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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.01058

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Levels of human Fis1 at the mitochondrial outer membrane regulate mitochondrial morphology

¹ Department of Biochemistry, La Trobe University, 3086 Melbourne, Australia
² Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA

View larger version (38K): [in a new window]   Fig. 1. Amino acid sequence alignment of Fis1 from different species. Residues in bold indicate conserved amino acids in three or more species. Asterisks (*) indicate residue conservation amongst all species. The predicted transmembrane domain of hFis1 is indicated (underlined).

View larger version (86K): [in a new window]   Fig. 2. GFP-hFis1 is targeted to the mitochondria. (A) COS-7 cells transfected with pOTC-GFP, a mitochondrially located protein, or GFP-hFis1 were counterstained with MitoTracker Red and analysed by fluorescence microscopy 24 hours after transfection. The far right panel depicts overlay images of the GFP and MitoTracker Red fluorescence. (B) High resolution confocal microscopy of mitochondrial tubules as seen in (A).

View larger version (51K): [in a new window]   Fig. 3. Characterization of hFis1. (A) hFis1 is located on the mitochondrial outer membrane. Samples of mitochondria (lane 1) and mitochondrial outer membrane vesicles (OMV; increased loading, lanes 2-5) were subjected to SDS-PAGE and western blot analysis using antibodies against the matrix located mtHsp70, the inner membrane complex I subunit NDUFA9, the outer membrane marker Tom40 and hFis1. (B) hFis1 is ubiquitously expressed. Isolated mitochondria from heart, brain, liver, testes, spleen and kidney rat tissues were subjected to SDS-PAGE and western blot analysis using Tom22 and hFis1 antibodies. (C) hFis1 displays a diffuse distribution along the mitochondrial surface. COS-7 cells were subjected to immunofluorescence assays using pre-immune serum, affinity-purified anti-hFis1 antibodies or mtHsp70 antibodies. (D) hFis1 is a mitochondrial protein with its N-terminus exposed to the cytosol. Isolated HeLa cell mitochondria (50 µg) were treated with or without externally added protease as indicated. Mitochondria were reisolated and subjected to SDS-PAGE and western blot analysis using mtHsp70, cytochrome c, Tom22 and hFis1 antibodies. (E) hFis1 is an integral membrane protein. HeLa cell mitochondria (100 µg) were extracted with 100 mM sodium carbonate, pH 11.5. After centrifugation at 18,000 g, the membrane pellet fraction (P; lane 1), and soluble supernatant fraction (S; lane 2) were subjected to SDS-PAGE and western analysis using antibodies directed against hFis1, mtHsp70 or Tom40.

View larger version (53K): [in a new window]   Fig. 4. hFis1 is targeted to the mitochondria via basic residues within its C-segment and its transmembrane domain. (A) Schematic representation of GFP-hFis1 fusion proteins and their subcellular location. (B-F) COS-7 cells expressing the GFP-hFis1 fusion proteins depicted in (A) were incubated with MitoTracker Red and the nuclear stain Hoechst 33258 before analysis by fluorescence microscopy.

View larger version (49K): [in a new window]   Fig. 5. Overexpression of hFis1 compromises steady state mitochondrial morphology. COS-7 cells overexpressing the GFP-hFis1 chimera were counterstained with MitoTracker Red and Hoechst 33258 and analysed by fluorescence microscopy. Cells expressing hFis1 mainly displayed one of two phenotypes: (A) fragmented mitochondria; or (B) mitochondrial aggregates in the vicinity of the nucleus. (C) Phenotype of cells expressing the control outer membrane protein Tom7. (D) High-resolution confocal microscopy of mitochondrial aggregates similar to those seen in (B) reveals that they are composed of small fragmented mitochondria. (E) Quantitation of mitochondrial aggregates in COS-7 cells overexpressing vector alone, Tom7 or hFis1 was monitored and quantified at 16 hours, 36 hours and 48 hours after transfection (n=3). (F) Quantitation of mitochondrial aggregates in >100 COS-7 cells co-expressing hFis1/Drp1 or hFis1/Drp1 (K38A) (n=3).

View larger version (33K): [in a new window]   Fig. 6. Complementation studies in S. cerevisiae. (A) Yeast fis1 cells expressing a mitochondrial targeted from of GFP as well as either vector alone (p-empty), vector expressing yeast Fis1p or hFis1 were subjected to DIC and fluorescence microscopy. (B) GFP-hFis1 is mis-targeted to the ER in yeast cells. By contrast, GFP-hFis1(N)-yFis1p(TM), a chimeric fusion protein containing the N-terminal cytosolic domain of hFis1 and the transmembrane domain and C-segment of yeast Fis1p, is targeted to the mitochondria but fails to complement in fis1 cells.

View larger version (69K): [in a new window]   Fig. 7. Knockdown of hFis1 in COS-7 cells leads to the formation of mitochondrial tubule extensions. COS-7 cells were transfected with vector alone (scrambled sequence; control) or vector encoding small interfering RNAs specific for hFis1 knockdown. (A) At 72 hours after transfection, mitochondria were isolated and subjected to SDS-PAGE and western blot analysis using antibodies specific for hFis1 and Tom40 (as control; left). The amount of hFis1 knockdown was quantified (right). (B) Representative mitochondrial morphologies observed in cells transfected with control and hFis1 small interfering RNAs as described in (A). Scale bar, 5 µm.

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