Cell Culture and Transfection Procedures

COS-7 cells were grown in DMEM with 10% FCS. Transfections were done by electroporation and cells were allowed to recover for 48 h. Methods for the VSV-G tsO45 GFP temperature shift experiment were described previously (Presley et al., 1997). The endocytosis experiments were performed by incubating cells at 37°C with serum-free medium and 10 μg/ml Bodipy FL low-density lipoprotein (LDL) or 20 μg/ml Bodipy FL transferrin (Molecular Probes, Inc.). The LDL-treated cells were processed for immunofluorescence after 25 min. The transferrin-treated cells were processed after 60 and 120 min.

Antibodies and Immunofluorescence Procedures

A 1150-bp fragment encoding the COOH-terminal half of dynamin-related protein was generated by PCR using primers that add an NcoI site to the 5′-end and a SalI site to the 3′-end. This fragment was recloned into the bacterial expression vector pET21d (Novagen Inc., Madison, WI), which adds six histidines to the carboxy terminus. The plasmid was expressed in Escherichia coli strain BL21DE3, and the 42-kD recombinant protein, which extends from the glutamic acid residue at position 359 to the carboxy-terminal end of Drp1, was purified by Ni-NTA chromatography. Chicken antisera were generated by Aves Labs (Tigard, OR) and then blot-purified with recombinant Drp1 protein fragment (Harlow and Lane, 1988). For Western blotting the proteins were size-fractionated on a 10% SDS-PAGE gel and transferred to nitrocellulose by standard procedures (Harlow and Lane, 1988). Blotted proteins were detected by chemiluminescence (Nycomed Amersham, Buckinghamshire, England). Protein extracts of human tissues were obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). Cells grown on coverslips were fixed for immunofluorescence with methanol/acetone or with paraformaldehyde. W.E. Balch (Scripps Research Institute) kindly provided anti-sec13 antibodies. The following primary antibodies were purchased: anti-HA (HA11), anti-dynamin and anti-myc (9E10) from BAbCO Inc. (Richmond, CA), anti-VSV-G and anti-β-COP from Sigma Chemical Co. (St. Louis, MO), anti-COXI from Molecular Probes, Inc. (Eugene, OR), anti-LAMPI from PharMingen (San Diego, CA), and anti-catalase from Calbiochem-Novabiochem Corp. (La Jolla, CA).

Expression Pattern and Subcellular Localization of Drp1

The Drp1 mRNAs in most human tissues are 2,800 and 4,800 nucleotides (Fig. ​(Fig.2)2) with the same coding sequence but different polyadenylation sites (not shown). This is different from rat, which has a dominant 4.6-kb mRNA (Yoon et al., 1998). Both of the human Drp1 mRNAs are slightly larger in brain, consistent with insertion of the alternative exon described above (Fig. ​(Fig.2,2, inset). The levels of Drp1 mRNAs are high in brain, moderate in skeletal and heart muscle, and low in other tissues (Fig. ​(Fig.2).2). Western blot analysis showed that brain Drp1 was slightly larger than nonneuronal Drp1, consistent with the insertion of an alternatively spliced exon (Fig. ​(Fig.33 A).

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Figure 2

Tissue-specific expression of human Drp1. A blot with 2 mg poly A+ RNA from each of the designated human tissues was hybridized with a radiolabeled Drp1 cDNA probe and processed for autoradiography. The mRNA sizes were estimated to be 2.8 and 4.8 kb, corresponding to the usage of different polyadenylation sites. Both mRNAs were ~100 nt longer in brain (inset shows a lighter exposure).

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Figure 3

(A) Immunoblot analysis of Drp1 in protein extracts from bovine brain, HeLa cells, and COS-7 cells, showing a prominent Drp1 band in brain. (B) Immunoblot analysis of untransfected COS-7 cells and COS-7 cells transfected with Drp1, showing overexpression of Drp1 in transfected cells. It is unclear whether the faint band in untransfected COS-7 cells represents a slightly larger splice variant of Drp1 or an unrelated protein that cross-reacts with the Drp1 antibody. (C) Indirect immunofluorescence of COS-7 cells with anti-Drp1 antibody. The brightly fluorescent cell on the right overexpresses Drp1, which indicates that this cell was a transfectant.

Endogenous Drp1 was difficult to detect by immunofluorescence. COS-7 cells that overexpress transfected Drp1 show primarily cytosolic immunofluorescence (Fig. ​(Fig.3,3, B and C). There was no detectable organelle association, even upon treatment with digitonin or streptolysin-O, which was intended to expose small amounts of Drp1 bound to organelles by washing out soluble protein (not shown). Two other studies of Drp1 immunofluorescence have also revealed cytosolic staining (Kamimoto et al., 1998; Shin et al., 1997). In a third study, a fraction of Drp1 was found to colocalize with ER, but most of the protein was cytosolic as well (Yoon et al., 1998).

Mutant Drp1 has no Effect on Secretory or Endocytic Pathways

Because of the suggestion that Drp1 might play a role in vesicular traffic (Imoto et al., 1998; Kamimoto et al., 1998; Shin et al., 1997; Yoon et al., 1998), we tested directly whether the transport function of the secretory pathway is impaired by transfecting mutant Drp1 into COS-7 cells. The K38A mutation in Drp1 is analogous to the dynamin K44A mutation that exhibits dominant interfering activity in transiently transfected cells (van der Bliek et al., 1993). We cotransfected K38A mutant Drp1 together with a temperature-sensitive mutant of VSV-G tagged with GFP (Presley et al., 1997). We monitored the progression of GFP fluorescence from ER to Golgi and onto plasma membrane. This experiment showed no difference in the transport of VSV-G through the secretory pathway of cells transfected either with mutant or with wild-type Drp1 (Fig. ​(Fig.4).4). In agreement with this finding, anti-Drp1 antibody did not affect ER vesicle formation in vitro (W.E. Balch, personal communication). We conclude that the secretory pathway was not affected by mutant Drp1.

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Figure 4

Absence of an effect of mutant Drp1 on the secretory pathway. The function of the secretory pathway was monitored by cotransfecting wild-type or mutant Drp1, together with a chimera containing VSV-G tsO45 fused to GFP (Presley et al., 1997). Overexpression of mutant or wild-type Drp1 was verified by double labeling with anti-Drp1 antibody (not shown). The VSV-G chimera was accumulated in the ER by growing transfected cells at 40°C. At 0 min the cells were transferred to the permissive temperature (32°C), allowing the VSV-G chimera to exit the ER. At 15 and 30 min, most of the label was concentrated in the Golgi, and at 60 min most of the label was transferred to the plasma membrane. There was no detectable difference between cells transfected with mutant or wild-type Drp1.

We also tested the endocytic pathway in cells that were transfected with mutant Drp1. The transfected cells were incubated with fluorescent LDL in order to assess endocytic function. Accumulation of fluorescence in lysosomes indicated successful transfer of LDL from the plasma membrane to endosomes and onto lysosomes. There was no difference between cells transfected with K38A mutant or with wild-type Drp1, while mutant dynamin clearly blocked LDL uptake (compare the transfected cells that are outlined in Fig. ​Fig.55 with adjacent untransfected cells). The distribution of fluorescently labeled transferrin, which is taken from the plasma membrane to early endosomes and then recycled back, was also no different in cells transfected with K38A mutant or wild-type Drp1 (not shown), demonstrating that endocytosis was not affected by mutant Drp1.

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Figure 5

Absence of an effect of mutant Drp1 on the endocytic pathway. The function of the endocytic pathway was monitored by following LDL uptake. Cells transfected with the indicated Drp1 or dynamin constructs were incubated at 37°C in serum-free medium with Bodipy-LDL. After 25 min the cells were washed and fixed for staining with anti-Drp1 or anti-dynamin antibody. Cells that overexpress mutant or wild-type Drp1 or dynamin were identified by double labeling with anti-Drp1 or anti-dynamin antibody as indicated by the gray outlines. LDL does not appear in the lysosomes of 70% of cells transfected with mutant dynamin (Dyn1-K44A), demonstrating the validity of this approach. There was no detectable difference in the distribution of lysosomes in cells transfected with wild-type Drp1 (Drp1-wt), the K38A mutant (Drp1-K38A), or in untransfected cells, showing that mutant Drp1 does not affect the endocytic pathway.

Transfected Drp1 Affects Mitochondria, but not Other Organelles

To test whether some other aspect of organelle function was affected by mutant Drp1, we investigated the morphology and subcellular distribution of all membrane-bounded organelles in COS-7 cells transfected with K38A mutant Drp1. The ER network was detected by immunostaining of cotransfected VSV-G to which we had attached an ER-retention signal (KKTN at the COOH terminus). Expression of Drp1 containing the K38A mutation had no visible effect on the reticulum (Fig. ​(Fig.6,6, compare 1 and 2). The ER bud sites of COPII-coated vesicles that appear clustered near the Golgi, were detected with anti-sec13 antibody (Bannykh et al., 1996). Their distribution was unaltered by Drp1 containing the K38A mutation (compare the transfected cells, which are outlined in of Fig. ​Fig.6,6, 3 and 4 with adjacent untransfected cells). The bud sites of COPI-coated vesicles, which were detected with anti-β-COP antibody, were localized to the Golgi. Their distribution was also unchanged by mutant Drp1 (Fig. ​(Fig.6,6, 5 and 6). Lysosomes, which were detected with anti-LAMP1 antibody, were also not affected (Fig. ​(Fig.6,6, 7 and 8), nor were early endosomes, detected by cotransfecting epitope-tagged rab5 (not shown) and peroxisomes, detected with anti-catalase antibody (not shown).

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Figure 6

Absence of an effect of dominant negative mutant Drp1 on organelles other than mitochondria. Cells that overexpress mutant or wild-type Drp1 were identified by double labeling with anti-Drp1 antibody as indicated by the gray outlines. The ER was detected with an anti-VSV–G antibody by staining cells that were cotransfected with Drp1 and VSV-G carrying an ER retention signal (KKTN at the COOH terminus). The inset shows that the reticulum extends to the cell periphery, both in cells transfected with wild-type (Drp1-wt) and with mutant (Drp1-K38A). COPII-coated vesicles which bud from the ER were detected with anti-sec13 antibody. COPI-coated vesicles, which bud from the Golgi, were detected with anti-β-COP antibody. Lysosomes were detected with anti-LAMP1 antibody. Endosomes detected by cotransfecting epitope-tagged rab5 and peroxisomes detected with anti-catalase antibody were also not affected (not shown).

We then tested whether Drp1 affects mitochondrial morphology by transfecting mutant Drp1 and staining with an antibody specific for mitochondria. In untransfected cells and in cells transfected with wild-type Drp1, the mitochondria are organized in a perinuclear array with tubular extensions that often reach to the cell periphery (compare the transfected cells, which are outlined in Fig. ​Fig.7,7, 1 and 2, with adjacent untransfected cells). When we transfected mutant Drp1, the mitochondrial network invariably collapsed into large perinuclear aggregates (Fig. ​(Fig.7).7). Occasionally, a few long tubules were retained, but even in those cells mitochondrial collapse was readily apparent (in Fig. ​Fig.7,7, 5 and 6′). The effect on mitochondrial morphology was the same with the nonneuronal isoform of Drp1 containing the K38A mutation (Fig. ​(Fig.7,7, 5 and 5′) and with the brain-specific isoform of Drp1 containing the K38A mutation (Fig. ​(Fig.7,7, 6 and 6′). In contrast to mutant Drp1, neither mutant nor wild-type forms of dynamin I altered the morphology of the mitochondrial network (Fig. ​(Fig.7,7, 3 and 4), which shows that the observed mitochondrial defect is specifically associated with overexpression of mutant Drp1.

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Figure 7

Effect of Drp1 on mitochondrial morphology. (A) COS-7 cells transfected with wild-type Drp1, wild-type dynamin, or mutant dynamin all show normal mitochondrial morphology. The cells were transfected with Drp1-wt (the nonneuronal isoform; 1), B-Drp1-wt (the brain-specific isoform of Drp1; 2), Dyn1-wt (dynamin I wild-type; 3) or Dyn1-K44A (dynamin I K44A, which blocks endocytosis [van der Bliek et al., 1993]; 4). The cells were double-labeled with a mitochondrial antibody (anti-COXI) and either anti-Drp1 or anti-dynamin antibody. Outlines of cells that are overexpressing Drp1 or dynamin are superimposed on the mitochondrial staining. Untransfected cells were not outlined. (B) COS-7 cells transfected with mutant Drp1 show collapsed mitochondria. Two examples of each condition are shown. The following constructs were tested: Drp1-K38A (the K38A mutant of the non-neuronal isoform; 5 and 5′), and B-Drp1-K38A (the K38A mutant of the brain specific isoform; 6 and 6′).

We thank the other lab members, G.S. Payne, I.V. Davydov, and P.J. Johnson for valuable suggestions and comments on the manuscript. We thank J. Shaw (University of Utah, Salt Lake City, UT) for sharing unpublished information and for many stimulating discussions. We thank J. Chen and M. Kang for assistance with cDNA cloning and sequence analysis. We thank W.E. Balch (Scripps Research Institute, La Jolla, CA) for testing our anti-Drp1 antibodies in an ER-budding assay, for anti-sec13 antibodies, and for VSV-G plasmid. We thank J.F. Presley and J. Lippincott-Schwartz (National Institutes of Health, Bethesda, MD) for the VSV-G/GFP plasmid and advice on performing the VSV-G transport assay.

This work was supported by grants from the National Institutes of Health (GM 51866), the American Heart Association, and the Cancer Research Coordinating Committee to A.M. van der Bliek. E. Smirnova received fellowships from the Myasthenia Gravis Foundation and the American Heart Association of Greater Los Angeles.