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First published online 20 May 2003
doi: 10.1242/jcs.00479

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Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells

Ansgar Santel*,1, Stephan Frank2,4, Brigitte Gaume2, Michael Herrler3, Richard J. Youle2 and Margaret T. Fuller1,{ddagger}

1 Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
2 Biochemistry Section, Surgical Neurology Branch, NINDS, National Institutes of Health, Bethesda, MD 20892, USA
3 BD Biosciences CLONTECH, Palo Alto, CA 94303, USA
4 Department of Neuropathology, University of Bonn, 53105 Bonn, Germany



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  		Fig. 1. Human mitofusin homologues Mfn1 and Mfn2 aligned with Drosophila Fzo (ClustalW and Boxshade). Black shading: amino acid identity. Gray shading: amino acid similarity. Asterisks: G1, G2, G3, G4 (four motifs of the signature GTPase domain). Thick brackets: region used to generate Mfn1-specific antibodies. Parentheses: internal peptide sequence used as imunogen for Mfn2-specific antibodies.

 


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  		Fig. 2. Differential expression of Mfn1 and Mfn2 mRNAs in human tissues. (A) Human multiple tissue northern (Clontech) probed with Mfn1- or Mfn2-specific probes or actin (loading control). (B) RNA-chip carrying human polyA-mRNA from 128 different tissues or cell types. Arrows and boxes highlight cases of differential expression between Mfn1 and Mfn2. Elevated Mfn2 mRNA in heart tissues (double arrows).

 


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  		Fig. 3. Mfn1 and Mfn2 protein expression in different cell lines, human and mouse tissues. (A) Extracts from COS-7 cells transiently transfected with various Mfn1 (GFP-Mfn1, Mfn1, antisense-Mfn1) and Mfn2- (Mfn2-myc and Mfn2{Delta}1-96-myc) expression constructs (as indicated) analyzed by western blotting using anti-Mfn1 or anti-myc antibodies. (B) Human Mfn1 protein detected as an 86 kDa band (arrow) in extracts from adult human heart, kidney (both from Clontech) and postnuclear supernatant from HeLa cell extracts probed with anti-Mfn1. Unspecific crossreacting band (asterisk). Same gel probed with anti-Tom40 antibodies as loading control (lower panel). (C) Mfn2 is abundantly expressed in human heart. Total cell extracts from yeast containing pGal1::Mfn2 plasmid and grown in either dextrose- or galactose-containing medium (left two lanes). Protein extracts from human kidney or heart (right two lanes) probed with Mfn2-specific polyclonal antibodies (two different exposures shown). Mfn2 runs as a doublet at around 86 kDa (arrow). Parallel loadings of same extracts probed with anti-Mfn1 antibodies, or anti-Tom40 and anti-actin as loading controls (lower three panels).

 


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  		Fig. 4. Mfn1 co-fractionates with mitochondria and participates in a high molecular weight complex. (A) Mfn1 co-fractionated with the mitochondrial outer membrane proteins TOM40 and porin. Postnuclear protein extracts from HeLa cells and mouse heart were fractionated by differential centrifugation, subjected to SDS-PAGE, and then western blots were probed with anti-Mfn1, anti-TOM40, anti-porin and anti-actin. Total, postnuclear extract. PMS, postmitochondrial supernatent, depleted of mitochondria. Mito, crude mitochondrial pellet. (B) Migration of Mfn1 in a 350 kDa complex. Mitochondrial extracts derived from human HL-60 cells were solubilized with digitonin and fractionated through a Superose-6 gel filtration column. Fractions were analyzed by western blotting using anti-Mfn1 antibodies, void volume to the left; mobility of marker proteins (arrows).

 


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  		Fig. 5. Overexpression of Mfn1 in COS-7 cells caused formation of grape-like clusters with enlarged mitochondria. COS-7 cells transiently transfected with (A,C-E) GFP-Mfn1 or (B) untagged Mfn1 expression constructs. (A) Confocal image of live COS-7 cells. Mitochondria visualized with Mitotracker (red). Transfected cells expressing GFP-Mfn1 (arrows, green). (B) Cells fixed and stained with anti-Mfn1 (green) and counterstained with anti-cytochrome c (red). Transfected cell with typical grape-like perinuclear cluster of mitochondria. (arrow; C-E) High magnification view of cluster-of-grape-like mitochondrial network formed after transient transfection with GFP-Mfn1 (green); mitochondrial intermembrane space labeled with anti-cytochrome c (red). (F) Electron microscope image showing cluster-of-grape-like mitochondrial network in COS-7 cell transiently transfected with GFP-Mfn1 and processed for immunogold-labeling using anti-GFP antibodies. Note the characteristic central space surrounded by tightly packed mitochondria with enlarged mitochondria at the periphery.

 


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  		Fig. 6. Dependence of Mfn1-induced mitochondrial cluster formation on a wild type Mfn1 GTPase domain. (A) Diagram of wildtype and mutant GFP-Mfn1 expression constructs. (B-E) Immunofluorescence images of COS-7 cells showing GFP (green) and counterstained with anti-cytochrome C (red). (B) Cells transiently transfected with wildtype GFP-Mfn1 (arrows), showing typical clustered networks with large mitochondria (red) around the periphery. (C,D) Cells from culture transiently transfected with mutant GFP-Mfn1K88T showing that the majority of transfected cells have normal appearing mitochondrial arrays (arrowheads) Higher magnification view. Some transfected cells (30%) still had clustered mitochondria (arrow), although they did not usually show the cluster-of-grape morphology. Instead, the GFP and anti-cytochome c commonly colocalized and the clustered mitochondria commonly lacked the large regions stained with anti-cytochome c but not GFP. (E) COS-7 cells transiently transfected with an N-terminal deletion mutant GFP-Mfn1{Delta}GTPase lacking the entire GTPase domain. Most transfected cells had clustered mitochondria in which the GFP and anti-cytochome c colocalized and did not show grape-like clusters (arrow).

 


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  		Fig. 7. Overexpression of the GTPase G2 motif mutant GFP-Mfn1T109A caused mitochondrial fragmentation. (A) Live COS-7 cells nine hours after transient transfection with GFP-Mfn1T109A (green) counterstained with Mitotracker (red). Arrow, transfected cell. High magnification view of fragmented mitochondria, showing GFP-Mfn1T109A around the surface of individual mitochondria stained with mitotracker (Inset). (B) COS-7 cells transfected with GFP-Mfn1T109A, showing effects of low (arrow) or high (double arrow) levels of GFP-Mfn1T109A expression. (C) Example from COS-7 cell culture co-transfected with GFP-Mfn1T109A and wildtype Mfn1. Samples in B and C imaged by fluorescence of the GFP moiety of the GFP-Mfn1T109A fusion protein. (D) COS-7 cells from culture co-transfected with GFP-Mfn1T109A (green) and HIS-tagged wildtype Mfn1 (Mfn1-V5/HIS) (red, immunofluorescence using anti-HIS-tag antibodies also labels nuclei). Fine mitochondrial network in a cell expressing GFP-Mfn1T109A with low levels of Mfn1-V5/HIS (double arrow). Grape-like mitochondrial cluster in a cell co-expressing GFP-Mfn1T109A and high levels of V5/HIS-tagged Mfn1 (arrow). Lower panels: GFP-Mfn1T109A-transfected cell with no detectable Mfn1-V5/HIS protein, from the same co-transfection experiment.

 


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  		Fig. 8. Drp1K38A blocks mitochondrial fragmentation in cells expressing Mfn1T109A. Mitochondrial morphology visualized by GFP-fluorescence in representative doubly-transfected COS-7 cells expressing GFP-Mfn1T109A (green) and HA-tagged Drp1K38A (red dot-like structures counterstained with anti-HA).

 

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© The Company of Biologists Ltd 2003