Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M607279200 on February 16, 2007 Originally published In Press as doi:10.1074/jbc.M607279200 on February 14, 2007

J. Biol. Chem., Vol. 282, Issue 15, 11521-11529, April 13, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/15/11521    most recent
M607279200v2
M607279200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Taguchi, N.
Right arrow Articles by Mihara, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Taguchi, N.
Right arrow Articles by Mihara, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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

Naoko Taguchi, Naotada Ishihara, Akihiro Jofuku, Toshihiko Oka, and Katsuyoshi Mihara1

From the Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan

Received for publication, August 1, 2006 , and in revised form, January 9, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondria are double membrane organelles changing their size and position in their dynamic movement (1-6). 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 (7-10). Mitofusin (Mfn, in mammals) and Fzo1 (in yeast) proteins are mitochondrial outer membrane GTPases that are essential for mitochondrial fusion (11-14). The dynamin-related GTPase OPA1 and a yeast homolog Mgm1, localized in the mitochondrial intermembrane space, are also required for fusion (15-17). These mitochondrial fusion proteins have important roles in mitochondrial function, as respiration is defective in fzo1-or mgm1-mutated yeast cells and Mfn- or OPA1-deficient mouse embryonic fibroblasts (12, 15, 17). 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 (18-20). Other dynamin-related proteins, Drp1 (mammals) and Dnm1 (yeast), participate in mitochondrial fission, and their mutation leads to highly elongated filamentous mitochondria structures (8, 21, 22). 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 (23-28). Dissipation of the mitochondrial membrane potential and apoptotic stimuli induce mitochondrial fragmentation by inhibiting fusion or stimulating fission (10, 29). 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 (30). The inheritance of mitochondria is well investigated in budding yeast by genetic and morphologic studies (6, 31). Yeast mitochondria form a network of interconnected tubules along actin filaments, and mitochondrial tubules are transported into the growing daughter buds (31). The mitochondrial movement depends on actin cables, class V myosin, Rab family GTPase Ypt11, and mitochondrial surface protein Mmr1 (31, 32). Mmm1, Mdm10, and Mdm12 were identified from the mitochondrial inheritance mutants, and they form a complex on the mitochondrial outer membrane (6). 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 (6, 8, 22, 25). On the other hand, mitochondrial fragmentation is observed at specific stages in meiosis and sporulation (33). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Monoclonal antibodies against FLAG (M2, Sigma), Drp1 (D80320 [GenBank] , 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 (10). The YFP2-{alpha}-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 (10) 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).

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 (26). 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 (34). 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 beta-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 [{gamma}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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.


Figure 1
View larger version (68K):
[in this window]
[in a new window]

  		FIGURE 1.
Mitochondrial morphology in mitotic cells. A, HeLa cells transfected with su9-RFP (a-e and k-t) were synchronized in mitosis using thymidine as described under "Experimental Procedures." After fixation, nuclei were stained by DAPI (f-j and k-o) and analyzed by fluorescence microscopy. Mitochondria, nucleus, and merged images are shown in each stage of the cell cycle. The boxed regions in k-o are magnified (p-t). The number of cells with tubular mitochondria (tubular), intermediate mitochondrial structures (intermediate), or fragmented mitochondria (fragmented) was counted. The number of cells with the indicated mitochondrial morphology is shown as a percentage (bottom table). More than 100 cells of three independent experiments in each stage were counted. B, mitotic cells were prepared as in described for A and analyzed by confocal microscopy. Top (a-e), middle (f-j), and bottom (k-o) fractions of the compiled confocal section images are shown. The boxed regions in k-o are magnified (p-t). Note that movie files of three-dimensional images are available in the supplemental data. See "Experimental Procedures" for details.

 
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 (10, 21). 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.


Figure 2
View larger version (82K):
[in this window]
[in a new window]

  		FIGURE 2.
Inactivation 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).

 


Figure 3
View larger version (50K):
[in this window]
[in a new window]

  		FIGURE 3.
Drp1 is phosphorylated by Cdk1/cyclin B in mitosis. A, HeLa cells were synchronized in the G1/S phase or M phase (see "Experimental Procedures"). Total cell extracts were subjected to immunoprecipitation by anti-Drp1 antibodies. Immunoprecipitated proteins were treated with the indicated concentrations of calf intestinal alkaline phosphatase (CIP) at 37 °C for 1 h. The samples were analyzed by immunoblotting using anti-Drp1 antibodies. B, in vitro protein kinase assay was performed using purified recombinant His-Drp1 or bovine serum albumin (BSA) as substrates and immunoisolated cdk1/cyclin B as the enzyme. They were incubated in protein kinase buffer at 30 °C for 0.5 or 2 h. The samples were analyzed by SDS-PAGE and subsequent Coomassie Brilliant Blue (CBB) staining and autoradiography. C, in vitro protein kinase assay was performed using purified recombinant Cdk1/cyclin B (as in B) and analyzed by autoradiography. U, units.

 
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) (21). 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.

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 (34), 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 [{gamma}-32P]ATP. Recombinant Drp1 was phosphorylated by the isolated Cdk1/cyclin B, as analyzed by autoradiography, and the band shift was also observed by Coomassie Brilliant Blue staining, suggesting that Drp1 is a substrate of Cdk1/cyclin B (Fig. 3B). To confirm whether Cdk1/cyclin B directly phosphorylates Drp1, we examined the protein kinase reaction using purified recombinant Cdk1/cyclin B (Fig. 3C). Drp1 was also phosphorylated by purified Cdk1/cyclin B, indicating that it acted as a responsible kinase of Drp1.

Serine 585 of Rat Drp1 Is Phosphorylated by Cdk1/cyclin B—Cdk1/cyclin B is a Ser/Thr protein kinase that recognizes the consensus motif (Ser/Thr)-Pro-Xaa-(Arg/Lys) of substrates; Arg/Lys at the +3 position is preferred but not essential for protein kinase recognition (35, 36). Rat Drp1 has four potential recognition sites for Cdk1/cyclin B, Ser-71, -126, -136, and -585; therefore, we constructed four Ser->Ala mutants, S71A, S126A, S136A, and S585A. The FLAG-tagged Drp1 mutant proteins were expressed in HeLa cells and synchronized at the mitotic phase (Fig. 4B). Immunoblots using anti-FLAG antibody revealed that the three Drp1 mutants, S71A, S126A, and S136A, as well as wild-type Drp1, were shifted upward, suggesting that these mutants were normally phosphorylated. In contrast, the S585A mutant in mitotic cells was detected as a single band with mobility similar to that of wild-type Drp1 in the G1/S phase, indicating that the phosphorylation at Ser-585 was abolished by this mutation. Recombinant Drp1wt and the Ser->Ala mutants, except for Drp1S585A, were phosphorylated in vitro by immunoisolated or recombinant Cdk1/cyclin B (Fig. 4, C-E), indicating that Drp1 was mitotically phosphorylated on Ser-585 by Cdk1/cyclin B.

To further confirm the mitotic phosphorylation of Drp1, the antibodies specifically recognizing phosphorylation at Ser-585 in Drp1 (anti-p-Drp1) were prepared by immunizing rabbits with phosphorylated synthetic peptide (Fig. 4A). The amino acid sequence of the antigen peptide is completely conserved in human Drp1, and Ser-585 in rat Drp1 corresponds to Ser-590 in human Drp1 isoform 2 (710 amino acid residues). Drp1 was immunoisolated using anti-Drp1 and then analyzed by immunoblotting using anti-p-Drp1 (Fig. 4F). A significant amount of phosphorylated endogenous Drp1 was detected in the mitotic cells (Fig. 4, F and G). Phosphorylation of exogenously expressed Drp1wt (but not Drp1S585A) was also observed in mitotic cells. Together, these results strongly supported mitotic phosphorylation of Drp1 at Ser-585. Of note, the endogenous and exogenously expressed Drp1wt were also phosphorylated at Ser-585 to a lower but significant extent in the G1/S phase cells (Fig. 4, F and G), suggesting that some protein kinase(s) other than Cdk1/cyclin B is involved.


Figure 4
View larger version (60K):
[in this window]
[in a new window]

  		FIGURE 4.
Ser-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.

 
Phosphorylation of Drp1 at Ser-585 Stimulates Mitochondrial Fission—The sequence alignment with dynamin suggested that Ser-585 of Drp1 was present in the region with homology to the GTPase effector domain. The GTPase effector domain stimulates the intra- or intermolecular interaction of dynamin and stimulates GTP hydrolysis activity (37). To examine the effect of the S585A mutation on GTPase activity, the GTP hydrolysis activity of the recombinant Drp1 proteins was analyzed using [{alpha}-32P]GTP. Drp1S585A mutant and wild-type Drp1 exhibited indistinguishable GTPase activity, suggesting that Ser-585 was not critical for the GTP hydrolysis activity of Drp1 (data not shown).


Figure 5
View larger version (84K):
[in this window]
[in a new window]

  		FIGURE 5.
Phosphorylated 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).

 
To investigate the effect of Drp1 phosphorylation, the phosphorylation-negative mutant Drp1S585A was expressed in HeLa cells. In the mitotic cells expressing Drp1S585A, mitochondrial fragmentation was significantly repressed compared with the cells expressing wild-type Drp1 (Fig. 5, A and B). As expected, the cells with filamentous mitochondria were increased upon expression of the Drp1S585A mutant. These results suggest that phosphorylation at Ser-585 stimulates mitochondrial fission in mitosis.

In this experiment, the effect of exogenously expressed Drp1 was examined in the presence of endogenous Drp1. We therefore examined the effect of Drp1S585A in Drp1-repressed cells. Human Drp1 was knocked down in HeLa cells by the specific siRNA as described in the legend to Fig. 2, and then rat Drp1wt or Drp1S585A was expressed (Fig. 5, C-F). As described above, the repression of endogenous Drp1 inhibited mitochondrial fragmentation in mitotic cells, and the tubular mitochondria were maintained throughout the mitotic phase (Fig. 2, B-E). Expression of Drp1wt restored the mitotic mitochondrial fragmentation as in control RNAi cells (Fig. 5, C and D, compared with Fig. 2B). Morphometric analysis revealed that the number of short (<2 µm in length) mitochondria in early and late mitotic cells reached ~80 and ~50%, respectively (Fig. 5F). In marked contrast, the mitochondrial fragmentation was clearly affected by exogenous expression of Drp1S585A; the fragmented mitochondria with <2 µm decreased to ~50 and ~20% in the early mitotic and late mitotic phases, respectively. (Fig. 5; C, D, and F). These results suggest that phosphorylation at Ser-585 in mitosis stimulates mitochondrial fission.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In mitosis, organelles are inherited to daughter cells beyond dynamic changes of the membrane structures (7, 38, 39). The nuclear envelope and Golgi apparatus are broken down in early mitotic phase, transmitted to the daughter cells, and then reconstructed at a late stage of mitosis. Factors involved in Golgi assembly, GRASP65 (40), GM130 (41), and p47 (42), are phosphorylated during mitosis and stimulate mitotic Golgi disassembly. The inactivation of GRASP65 blocks mitotic Golgi fragmentation as well as mitotic entry (40). Here we examined the mitochondrial dynamics in mitotic HeLa cells. In mitosis, mitochondria were fragmented in a Drp1-dependent manner and transmitted to daughter cells, and then they regained their filamentous structures. Drp1 was specifically phosphorylated at Ser-585 by Cdk1/cyclin B, which stimulated the mitochondrial fission in mitosis. From these results, we concluded that mitochondrial morphology is regulated by Drp1-dependent mitochondrial fission in mitotic cells. To our knowledge this is the first demonstration for Drp1-dependent mitochondrial fragmentation in the early mitotic phase.

Previous studies have demonstrated that the filamentous mitochondria in the G1 phase become shorter after the S phase in fibroblasts (43) and osteosarcoma cells (44). The dynamics of mitochondrial morphology in mitosis, however, have not been analyzed sufficiently. In the present study, we used video microscopy to examine mitochondrial dynamics in mitotic HeLa cells. HeLa cells have elongated and connected mitochondrial network structures, and mitochondria are fragmented in the mitotic phase. As expected, Drp1 was required for the mitotic mitochondrial fragmentation. In yeast, filamentous mitochondria are maintained and distributed to the daughter buds in mitosis, and the yeast homolog Dnm1 is not essential for mitosis (8, 22). In mitotic HeLa cells, fragmented mitochondria are distributed throughout the cytoplasm and almost equally transmitted to the two daughter cells. When Drp1 is inactivated, however, the mitochondria are not transmitted to the daughter cells equally. It is possible that mitochondrial fragmentation is not a checkpoint in mitotic entry, which is different from the fragmentation of the Golgi apparatus. Drp1-mediated mitochondrial fragmentation might allow mitochondria to disperse randomly throughout the cytoplasm and help ensure equal delivery of the mitochondria to the two daughter cells.

Drp1 was phosphorylated in mitosis by Cdk1/cyclin B, one of the major protein kinases involved in mitosis (35). The phosphorylation of Drp1 by cdk1/cyclin B stimulates mitochondrial fission activity. The phosphorylation consensus motif is conserved among mammalian Drp1 proteins, but not in yeast Dnm1, consistent with the notion that the filamentous mitochondrial networks are maintained throughout mitosis in yeast cells (7, 31, 32). Although substitution with an Asp residue is usually used to mimic a phosphorylated Ser residue, we did not observe any visible differences by the expression of Drp1S585D compared with wild-type Drp1 in mitochondrial morphology or in GTP hydrolysis activity (data not shown). It remains unknown how phosphorylation stimulates the mitochondrial fission activity of Drp1. Dynamin is phosphorylated by the Cdk family protein Cdk5 at their C-terminal Pro-rich domain (45, 46). This phosphorylation affects the complex formation with their partner proteins containing amphiphysin I and endophilin I, which regulates endocytic activity in neuronal cells (46, 47). Although the C-terminal Pro-rich region is not conserved in Drp1, five Pro residues are present near the Drp1 phosphorylation site (see Fig. 4A). Furthermore, the endophilin family protein endophilin B1 acting downstream of Drp1 is targeted from the cytoplasm to the mitochondrial fission foci to be involved in the fission reaction (48). Together, it is possible that the Cdk1/cyclin B-dependent Drp1 phosphorylation stimulates the interaction with endophilin B1 or other unidentified partner protein(s). Further analysis of the physiologic significance of the Drp1 phosphorylation will lead to new insights into the regulation of mitochondrial fission in response to cellular signaling and differentiation. Finally, it should be noted that we and others have previously demonstrated that treatment of cultured cells with nocodazole does not induce mitochondrial fragmentation (10, 49), suggesting that mitochondrial fragmentation in the mitotic phase is independent of disassembly of cytoplasmic microtubules (Fig. 1A, b and c).


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Science, and Culture of Japan, Core Research from Evolutional Science and Technology, and Takara Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1A-C and Movies 1-5. Back

1 To whom correspondence should be addressed: Dept. of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1, Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan. Tel.: 81-92-642-4709; Fax: 81-92-642-6183; E-mail: mihara{at}cell.med.kyushu-u.ac.jp.

2 The 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. Back


   ACKNOWLEDGMENTS
 
The plasmid for YFP-{alpha}-tubulin was a generous gift from K. Ohashi and K. Mizuno (Tohoku University). We are grateful to H. Nishijima and H. Nishitani (Kyushu University) for their generous help and advice regarding the phosphorylation and cell cycle analyses. We extend our thank to S. Yoshimura and N. Nakamura (Kanazawa University) for helpful discussions, and to T. Yoshimori (Osaka University) for helpful advice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yaffe, M. P. (1999) Science 283, 1493-1497[Abstract/Free Full Text]
  2. Griparic, L., and van der Bliek, A. M. (2001) Traffic 2, 235-244[CrossRef][Medline] [Order article via Infotrieve]
  3. Mozdy, A. D., and Shaw, J. M. (2003) Nat. Rev. Mol. Cell Biol. 4, 468-478[CrossRef][Medline] [Order article via Infotrieve]
  4. Westermann, B. (2003) Biochim. Biophys. Acta 1641, 195-202[Medline] [Order article via Infotrieve]
  5. Chen, H., and Chan, D. C. (2004) Curr. Top. Dev. Biol. 59, 119-144[Medline] [Order article via Infotrieve]
  6. Okamoto, K., and Shaw, J. M. (2005) Annu. Rev. Genet. 39, 503-536[CrossRef][Medline] [Order article via Infotrieve]
  7. Nunnari, J., Marshall, W. F., Straight, A., Murray, A., Sedat, J. W., and Walter, P. (1997) Mol. Biol. Cell 8, 1233-1242[Abstract]
  8. Bleazard, W., McCaffery, J. M., King, E. J., Bale, S., Mozdy, A., Tieu, Q., Nunnari, J., and Shaw, J. M. (1999) Nat. Cell Biol. 1, 298-304[CrossRef][Medline] [Order article via Infotrieve]
  9. Sesaki, H., and Jensen, R. E. (1999) J. Cell Biol. 147, 699-706[Abstract/Free Full Text]
  10. Ishihara, N., Jofuku, A., Eura, Y., and Mihara, K. (2003) Biochem. Biophys. Res. Commun. 301, 891-898[CrossRef][Medline] [Order article via Infotrieve]
  11. Hales, K. G., and Fuller, M. T. (1997) Cell 90, 121-129[CrossRef][Medline] [Order article via Infotrieve]
  12. Hermann, G. J., Thatcher, J. W., Mills, J. P., Hales, K. G., Fuller, M. T., Nunnari, J., and Shaw, J. M. (1998) J. Cell Biol. 143, 359-373[Abstract/Free Full Text]
  13. Chen, H., Detmer, S. A., Ewald, A. J., Griffin, E. E., Fraser, S. E., and Chan, D. C. (2003) J. Cell Biol. 160, 189-200[Abstract/Free Full Text]
  14. Eura, Y., Ishihara, N., Yokota, S., and Mihara, K. (2003) J. Biochem. (Tokyo) 134, 333-344[Abstract/Free Full Text]
  15. Shepard, K. A., and Yaffe, M. P. (1999) J. Cell Biol. 144, 711-720[Abstract/Free Full Text]
  16. Wong, E. D., Wagner, J. A., Gorsich, S. W., McCaffery, J. M., Shaw, J. M., and Nunnari, J. (2000) J. Cell Biol. 151, 341-352[Abstract/Free Full Text]
  17. Chen, H., Chomyn, A., and Chan, D. C. (2005) J. Biol. Chem. 280, 26185-26192[Abstract/Free Full Text]
  18. Alexander, 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., and Wissinger, B. (2000) Nat. Genet. 26, 211-215[CrossRef][Medline] [Order article via Infotrieve]
  19. Delettre, 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., and Hamel, C. P. (2000) Nat. Genet. 26, 207-210[CrossRef][Medline] [Order article via Infotrieve]
  20. Zuchner, 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., and Vance, J. M. (2004) Nat. Genet. 36, 449-451[CrossRef][Medline] [Order article via Infotrieve]
  21. Smirnova, E., Shurland, D. L., Ryazantsev, S. N., and van der Bliek, A. M. (1998) J. Cell Biol. 143, 351-358[Abstract/Free Full Text]
  22. Otsuga, D., Keegan, B. R., Brisch, E., Thatcher, J. W., Hermann, G. J., Bleazard, W., and Shaw, J. M. (1998) J. Cell Biol. 143, 333-349[Abstract/Free Full Text]
  23. Yoon, Y., Krueger, E. W., Oswald, B. J., and McNiven, M. A. (2003) Mol. Cell Biol. 23, 5409-5420[Abstract/Free Full Text]
  24. Stojanovski, D., Koutsopoulos, O. S., Okamoto, K., and Ryan, M. T. (2004) J. Cell Sci. 117, 1201-1210[Abstract/Free Full Text]
  25. Griffin, E. E., Graumann, J., and Chan, D. C. (2005) J. Cell Biol. 170, 237-248[Abstract/Free Full Text]
  26. Jofuku, A., Ishihara, N., and Mihara, K. (2005) Biochem. Biophys. Res. Commun. 333, 650-659[CrossRef][Medline] [Order article via Infotrieve]
  27. Karren, M. A., Coonrod, E. M., Anderson, T. K., and Shaw, J. M. (2005) J. Cell Biol. 171, 291-301[Abstract/Free Full Text]
  28. Naylor, K., Ingerman, E., Okreglak, V., Marino, M., Hinshaw, J. E., and Nunnari, J. (2006) J. Biol. Chem. 281, 2177-2183[Abstract/Free Full Text]
  29. Lee, Y. J., Jeong, S. Y., Karbowski, M., Smith, C. L., and Youle, R. J. (2004) Mol. Biol. Cell 15, 5001-5011[Abstract/Free Full Text]
  30. Posakony, J. W., England, J. M., and Attardi, G. (1977) J. Cell Biol. 74, 468-491[Abstract/Free Full Text]
  31. Boldogh, I. R., Yang, H. C., and Pon, L. A. (2001) Traffic 2, 368-374[CrossRef][Medline] [Order article via Infotrieve]
  32. Itoh, T., Toh-e, A., and Matsui, Y. (2004) EMBO J. 23, 2520-2530[CrossRef][Medline] [Order article via Infotrieve]
  33. Gorsich, S. W., and Shaw, J. M. (2004) Mol. Biol. Cell 15, 4369-4381[Abstract/Free Full Text]
  34. Nishijima, H., Nishitani, H., Seki, T., and Nishimoto, T. (1997) J. Cell Biol. 138, 1105-1116[Abstract/Free Full Text]
  35. Nigg, E. A. (1995) BioEssays 17, 471-480[CrossRef][Medline] [Order article via Infotrieve]
  36. Holmes, J. K., and Solomon, M. J. (1996) J. Biol. Chem. 271, 25240-25246[Abstract/Free Full Text]
  37. Hinshaw, J. E. (2000) Annu. Rev. Cell Dev. Biol. 16, 483-519[CrossRef][Medline] [Order article via Infotrieve]
  38. Shorter, J., and Warren, G. (2002) Annu. Rev. Cell Dev. Biol. 18, 379-420[CrossRef][Medline] [Order article via Infotrieve]
  39. Hetzer, M. W., Walther, T. C., and Mattaj, I. W. (2005) Annu. Rev. Cell Dev. Biol. 21, 347-380[CrossRef][Medline] [Order article via Infotrieve]
  40. Sutterlin, C., Hsu, P., Mallabiabarrena, A., and Malhotra, V. (2002) Cell 109, 359-369[CrossRef][Medline] [Order article via Infotrieve]
  41. Lowe, M., Rabouille, C., Nakamura, N., Watson, R., Jackman, M., Jamsa, E., Rahman, D., Pappin, D. J., and Warren, G. (1998) Cell 94, 783-793[CrossRef][Medline] [Order article via Infotrieve]
  42. Uchiyama, K., Jokitalo, E., Lindman, M., Jackman, M., Kano, F., Murata, M., Zhang, X., and Kondo, H. (2003) J. Cell Biol. 161, 1067-1079[Abstract/Free Full Text]
  43. Barni, S., Sciola, L., Spano, A., and Pippia, P. (1996) Biotech. Histochem. 71, 66-70[Medline] [Order article via Infotrieve]
  44. Margineantu, D. H., Cox, W. G., Sundell, L., Sherwood, S. W., Beechem, J. M., and Capaldi, R. A. (2002) Mitochondrion 1, 425-435[CrossRef][Medline] [Order article via Infotrieve]
  45. Tomizawa, K., Sunada, S., Lu, Y. F., Oda, Y., Kinuta, M., Ohshima, T., Saito, T., Wei, F. Y., Matsushita, M., Li, S. T., Tsutsui, K., Hisanaga, S., Mikoshiba, K., Takei, K., and Matsui, H. (2003) J. Cell Biol. 163, 813-824[Abstract/Free Full Text]
  46. Tan, T. C., Valova, V. A., Malladi, C. S., Graham, M. E., Berven, L. A., Jupp, O. J., Hansra, G., McClure, S. J., Sarcevic, B., Boadle, R. A., Larsen, M. R., Cousin, M. A., and Robinson, P. J. (2003) Nat. Cell Biol. 8, 701-710
  47. Solomaha, E., Szeto, F. L., Yousef, M. A., and Palfrey, H. C. (2005) J. Biol. Chem. 280, 23147-23156[Abstract/Free Full Text]
  48. Karbowski, M., Jeong, S. Y., and Youle, R. J. (2004) J. Cell Biol. 166, 1027-1039[Abstract/Free Full Text]
  49. Karbowski, M., Spodnik, J. H., Teranishi, M., Wozniak, M., Nishizawa, Y., Usukura, J., and Wakabayashi, T. (2001) J. Cell Sci. 114, 281-291[Abstract]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
M. Liesa, M. Palacin, and A. Zorzano
Mitochondrial Dynamics in Mammalian Health and Disease
Physiol Rev, July 1, 2009; 89(3): 799 - 845.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
J. Zhao, T. Liu, S.-B. Jin, N. Tomilin, J. Castro, O. Shupliakov, U. Lendahl, and M. Nister
The novel conserved mitochondrial inner-membrane protein MTGM regulates mitochondrial morphology and cell proliferation
J. Cell Sci., July 1, 2009; 122(13): 2252 - 2262.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Zunino, E. Braschi, L. Xu, and H. M. McBride
Translocation of SenP5 from the Nucleoli to the Mitochondria Modulates DRP1-dependent Fission during Mitosis
J. Biol. Chem., June 26, 2009; 284(26): 17783 - 17795.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. K. Dagda, R. A. Merrill, J. T. Cribbs, Y. Chen, J. W. Hell, Y. M. Usachev, and S. Strack
The Spinocerebellar Ataxia 12 Gene Product and Protein Phosphatase 2A Regulatory Subunit B{beta}2 Antagonizes Neuronal Survival by Promoting Mitochondrial Fission
J. Biol. Chem., December 26, 2008; 283(52): 36241 - 36248.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
S. Arnold, G. W. de Araujo, and C. Beyer
Gender-specific regulation of mitochondrial fusion and fission gene transcription and viability of cortical astrocytes by steroid hormones
J. Mol. Endocrinol., November 1, 2008; 41(5): 289 - 300.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. M. Cereghetti, A. Stangherlin, O. M. de Brito, C. R. Chang, C. Blackstone, P. Bernardi, and L. Scorrano
Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria
PNAS, October 14, 2008; 105(41): 15803 - 15808.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
X.-J. Han, Y.-F. Lu, S.-A. Li, T. Kaitsuka, Y. Sato, K. Tomizawa, A. C. Nairn, K. Takei, H. Matsui, and M. Matsushita
CaM kinase I{alpha}-induced phosphorylation of Drp1 regulates mitochondrial morphology
J. Cell Biol., August 11, 2008; 182(3): 573 - 585.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
S. Tamai, H. Iida, S. Yokota, T. Sayano, S. Kiguchiya, N. Ishihara, J.-I. Hayashi, K. Mihara, and T. Oka
Characterization of the mitochondrial protein LETM1, which maintains the mitochondrial tubular shapes and interacts with the AAA-ATPase BCS1L
J. Cell Sci., August 1, 2008; 121(15): 2588 - 2600.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
D.-F. Suen, K. L. Norris, and R. J. Youle
Mitochondrial dynamics and apoptosis
Genes & Dev., June 15, 2008; 22(12): 1577 - 1590.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
S. Gandre-Babbe and A. M. van der Bliek
The Novel Tail-anchored Membrane Protein Mff Controls Mitochondrial and Peroxisomal Fission in Mammalian Cells
Mol. Biol. Cell, June 1, 2008; 19(6): 2402 - 2412.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
T. Oka, T. Sayano, S. Tamai, S. Yokota, H. Kato, G. Fujii, and K. Mihara
Identification of a Novel Protein MICS1 that is Involved in Maintenance of Mitochondrial Morphology and Apoptotic Release of Cytochrome c
Mol. Biol. Cell, June 1, 2008; 19(6): 2597 - 2608.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Westermann
Molecular Machinery of Mitochondrial Fusion and Fission
J. Biol. Chem., May 16, 2008; 283(20): 13501 - 13505.
[Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. C. Poole, R. E. Thomas, L. A. Andrews, H. M. McBride, A. J. Whitworth, and L. J. Pallanck
The PINK1/Parkin pathway regulates mitochondrial morphology
PNAS, February 5, 2008; 105(5): 1638 - 1643.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C.-R. Chang and C. Blackstone
Cyclic AMP-dependent Protein Kinase Phosphorylation of Drp1 Regulates Its GTPase Activity and Mitochondrial Morphology
J. Biol. Chem., July 27, 2007; 282(30): 21583 - 21587.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. Brooks, Q. Wei, L. Feng, G. Dong, Y. Tao, L. Mei, Z.-J. Xie, and Z. Dong
Bak regulates mitochondrial morphology and pathology during apoptosis by interacting with mitofusins
PNAS, July 10, 2007; 104(28): 11649 - 11654.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/15/11521    most recent
M607279200v2
M607279200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Taguchi, N.
Right arrow Articles by Mihara, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Taguchi, N.
Right arrow Articles by Mihara, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement