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. 2001 Aug;12(8):2245-56.
doi: 10.1091/mbc.12.8.2245.

Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells

E Smirnova  1 , L Griparic, D L Shurland, A M van der Bliek
Affiliations
Free PMC article

Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells

E Smirnova et al. Mol Biol Cell. 2001 Aug.
Free PMC article

Abstract

Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

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Figures

Figure 1
Figure 1
Effect of temperature-sensitive mutations in Drp1 on mitochondrial morphology. (A and B) Cells transfected with Drp1(V41F) and grown at 30°C (A) or 40°C (B). (C and D) Cells transfected with Drp1(G281D) and grown at 30°C (C) or 40°C (D). The transfected cells were identified by immunofluorescence with anti-Drp1 antibody (outlined cells). Mitochondrial morphology was monitored by staining with Mitotracker. A and B each also show an untransfected cell, to illustrate that the growth temperature does not appreciably affect mitochondrial distribution in cells without mutant Drp1.
Figure 2
Figure 2
Range of mitochondrial distribution defects induced by different mutations in Drp1. The left column of images shows transfected cells that were not treated with nocodazole. The remaining three columns show different examples of transfected cells that were treated with nocodazole. (A–A‴) COS-7 cells transfected with a wild-type Drp1 construct. (B–B‴) Cells transfected with a Tom20::GFP construct, which was used as a negative a control, because it causes mitochondrial clustering without affecting connectivity. (C–C‴) Cells transfected with a Drp1(K38A) construct. (D–D‴) Cells transfected with a Drp1(V41F) construct. The cell shown in D′ was grown at 30°C, whereas the cells shown in D" and D‴ were grown at 40°C. (E–E‴) Cells transfected with a Drp1(T59A) construct. The inset in E" shows an enlargement of a net formed by the mitochondria in this cell. (F–F‴) Cells transfected with a Drp1(G281D) construct and grown at 30°C. (G–G‴) Cells transfected with a Drp1(G281D) construct and grown at 40°C.
Figure 3
Figure 3
Morphology of the endoplasmic reticulum is not affected by Drp1(T59A). (A and B) Cells transfected with Drp1(T59A) alone. (C and D) A cell cotransfected with Drp1(T59A) and an exogenous ER-marker (VSV-G::KKTN), which we know from previous experiments helps to visualize the reticular nature of the ER (Smirnova et al., 1998). The transfected cells were identified by staining with anti-Drp1 antibody (A and C). The top cell in A shows endogenous levels of Drp1, whereas the bottom cell shows overexpression of Drp1, as does the cell in C. ER morphology was observed by staining with antiprotein disulfide isomerase antibody (B) or with anti-VSV-G antibody (D). The intensity and morphology of ER were unaffected by Drp1(T59A) (compare the staining pattern of the untransfected cell at the top of B with the staining pattern of the transfected cell at the bottom). The reticular nature of the ER also appears unaffected (compare the staining pattern in D with similar results obtained previously with wild-type and Drp1(K40A) transfected cells (Smirnova et al., 1998)).
Figure 4
Figure 4
Localization of endogenous Drp1 as determined by immunofluorescence. (A) Mitochondria stained with MitoTracker. (B) Endogenous Drp1 detected by immunofluorescence with anti-Drp1 antibody. (C) Merged image with Drp1 staining shown green and mitochondrial staining shown in red. (D) Enlargement of the boxed area in C. Arrows point to Drp1 spots that appear to transect the mitochondria. (E and F) Lack of colocalization of ER and Drp1. (E) ER staining pattern obtained with anti-ribophorin-I antibody. (F) Staining pattern obtained with anti-Drp1 antibody. (G) Merged image showing Drp1 staining in green and ER staining in red. (H) Enlargement of the boxed area in G. Bar, 5 μm.
Figure 5
Figure 5
Localization of Drp1 determined by subcellular fractionation of bovine brain. Lane 1, crude extract; lane 2, postnuclear supernatant (S1); lane 3, medium speed supernatant (S2); lane 4, medium-speed pellet (P2); lane 5, mitochondrial fractions after further purification on a Percoll gradient; lane 6, high-speed supernatant (S3); and lane 7, high-speed pellet (P3). The subcellular fractions were probed with antibody for Drp1, for a cytosolic marker (tubulin), and for mitochondria (39-kDa subunit of OxPhos complex I). The number of volume equivalents needed to load equal amounts of protein (75 μg/lane) was 1× for lane 1, 3× for lane 2 and 3, 43× for lane 4, 21× for lane 5, 3× for lane 6, and 75× for lane 7. Densitometry and adjustment for volume equivalents shows that ∼3% of Drp1 is in the mitochondrial fractions (lanes 4 and 5), 2% is in the high-speed pellet and the rest is in the supernatant. This fractionation experiment was replicated five times, each time giving similar results.
Figure 6
Figure 6
(A) Localization of a yellow-fluorescent protein (YFP)::Drp1 fusion protein. COS-7 and C2C12 cells were transfected with a YFP::Drp1 construct (green) and a construct encoding cyan-fluorescent protein that was targeted to the mitochondrial matrix (red). Insets show enlargements of peripheral portions of the cells. The images show that Drp1 is largely diffuse throughout the cytosol or localized to spots that are on mitochondria. (B) Time-lapse photography of a C2C12 cell with YFP::Drp1 in spots on mitochondria. C2C12 cells were cotransfected with a YFP::Drp1 construct (green) and a construct encoding cyan-fluorescent protein that was targeted to the mitochondrial matrix (red) and photographed at 5-s intervals. The arrows point to a division event preceded by a YFP::Drp1 spot. The two arrows at later time points indicate the two mitochondrial ends that are formed by mitochondrial division.
Figure 7
Figure 7
In vitro formation of ring-like structures by purified Drp1. (A) Electron micrograph of Drp1 incubated with 150 mM NaCl. (B) Drp1 incubated with GDP and AlF4−. Arrows point to the ring-like structures. (C) Examples of ring-like structures that were observed with GDP and AlF4−. The proteins were detected by negative staining with uranyl acetate.
Figure 8
Figure 8
Phenotypes caused by mutant Drp1 arranged in an allelic series. The drawings on the right depict the different mitochondrial morphologies that were observed with mutant Drp1. The brackets on the left indicate which morphologies were observed with each mutation. The overlap of phenotypes obtained with different mutations in Drp1 made it possible to rank those phenotypes from weak (a closed network of mitochondria) through strong (a collapsed network) to very strong (cell lysis). The weakest phenotype is most directly linked to the primary defect, indicating that this defect is a block in mitochondrial division. The stronger phenotypes may be due to secondary defects.
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