morphologies were analyzed. The TPR mutants (L42P, L58P, L77P, and L91P) in Myc-hFis1[32-152] significantly decreased the mitochondrial swelling phenotype of Myc-hFis1[32-152] although L42P was less effective, indicating that DLP1 binding to the hFis1 TPR is necessary for mitochondrial swelling. The L110P mutation had no effect on mitochondrial swelling. (B) Co-expression of the GTPase-defective DLP1-K38A with Myc-hFis1[32-152] did not reduce the mitochondrial swelling, but mitochondrial fragmentation by Myc-hFis1-FL was greatly decreased by DLP1-K38A, indicating that GTP hydrolysis by DLP1 is required for mitochondrial fission but not for the swelling. (C) Although mitochondrial swelling via 1-deletion (Myc-hFis1[32-152]) was reduced by the Bcl-2 co-expression, Bcl-2 had no effect on mitochondrial fragmentation induced by Myc-hFis1-FL, indicating that Bcl-2 does not directly participate in DLP1/hFis1-mediated mitochondrial fission.

View larger version (24K): [in a new window]   Fig. 7. Cytochrome c release and cell death are delayed in cells overexpressing Myc-hFis1[32-152]. Myc-hFis1[32-152] or Myc-hFis1-FL was co-expressed with GFP as a transfection marker and distributions of cytochrome c were analyzed by immunofluorescence. (A) Cytochrome c staining was greatly reduced and diffuse in cells expressing Myc-hFis1-WT, indicating cytochrome c release from mitochondria (a,a'). Cells expressing Myc-hFis1[32-152] contained cytochrome c in their swollen mitochondria (b,b'). T: transfected cells. Scale bar, 10 µm. (B) Cells with diffuse cytochrome c staining were scored at different times after transfection. Less than 25% of cells transfected with Myc-hFis1[32-152] released cytochrome c up to 72 hours post transfection, whereas 70% of cells expressing full-length hFis1 released cytochrome c at 72 hours. (C) Frequencies of cells expressing full-length hFis1, hFis1[32-152], or both Bcl-2 and hFis1[32-152] were scored at 24, 48 and 72 hours post transfection and presented as percent cell survival at each time point. The frequency at 24 hours was given as initial survival (100%). Survival of cells expressing full-length hFis1 decreased to 20% after 72 hours, whereas that of hFis1[32-152] cells were 75-80% at the same time point. Co-expression of Bcl-2 with Myc-hFis1[32-152] increased cell survival to 95% after 72 hours.