Mitotic Phosphorylation of Dynamin-related GTPase Drp1 Participates in Mitochondrial Fission

Organelles are inherited to daughter cells beyond dynamic changes of the membrane structure during mitosis. Mitochondria are dynamic entities, frequently dividing and fusing with each other, during which dynamin-related GTPase Drp1 is required for the fission reaction. In this study, we analyzed mitochondrial dynamics in mitotic mammalian cells. Although mitochondria in interphase HeLa cells are long tubular network structures, they are fragmented in early mitotic phase, and the filamentous network structures are subsequently reformed in the daughter cells. In marked contrast, tubular mitochondrial structures are maintained during mitosis in Drp1 knockdown cells, indicating that the mitochondrial fragmentation in mitosis requires mitochondrial fission by Drp1. Drp1 was specifically phosphorylated in mitosis by Cdk1/cyclin B on Ser-585. Exogenous expression of unphosphorylated mutant Drp1S585A led to reduced mitotic mitochondrial fragmentation. These results suggest that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells. Organelles are inherited to daughter cells beyond dynamic changes of the membrane structure during mitosis. Mitochondria are dynamic entities, frequently dividing and fusing with each other, during which dynamin-related GTPase Drp1 is required for the fission reaction. In this study, we analyzed mitochondrial dynamics in mitotic mammalian cells. Although mitochondria in interphase HeLa cells are long tubular network structures, they are fragmented in early mitotic phase, and the filamentous network structures are subsequently reformed in the daughter cells. In marked contrast, tubular mitochondrial structures are maintained during mitosis in Drp1 knockdown cells, indicating that the mitochondrial fragmentation in mitosis requires mitochondrial fission by Drp1. Drp1 was specifically phosphorylated in mitosis by Cdk1/cyclin B on Ser-585. Exogenous expression of unphosphorylated mutant Drp1S585A led to reduced mitotic mitochondrial fragmentation. These results suggest that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells. Mitochondria are double membrane organelles changing their size and position in their dynamic movement (1Yaffe M.P. Science. 1999; 283: 1493-1497Crossref PubMed Scopus (417) Google Scholar, 2Griparic L. van der Bliek A.M. Traffic. 2001; 2: 235-244Crossref PubMed Scopus (114) Google Scholar, 3Mozdy A.D. Shaw J.M. Nat. Rev. Mol. Cell Biol. 2003; 4: 468-478Crossref PubMed Scopus (105) Google Scholar, 4Westermann B. Biochim. Biophys. Acta. 2003; 1641: 195-202Crossref PubMed Scopus (44) Google Scholar, 5Chen H. Chan D.C. Curr. Top. Dev. Biol. 2004; 59: 119-144Crossref PubMed Scopus (141) Google Scholar, 6Okamoto K. Shaw J.M. Annu. Rev. Genet. 2005; 39: 503-536Crossref PubMed Scopus (594) Google Scholar). Mitochondrial morphology varies in response to the environment and cellular differentiation; mitochondria form connected and filamentous network structures in most fibroblasts, are arranged along the myofibrils in skeletal muscle, and are coiled around the flagella in sperm. Mitochondrial morphology is maintained in a dynamic balance between fusion and fission (7Nunnari J. Marshall W.F. Straight A. Murray A. Sedat J.W. Walter P. Mol. Biol. Cell. 1997; 8: 1233-1242Crossref PubMed Scopus (402) Google Scholar, 8Bleazard W. McCaffery J.M. King E.J. Bale S. Mozdy A. Tieu Q. Nunnari J. Shaw J.M. Nat. Cell Biol. 1999; 1: 298-304Crossref PubMed Scopus (592) Google Scholar, 9Sesaki H. Jensen R.E. J. Cell Biol. 1999; 147: 699-706Crossref PubMed Scopus (431) Google Scholar, 10Ishihara N. Jofuku A. Eura Y. Mihara K. Biochem. Biophys. Res. Commun. 2003; 301: 891-898Crossref PubMed Scopus (224) Google Scholar). Mitofusin (Mfn, in mammals) and Fzo1 (in yeast) proteins are mitochondrial outer membrane GTPases that are essential for mitochondrial fusion (11Hales K.G. Fuller M.T. Cell. 1997; 90: 121-129Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 12Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (426) Google Scholar, 13Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1783) Google Scholar, 14Eura Y. Ishihara N. Yokota S. Mihara K. J. Biochem. (Tokyo). 2003; 134: 333-344Crossref PubMed Scopus (310) Google Scholar). The dynamin-related GTPase OPA1 and a yeast homolog Mgm1, localized in the mitochondrial intermembrane space, are also required for fusion (15Shepard K.A. Yaffe M.P. J. Cell Biol. 1999; 144: 711-720Crossref PubMed Scopus (147) Google Scholar, 16Wong E.D. Wagner J.A. Gorsich S.W. McCaffery J.M. Shaw J.M. 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The fusion proteins are also crucial for neuronal development, because Mfn2 and OPA1 are causal gene products of neuropathy, such as Charcot Marie tooth neuropathy type 2A and dominant optic atrophy type I, respectively (18Alexander C. Votruba M. Pesch U.E. Thiselton D.L. Mayer S. Moore A. Rodriguez M. Kellner U. Leo-Kottler B. Auburger G. Bhattacharya S.S. Wissinger B. Nat. Genet. 2000; 26: 211-215Crossref PubMed Scopus (1056) Google Scholar, 19Delettre C. Lenaers G. Griffoin J.M. Gigarel N. Lorenzo C. Belenguer P. Pelloquin L. Grosgeorge J. Turc-Carel C. Perret E. Astarie-Dequeker C. Lasquellec L. Arnaud B. Ducommun B. Kaplan J. Hamel C.P. Nat. Genet. 2000; 26: 207-210Crossref PubMed Scopus (1149) Google Scholar, 20Zuchner S. Mersiyanova I.V. Muglia M. Bissar-Tadmouri N. Rochelle J. Dadali E.L. Zappia M. Nelis E. Patitucci A. Senderek J. Parman Y. Evgrafov O. Jonghe P.D. Takahashi Y. Tsuji S. Pericak-Vance M.A. Quattrone A. Battaloglu E. Polyakov A.V. Timmerman V. Schroder J.M. Vance J.M. Nat. Genet. 2004; 36: 449-451Crossref PubMed Scopus (1233) Google Scholar). Other dynamin-related proteins, Drp1 (mammals) and Dnm1 (yeast), participate in mitochondrial fission, and their mutation leads to highly elongated filamentous mitochondria structures (8Bleazard W. McCaffery J.M. King E.J. Bale S. Mozdy A. Tieu Q. Nunnari J. Shaw J.M. Nat. Cell Biol. 1999; 1: 298-304Crossref PubMed Scopus (592) Google Scholar, 21Smirnova E. Shurland D.L. Ryazantsev S.N. van der Bliek A.M. J. Cell Biol. 1998; 143: 351-358Crossref PubMed Scopus (578) Google Scholar, 22Otsuga D. Keegan B.R. Brisch E. Thatcher J.W. Hermann G.J. Bleazard W. Shaw J.M. J. Cell Biol. 1998; 143: 333-349Crossref PubMed Scopus (332) Google Scholar). The partner proteins, mitochondrial outer membrane Fis1 and peripheral Mdv1 and Caf4, interact with Drp1/Dnm1 on the mitochondrial surface, although the homologs of WD motif proteins Mdv1 and Caf4 have not been found in mammalian cells (23Yoon Y. Krueger E.W. Oswald B.J. McNiven M.A. Mol. Cell Biol. 2003; 23: 5409-5420Crossref PubMed Scopus (638) Google Scholar, 24Stojanovski D. Koutsopoulos O.S. Okamoto K. Ryan M.T. J. Cell Sci. 2004; 117: 1201-1210Crossref PubMed Scopus (259) Google Scholar, 25Griffin E.E. Graumann J. Chan D.C. J. Cell Biol. 2005; 170: 237-248Crossref PubMed Scopus (200) Google Scholar, 26Jofuku A. Ishihara N. Mihara K. Biochem. Biophys. Res. Commun. 2005; 333: 650-659Crossref PubMed Scopus (63) Google Scholar, 27Karren M.A. Coonrod E.M. Anderson T.K. Shaw J.M. J. Cell Biol. 2005; 171: 291-301Crossref PubMed Scopus (70) Google Scholar, 28Naylor K. Ingerman E. Okreglak V. Marino M. Hinshaw J.E. Nunnari J. J. Biol. Chem. 2006; 281: 2177-2183Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Dissipation of the mitochondrial membrane potential and apoptotic stimuli induce mitochondrial fragmentation by inhibiting fusion or stimulating fission (10Ishihara N. Jofuku A. Eura Y. Mihara K. Biochem. Biophys. Res. Commun. 2003; 301: 891-898Crossref PubMed Scopus (224) Google Scholar, 29Lee Y.J. Jeong S.Y. Karbowski M. Smith C.L. Youle R.J. Mol. Biol. Cell. 2004; 15: 5001-5011Crossref PubMed Scopus (851) Google Scholar). The molecular mechanisms of the regulation of mitochondrial fusion and fission, however, are not well understood. Mitochondria proliferate by growth and division of pre-existing mitochondria (30Posakony J.W. England J.M. Attardi G. J. Cell Biol. 1977; 74: 468-491Crossref PubMed Scopus (136) Google Scholar). The inheritance of mitochondria is well investigated in budding yeast by genetic and morphologic studies (6Okamoto K. Shaw J.M. Annu. Rev. Genet. 2005; 39: 503-536Crossref PubMed Scopus (594) Google Scholar, 31Boldogh I.R. Yang H.C. Pon L.A. Traffic. 2001; 2: 368-374Crossref PubMed Scopus (48) Google Scholar). Yeast mitochondria form a network of interconnected tubules along actin filaments, and mitochondrial tubules are transported into the growing daughter buds (31Boldogh I.R. Yang H.C. Pon L.A. Traffic. 2001; 2: 368-374Crossref PubMed Scopus (48) Google Scholar). The mitochondrial movement depends on actin cables, class V myosin, Rab family GTPase Ypt11, and mitochondrial surface protein Mmr1 (31Boldogh I.R. Yang H.C. Pon L.A. Traffic. 2001; 2: 368-374Crossref PubMed Scopus (48) Google Scholar, 32Itoh T. Toh-e A. Matsui Y. EMBO J. 2004; 23: 2520-2530Crossref PubMed Scopus (89) Google Scholar). Mmm1, Mdm10, and Mdm12 were identified from the mitochondrial inheritance mutants, and they form a complex on the mitochondrial outer membrane (6Okamoto K. Shaw J.M. Annu. Rev. Genet. 2005; 39: 503-536Crossref PubMed Scopus (594) Google Scholar). Mitochondrial filamentous structures are maintained through the mitotic stages, and long mitochondria are transported to the daughter buds. None of the known mitochondrial fission factors, Dnm1, Fis1, Mdv1, nor Caf4, is essential for mitochondrial inheritance or cell viability (6Okamoto K. Shaw J.M. Annu. Rev. Genet. 2005; 39: 503-536Crossref PubMed Scopus (594) Google Scholar, 8Bleazard W. McCaffery J.M. King E.J. Bale S. Mozdy A. Tieu Q. Nunnari J. Shaw J.M. Nat. Cell Biol. 1999; 1: 298-304Crossref PubMed Scopus (592) Google Scholar, 22Otsuga D. Keegan B.R. Brisch E. Thatcher J.W. Hermann G.J. Bleazard W. Shaw J.M. J. Cell Biol. 1998; 143: 333-349Crossref PubMed Scopus (332) Google Scholar, 25Griffin E.E. Graumann J. Chan D.C. J. Cell Biol. 2005; 170: 237-248Crossref PubMed Scopus (200) Google Scholar). On the other hand, mitochondrial fragmentation is observed at specific stages in meiosis and sporulation (33Gorsich S.W. Shaw J.M. Mol. Biol. Cell. 2004; 15: 4369-4381Crossref PubMed Scopus (70) Google Scholar). Mutant cells deficient in mitochondrial fission affect the uniform distribution of mitochondria into spores, which results in an increased number of inviable spores, although mitochondrial fission is not required for spore formation itself. It is less clear, however, how the interconnected mitochondrial network structures are inherited to mammalian daughter cells. In this study, we analyzed mitochondrial dynamics and inheritance in mitotic mammalian cells. The interconnected mitochondrial network structures in interphase HeLa cells became fragmented in the early mitotic phase and stochastically segregated into two daughter cells. Finally, the filamentous mitochondria reformed in the daughter cells. The mitotic fragmentation required the mitochondrial fission factor Drp1. Furthermore, Drp1 was specifically phosphorylated by mitosis-promoting factor (MPF, Cdk1/cyclin B), which stimulated mitotic mitochondrial fragmentation. The results demonstrated that mitochondrial morphology is regulated by Drp1-dependent mitochondrial fission in mitotic cells. Materials—Monoclonal antibodies against FLAG (M2, Sigma), Drp1 (D80320, Transduction Laboratories, San Jose, CA), and cyclin B1 (sc-245, Santa Cruz Biotechnology, Santa Cruz, CA) were purchased from the indicated companies. Rabbit polyclonal antibodies against phosphorylated Drp1 were prepared using phosphorylated synthetic peptide IPIM-PASPQKGHAVC (the underlined Ser residue was phosphorylated). Purified Cdk1/cyclin B was purchased from New England Biochemical (Beverly, MA) (P6020S). Calf alkaline phosphatase was purchased from New England Biochemical (M0290S). Protein kinase A inhibitor was purchased from Sigma (P5990). Plasmids—The mammalian su9-RFP expression plasmid has been described previously (10Ishihara N. Jofuku A. Eura Y. Mihara K. Biochem. Biophys. Res. Commun. 2003; 301: 891-898Crossref PubMed Scopus (224) Google Scholar). The YFP 2The abbreviations used are: YFP, yellow fluorescent protein; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; RNAi, RNA interference; siRNA, small interfering RNA; DAPI, 4′,6-diamidino-2-phenylindole; wt, wild type. -α-tubulin expression plasmid was a generous gift from K. Ohashi and K. Mizuno, Tohoku University. The cDNA (encoding 705 amino acid residues) of rat Drp1 (10Ishihara N. Jofuku A. Eura Y. Mihara K. Biochem. Biophys. Res. Commun. 2003; 301: 891-898Crossref PubMed Scopus (224) Google Scholar) was subcloned into pEGFP-N1 (for Drp1-GFP), p3xFLAG-CMV-10 (for FLAG-Drp1), or pET28a (for His-Drp1), respectively. All rat Drp1 mutants were prepared by PCR. Cell Culture, Synchronization, Transfection, and Analysis—HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum under 5% CO2 at 37 °C. DNA transfection was performed using Lipofectamine (Invitrogen) as recommended by the manufacturer. The cells were fixed with 4% paraformaldehyde for fluorescence microscopy. For live cell imaging, HeLa cells cultured in a glass-bottomed dish at 37 °C were analyzed by a model IX81 fluorescence microscope (Olympus, Tokyo, Japan) with a cooled charge-coupled device camera (Roper Scientific, Tokyo, Japan). For visualizing mitotic cells, HeLa cells were treated with 2.5 mm thymidine for 16-24 h and released from G1/S phase in DMEM for 2-4 h (first thymidine). The cells were transfected with plasmids by Lipofectamine, treated with thymidine again for 16-24 h, and then cultured in DMEM for 12-18 h for analyzing mitotic cells. For preparing mitotic cell lysates, cells were treated with 2.5 mm thymidine for 16-24 h, released from G1/S phase in DMEM for 8 h, and then treated with 0.2 μm nocodazole for 6-8 h. Morphometric Analysis of Mitochondrial Morphology—Synchronized mitotic HeLa cells as described above were fixed and stained. Serial z-sectioned images (Fig. 1B from top to bottom) of the cells were obtained by confocal microscopy (Radiance 2000, Bio-Rad). The images of serial sections within the top one-fourth (top or peripheral), middle one-fourth (middle), or bottom one-fourth (bottom) of the cell depth were combined to reconstruct respective projection images (Figs. 1B, 2D, and 5E). Mitochondria in the top fraction were traced, and the size distribution (in μm; 0-1, 1-2, 2-3, 3-4, 4-5, and longer than 5) was quantified using Metamorph software (Roper) and shown as a percentage (Figs. 2E and 5F).FIGURE 2Inactivation of Drp1 induces morphologic changes of the mitochondria in the mitotic phase. A, knockdown of endogenous Drp1. siRNA specific for Drp1 or GFP (control) was transfected to HeLa cells. Total cell extracts were subjected to SDS-PAGE and subsequent immunoblot analysis using the indicated antibodies. TOM40, a mitochondrial protein as a loading marker. B, GFP (control) or Drp1-specific siRNA was transfected to HeLa cells and synchronized to M phase as described in the legend to Fig. 1A. The percentage of cells with the indicated mitochondrial morphology was counted as described in the legend to Fig. 1A. Control RNAi (gray bar) and Drp1 RNAi (black bar) cells in each stage (n > 100 of three independent experiments) were counted. C, Drp1 siRNA was transfected to HeLa cells, and mitochondrial morphology in each stage was analyzed by fluorescence microscopy as described in the legend to Fig. 1A. D, GFP (control) or Drp1-specific siRNA was transfected to HeLa cells, which were synchronized to the M phase and analyzed by confocal microscopy as in Fig. 1B. Compiled confocal images in cell periphery fractions are shown. E, quantification of mitochondrial fragmentation in the mitotic cells. GFP (control) or Drp1 siRNA was transfected to HeLa cells, which were synchronized to the M phase and analyzed by confocal microscopy as described for D. The mitochondrial length in peripheral fractions was analyzed by Metamorph software as described under “Experimental Procedures.” Control RNAi is as follows: interphase, n = 1070 (4 cells); prophase, n = 696 (4 cells); metaphase, n = 461 (3 cells); anaphase, n = 952 (8 cells); telophase, n = 542 (3 cells). Drp1 RNAi is as follows: interphase, n = 1156 (11 cells); prophase, n = 696 (4 cells); metaphase, n = 333 (4 cells); anaphase, n = 597 (6 cells); telophase, n = 525 (5 cells).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Phosphorylated Drp1 stimulates mitochondrial fission in mitosis. A and B, HeLa cells expressing FLAG-tagged Drp1wt or Drp1S585A were synchronized to the mitotic phase, and mitochondrial morphology was analyzed by fluorescence microscopy as described for Fig. 2B. n > 100 of three independent experiments. C and D, endogenously expressed Drp1 was repressed by RNAi as in Fig. 2, and then FLAG-tagged Drp1wt or Drp1S585A was expressed in the Drp1-RNAi cells. Mitochondrial morphology in interphase cells, early mitotic cells (containing prophase and metaphase), or late mitotic cells (containing anaphase, telophase, and cytokinesis phase) was analyzed by fluorescence microscopy (n > 30 of three independent experiments). Percentage of the cells with dominantly tubular mitochondria (tubular) and with completely fragmented mitochondria (fragmented) in each phase is shown. E, FLAG-tagged Drp1wt or Drp1S585A was expressed in the Drp1-RNAi cells and analyzed by confocal microscopy as described for Fig. 1B. Mitochondrial morphology in the cell periphery fraction of compiled confocal sections is shown. F, mitochondrial length was quantified as described for Fig. 2E. Drp1wt-expressing cells are as follows: interphase, n = 962 (4 cells); early mitotic phase, n = 911 (4 cells); late mitotic cells, n = 471 (3 cells); Drp1S585A-expressing cells are as follows: interphase, n = 974 (4 cells); early mitotic phase, n = 993 (6 cells); late mitotic phase, n = 503 (3 cells).View Large Image Figure ViewerDownload Hi-res image Download (PPT) RNA Interference (RNAi)—For human Drp1 RNAi, 27-base nucleotides were chemically synthesized (5′-ACUAUUGAAGGAACUGCAAAAUAUA-dAdG-3′ and 5′-UAUAUUUUGCAGUUCCUUCAAUAGU-dAdT-3′). The annealed small interfering RNA (siRNA) was transfected to HeLa cells three times using Oligofectamine (Invitrogen) as described previously (26Jofuku A. Ishihara N. Mihara K. Biochem. Biophys. Res. Commun. 2005; 333: 650-659Crossref PubMed Scopus (63) Google Scholar). Note that the target sequence is specific for human Drp1 but not for rat Drp1. Protein Kinase Assay—Recombinant His-Drp1 proteins were expressed and purified by metal-chelating resin and further purified by ion exchange chromatography using Q-Fast Flow (Pharmacia, Uppsala, Sweden). The in vitro protein kinase reaction was analyzed as reported previously (34Nishijima H. Nishitani H. Seki T. Nishimoto T. J. Cell Biol. 1997; 138: 1105-1116Crossref PubMed Scopus (83) Google Scholar). Cdk1/cyclin B was isolated from mitotic HeLa cells as follows. Synchronized mitotic HeLa cells were solubilized by lysis buffer (40 mm Hepes-KOH buffer, pH 7.4, containing 60 mm β-glycerophosphate, 20 mm p-nitrophenylphosphate, 0.5 mm Na3VO4, 250 mm NaCl, 15 mm MgCl2, 1% Triton X-100, 5 mm dithiothreitol, and protease inhibitor mixture), and the lysate was incubated with anti-cyclin B1 and protein G-Sepharose, washed in lysis buffer, and then suspended in protein kinase buffer (20 mm Hepes-KOH buffer, pH 7.4, 15 mm EGTA, and 20 mm MgCl2). Purified His-Drp1 was incubated in protein kinase buffer containing 5 ng/ml protein kinase A inhibitor, 1 μm dithiothreitol, 50 μm ATP, and [γ-32P]ATP with purified or immunoisolated Cdk1/cyclin B for 30-120 min at 30 °C. The samples were analyzed by SDS-PAGE and subsequent Coomassie Brilliant Blue staining and autoradiography. Mitochondrial Dynamics in Mitotic HeLa Cells—Mitochondrial morphology is maintained under dynamic movement with a balance between fusion and fission. In most cultured fibroblasts, filamentous mitochondria form network structures in the cytoplasm. It is not well understood, however, how mammalian daughter cells inherit the interconnected mitochondria. Here we observed mitochondrial dynamics in mitotic HeLa cells (Fig. 1). The HeLa cells were synchronized at the G1/S phase using the double thymidine block procedure and subsequently released into thymidine-free medium to restart the cell cycle progression. Mitochondrial morphology was visualized using mitochondria-targeted DsRed (su9-RFP) in which mitochondrial-targeting sequence (1-69 residue segment) of Neurospora crassa FoF1-ATPase subunit 9 was fused to the N-terminal end of DsRed. First, we observed live images of mitochondrial dynamics in dividing cells using time-lapse video microscopy (supplemental Fig. S1A). The filamentous network structures of the mitochondria in the interphase cells changed to smaller fragmented structures in the early mitotic phase (supplemental Fig. S1A, a and b). Restoration of the filamentous structures began in the late mitotic phase (supplemental Fig. S1A, g and h), and the mitochondria transmitted to the daughter cells regained normal filamentous network structures. For further detailed analysis of the mitotic stages, the synchronized cells were fixed and counter-stained with DAPI (Fig. 1A). The filamentous network structures of the mitochondria (tubular) were observed in most of the interphase cells (75% of counted cells) (Fig. 1A, p). From prophase to anaphase (Fig. 1A, q-s), <20% of the cells had filamentous mitochondria, and the number of cells with predominantly fragmented mitochondria clearly increased (∼30%). About half of the early mitotic cells had shorter mitochondria compared with most of the mitochondria in the interphase cells (Fig. 1A, intermediate). In the late stages of mitosis (telophase and cytokinesis phases), there were more cells with tubular mitochondria (∼50%) (Fig. 1A, t). These results indicated that mitochondrial morphology was drastically changed in HeLa cells, and fragmentation of mitochondria occurred during the early mitotic phase. Thereafter, we analyzed statistically the size distribution of mitochondria in the mitotic cells. For this purpose, serial z-section images were obtained (from top to bottom) of the cells by confocal microscopy (supplemental Movies 1-5). The section images within the top one-fourth, middle one-fourth, or bottom one-fourth of the cell depth were compiled (top, middle, and bottom, respectively) and projected (Fig. 1B). In metaphase and anaphase, mitochondria were almost completely fragmented (morphometric quantification shown in Fig. 2E); images of the bottom fractions clearly show mitochondrial fragmentation (Fig. 1B), thus providing further support for the above conclusion. Mitochondrial Fragmentation in Mitotic Cells Depends on Drp1—Next, we focused on the function of the mitochondrial fission factor Drp1 in mitotic mitochondrial fragmentation (Fig. 2). The expression of dominant negative mutant Drp1K38A inhibited mitochondrial fission and resulted in mitochondrial elongation (10Ishihara N. Jofuku A. Eura Y. Mihara K. Biochem. Biophys. Res. Commun. 2003; 301: 891-898Crossref PubMed Scopus (224) Google Scholar, 21Smirnova E. Shurland D.L. Ryazantsev S.N. van der Bliek A.M. J. Cell Biol. 1998; 143: 351-358Crossref PubMed Scopus (578) Google Scholar). In live images, filamentous mitochondrial structures were maintained throughout mitosis in Drp1K38A-expressing cells (supplemental Fig. S1B, a-h), suggesting that Drp1 activity was crucial for mitochondrial fragmentation during mitosis. In these cells, the mitochondria accumulated near the pole region but were still transmitted to the daughter cells (supplemental Fig. S1B, b-h). We further examined the effect of Drp1 knockdown using specific siRNA (Fig. 2). In control RNAi cells, mitochondria were fragmented in the early mitotic phase (∼50%) (Fig. 2B and supplemental Fig. 1C, q and r), as in Fig. 1. In contrast, the elongated mitochondrial structure was maintained throughout mitosis in Drp1 knockdown cells (∼80%) (Fig. 2, B and C, q and r). These results suggest that, in mitosis, mitochondria are fragmented by a Drp1-dependent fission reaction. For statistic analysis of mitochondrial fragmentation, the length of mitochondria in mitotic cells was measured morphometrically as shown in Fig. 1B. Typical compiled images of the peripheral fraction are shown in Fig. 2D, and quantified data are shown in Fig. 2E. In control RNAi cells, the number of mitochondria <1 μm (∼5% in interphase) clearly increased in mitosis (∼30% in prophase and metaphase), and oppositely, the number of mitochondria longer than 4 μm (∼30% in interphase) markedly decreased in the prophase and metaphase. On the other hand, in Drp1-RNAi cells, the formation of small mitochondria <1 μm in the early mitotic phase was clearly impaired (∼10% in the prophase and metaphase). These results clearly indicated that Drp1 stimulates mitochondrial fission in the early mitotic phase. Drp1 Is Mitotically Phosphorylated by Cdk1/cyclin B—The antibody against Drp1 recognized at least three bands due to alternative splicing (Fig. 3A) (21Smirnova E. Shurland D.L. Ryazantsev S.N. van der Bliek A.M. J. Cell Biol. 1998; 143: 351-358Crossref PubMed Scopus (578) Google Scholar). Here, these Drp1 bands in mitotic cells had lower gel mobility compared with those from G1/S cells (Fig. 3A). Exogenously expressed FLAG-tagged Drp1, detected as a single band in G1/S cells, was also shifted upward in mitotic cells (Fig. 4B, wt). All three bands of endogenous Drp1 proteins immunoisolated from mitotic cells were shifted down by treatment with alkaline phosphatase (Fig. 3A), suggesting that Drp1 is phosphorylated in mitosis.FIGURE 4Ser-585 of rat Drp1 is phosphorylated in the mitotic phase. A, schematic drawing of rat Drp1 used in this study. Rat Drp1 has four potential Cdk1/cyclin B recognition sites. Rabbit polyclonal antibodies against phosphorylated Drp1 at Ser-585 (anti-p-Drp1) were prepared using phosphorylated synthetic peptide (IPIMPAS*PQKGHAV-C, Ser residue was phosphorylated (S*) and added with Cys residue to the C terminus; dotted underline). B, FLAG-tagged Drp1wt, Drp1S71A, Drp1S126A, Drp1S136A, or Drp1S585A was transfected in HeLa cells. These cells were synchronized in the G1/S or M phase, and total cell extracts were analyzed by immunoblotting using anti-FLAG antibodies. C, purified recombinant Drp1wt, Drp1S71A, Drp1S126A, Drp1S136A, and Drp1S585A were analyzed by in vitro protein kinase assay using immunoisolated Cdk1/cyclin B. D, in vitro protein kinase assay was performed as described for Fig. 3B. Purified recombinant His-Drp1wt, His-Drp1S585A, or bovine serum albumin (BSA) was used as the substrate and immunoisolated Cdk1/cyclin B as the enzyme. CBB, Coomassie Brilliant Blue. E, protein kinase reactions were performed as described for Fig. 3C using purified recombinant His-Drp1wt and His-Drp1S585A as substrates and recombinant Cdk1/cyclin B as the enzyme. F, left, HeLa cells were synchronized to the indicated phase and immunoprecipitated using anti-Drp1 antibodies. Immunoprecipitated proteins were analyzed with immunoblotting using anti-phosphorylated Drp1 (top panel) or anti-Drp1 (bottom panel). Right, FLAG-tagged Drp1wt or FLAG-tagged Drp1S585A was expressed in HeLa cells. Whole cell extracts from synchronized cells were immunoprecipitated using anti-FLAG antibodies. These samples were analyzed by immunoblotting using anti-phosphorylated Drp1 peptide (top panel) or anti-Drp1 (bottom panel) antibodies. G, phosphorylated Drp1 as described for F was quantified by LAS3000. Shown are the results of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the protein kinase responsible for Drp1 phosphorylation, we used an in vitro phosphorylation assay (Fig. 3B). Cdk1/cyclin B (MPF) was immunoisolated from the mitotic HeLa cells using antibodies against cyclin B (34Nishijima H. Nishitani H. Seki T. Nishimoto T. J. Cell Biol. 1997; 138: 1105-1116Crossref PubMed Scopus (83) Google Scholar), and we confirmed that the isolated Cdk1/cyclin B had histone H1 kinase activity (data not shown). We used the immunoisolated Cdk1/cyclin B for phosphorylation of recombinant Drp1 with [γ