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. 1999 Nov 15;147(4):699-706.
doi: 10.1083/jcb.147.4.699.

Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape

H Sesaki  1 , R E Jensen
Affiliations

Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape

H Sesaki et al. J Cell Biol. .

Abstract

In yeast, mitochondrial division and fusion are highly regulated during growth, mating and sporulation, yet the mechanisms controlling these activities are unknown. Using a novel screen, we isolated mutants in which mitochondria lose their normal structure, and instead form a large network of interconnected tubules. These mutants, which appear defective in mitochondrial division, all carried mutations in DNM1, a dynamin-related protein that localizes to mitochondria. We also isolated mutants containing numerous mitochondrial fragments. These mutants were defective in FZO1, a gene previously shown to be required for mitochondrial fusion. Surprisingly, we found that in dnm1 fzo1 double mutants, normal mitochondrial shape is restored. Induction of Dnm1p expression in dnm1 fzo1 cells caused rapid fragmentation of mitochondria. We propose that dnm1 mutants are defective in the mitochondrial division, an activity antagonistic to fusion. Our results thus suggest that mitochondrial shape is normally controlled by a balance between division and fusion which requires Dnm1p and Fzo1p, respectively.

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Figures

Figure 1
Figure 1
Mutants defective in mitochondrial shape. (A) Screen to isolate mutants defective in mitochondrial morphology. (a) Mitochondria were visualized by COX4-GFP. (b–d) Individual mutants were isolated using a micropipette. The arrow indicates a potential mutant cell. (B) Wild-type YHS2 and a representative from each class of mutants were grown in YPD to log phase and then stained with 1 μg/ml 3,3′dihexyloxacarbocyanine (DiOC6) (Molecular Probes) to enhance mitochondrial fluorescence. Confocal images of cells are shown. (C) dnm1Δ cells contain an interconnected network of mitochondria. Wild-type (BY4733) and isogenic dnm1Δ cells expressing COX4-GFP (pHS12), were grown in YPGal to log phase. Fluorescence (COX4-GFP) and differential interference contrast (DIC) images are shown. Bars: (A) 10 μm; (B) 2 μm; (C) 3 μm.
Figure 2
Figure 2
dnm1Δ fzo1Δ cells have normal-shaped mitochondria. Wild-type (BY4733) and isogenic deletion strains (dnm1Δ, fzo1Δ, and dnm1Δ fzo1Δ) expressing COX4-GFP (pHS12), were grown in YPGal to log phase and examined by fluorescence microscopy. Bars, 2 μm.
Figure 3
Figure 3
Expression of Dnm1p causes fragmentation of mitochondria in dnm1Δ fzo1Δ cells. dnm1Δ fzo1Δ cells carrying pCOX4-GFP (pHS12) and pGAL1-DNM1-HA (pHS15) were pregrown in raffinose medium, centrifuged and resuspended to an OD600 of 0.2 in galactose medium (SGS) to induce Dnm1p-HA expression. Cells were examined for mitochondrial shape (n = 100) or used to prepare total protein at the indicated timepoints. (A) Mitochondrial shape was classified into the following four groups: tubules (○), partially fragmented tubules (□), fragments (▴), and other (⋄). (B) Total protein was extracted as described ( Yaffe and Schatz 1984) and subjected to Western blot analysis using antibodies to the HA epitope ( Field et al. 1988), or hexokinase (Davis, A., unpublished data) followed by chemiluminescence (Pierce). Relative amounts of Dnm1p-HA (+) were quantitated and plotted in A.
Figure 4
Figure 4
A Dnm1p-GFP preferentially localizes to site of mitochondrial division. (A) dnm1Δ strain YHS19 was transformed with pHS20 expressing Dnm1p-GFP. Cells were grown in SGal medium, labeled with 0.1 μM MitoTracker Red CMXRos (Molecular Probes) and then examined under the fluorescence microscope. Merged images taken in the red (Mitotracker) and green (GFP) channels are shown. (B and C) dnm1Δ fzo1Δ diploid cells carrying pGAL1-DNM1-GFP were pregrown in YPglycerol/ethanol medium, centrifuged and grown in galactose medium (SGS) for 1–2 h. Cells were then stained with MitoTracker and examined for mitochondrial localization of Dnm1p-GFP. Two representative cells are shown. Bar, 3 μm.
Figure 5
Figure 5
dnm1Δ fzo1Δ cells are defective in mitochondrial fusion. (A) MATa cells that express CS1-GFP from the GAL1 promoter of pCLbGFP were grown in galactose-containing medium (SGS). MATα cells lacking the plasmid were grown in glucose medium (YPD). MATa and α cells carrying the indicated genotypes were mated on YPD for 3.5 h, and then stained with 0.1 μM MitoTracker. The distribution of MitoTracker and GFP in representative zygotes containing a medial bud (asterisks) are shown. Arrows indicate three clusters of mitochondria lacking CS1-GFP in fzo1Δ zygotes. (B) Wild-type and dnm1Δ fzo1Δ MATa cells were treated with ethidium bromide to induce loss of mtDNA ( Fox et al. 1991), transformed with pCLbGFP, and then grown in galactose-containing medium (SGS). Wild-type and dnm1Δ fzo1Δ MATα cells containing mtDNA, but lacking the plasmid, were grown in YPD. Cells were mated on YPD and stained with 0.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes). A representative zygote formed by mating between wild-type cells or between dnm1Δ fzo1Δ mutants is shown. Fluorescence (GFP or DAPI), differential interference contrast (DIC) and merged images are shown. Mating mixtures were also incubated for an additional 24 h at 30°C on YPD, and representative diploids are shown. N indicates nuclear DNA staining. Bar, 3 μm.
Figure 5
Figure 5
dnm1Δ fzo1Δ cells are defective in mitochondrial fusion. (A) MATa cells that express CS1-GFP from the GAL1 promoter of pCLbGFP were grown in galactose-containing medium (SGS). MATα cells lacking the plasmid were grown in glucose medium (YPD). MATa and α cells carrying the indicated genotypes were mated on YPD for 3.5 h, and then stained with 0.1 μM MitoTracker. The distribution of MitoTracker and GFP in representative zygotes containing a medial bud (asterisks) are shown. Arrows indicate three clusters of mitochondria lacking CS1-GFP in fzo1Δ zygotes. (B) Wild-type and dnm1Δ fzo1Δ MATa cells were treated with ethidium bromide to induce loss of mtDNA ( Fox et al. 1991), transformed with pCLbGFP, and then grown in galactose-containing medium (SGS). Wild-type and dnm1Δ fzo1Δ MATα cells containing mtDNA, but lacking the plasmid, were grown in YPD. Cells were mated on YPD and stained with 0.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes). A representative zygote formed by mating between wild-type cells or between dnm1Δ fzo1Δ mutants is shown. Fluorescence (GFP or DAPI), differential interference contrast (DIC) and merged images are shown. Mating mixtures were also incubated for an additional 24 h at 30°C on YPD, and representative diploids are shown. N indicates nuclear DNA staining. Bar, 3 μm.
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References

    1. Adams A., Gottschling D., Kaiser C., Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press; Plainview, NY : 1997.
    1. Bereiter-Hahn J., Voth M. Dynamics of mitochondria in living cellsshape changes, dislocations, fusion, and fission of mitochondria. Microsc. Res. Tech. 1994;27:198–219 . - PubMed
    1. Brachmann C.B., Davies A., Caputo E., Li J., Hieter P., Boeke J.D. Designer deletion strains from Saccharomyces cerevisiae 288Ca useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998;14:115–132 . - PubMed
    1. Burgess S.M., Delannoy M., Jensen R.E. MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria. J. Cell Biol. 1994;126:1375–1391 . - PMC - PubMed
    1. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I.A., Lerner R.A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 1988;8:2159–2165 . - PMC - PubMed

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