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Originally published In Press as doi:10.1074/jbc.M504282200 on September 14, 2005

J. Biol. Chem., Vol. 280, Issue 45, 37901-37907, November 11, 2005
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Local Change in Phospholipid Composition at the Cleavage Furrow Is Essential for Completion of Cytokinesis*

Kazuo Emoto{ddagger}§1, Hironori Inadome{ddagger}, Yasunori Kanaho§, Shuh Narumiya¶, and Masato Umeda{ddagger}§2

From the {ddagger}Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan, §The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, and Department of Pharmacology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8315, Japan

Received for publication, April 19, 2005 , and in revised form, August 23, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell division ends up with the membrane separation of two daughter cells, presumably by a membrane fusion that requires dynamic changes of the distribution and the composition of membrane lipids. We have previously shown that a membrane lipid phosphatidylethanolamine (PE) is exposed on the cell surface of the cleavage furrow during late cytokinesis and that this PE movement is involved in regulation of the contractile ring disassembly. Here we show that immobilization of cell surface PE by a PE-binding peptide blocks the RhoA inactivation in the late stage of cytokinesis. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), but not other RhoA effectors, is co-localized with RhoA in the peptide-treated cells. Indeed, PIP5K and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are localized to the cleavage furrow of normally dividing cells. Both overexpression of a kinase-deficient PIP5K mutant and microinjection of anti-PI(4,5)P2 antibodies compromise cytokinesis by preventing local accumulation of PI(4,5)P2 in the cleavage furrow. These findings demonstrate that the localized production of PI(4,5)P2 is required for the proper completion of cytokinesis and that the possible formation of a unique lipid domain in the cleavage furrow membrane may play a crucial role in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytokinesis creates membrane barriers between the two daughter cells that complete the cell cycle (1-3). After segregation of condensed chromosomes toward opposite poles in late anaphase, the contractile ring containing actin and myosin assembles in the center of the cell. The ring then contracts to furrow the overlaying plasma membrane until the furrow membrane comes into contact with the central spindle. At the end of cytokinesis, both the central spindle and the contractile ring disassemble, followed by the homotypic fusion of the two facing plasma membranes and then cell separation.

The small GTPase RhoA cycles the GDP-bound inactive and the GTP-bound active forms and plays a central role in cytoskeletal rearrangement during cytokinesis (4, 5). Transient accumulation and activation of RhoA in the cleavage furrow is required for induction, maintenance, and constriction of the contractile ring, which is affected by action on a variety of downstream effectors, such as citron-kinase (6, 7), ROCK3/Rho-associated kinase/Rho-kinase (8), and mammalian homlogue of Drosophila diaphanous (9). In late cytokinesis, the down-regulation of Rho activity at the midbody region likely causes actin depolymerization needed for contractile ring disassembly, whereas RhoA activation is still required for reconstitution of the focal adhesions and stress fibers in the daughter cells after cytokinesis (10-12). Recent studies have identified two proteins, Cyk-4/MgcRacGAP (13, 14) and Nir2 (15), involved in the down-regulation of the RhoA after cleavage furrow ingression. During cytokinesis, both Cyk-4/MgcRacGAP and Nir2 are recruited to the cleavage furrow and presumably suppress the RhoA activity in the furrow through the GAP activity (13, 14) or the Rhoinhibitory function (15), respectively.

Recent studies indicate that new membrane insertion by the targeted vesicle fusion frequently occurs at the leading edge of the cleavage furrow (1, 16-20). This membrane insertion plays a role in furrowing (16-18) as well as scission of the residual bridge between daughter cells (19). A variety of membrane transactions appears important during cleavage, because mutations in proteins affecting endocytosis, secretions, lysosomal trafficking, or recycling endosomes cause cytokinesis or cellularization defects in various organisms (17, 19-21). Because efficient membrane fusion events require particular lipids as well as proteins for vesicle targeting and subsequent membrane merging (22), specific membrane lipids are likely recruited to the cleavage furrow membrane and play a role in the completion of cytokinesis. Indeed, previous studies suggest that a particular lipid domain is likely formed on the cleavage furrow membrane of dividing eggs (23, 24); however, the compositions and functions of this lipid domain have been unknown.

Previously, using a peptide that binds specifically to phosphatidylethanolamine (PE)3 on biological membranes (25), we have shown that PE is exposed on the cell surface of the cleavage furrow during late cytokinesis (26). Treatment of the dividing cells by the PE-binding peptide immobilizes the cell surface PE and inhibits disassembly of the contractile ring, resulting in formation of a long cytoplasmic bridge between the daughter cells (27). These observations indicate that the redistribution of PE on the cleavage furrow membrane plays a critical role in controlling the disassembly of the contractile ring.

In this study, we showed that PE-binding peptide inhibited the down-regulation of RhoA in the cleavage furrow. We also found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is localized on the inside of the furrow membrane, probably through specific recruitment of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to the cleavage furrow. Furthermore, this local production of PI(4,5)P2 plays a crucial role in completion of cytokinesis during late stage of cytokinesis. These data provide a potential role of a unique lipid domain at the cleavage furrow membrane in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Ro09-0198 (Ro peptide) was biotinylated and then conjugated to streptavidin as described previously (26). Chinese hamster ovary (CHO)-K1 fibroblasts were obtained from the American Type Culture Collection (ATCC CCL 61). R-41, a CHO-K1-derived mutant cell line, was established as a variant that is resistant to Ro peptide as described previously (28).

Incubation of Mitotic Cells with PE-binding Peptide—Mitotic cell populations were isolated from monolayer cultures grown in 850-cm2 plastic roller bottles (Falcon) as described previously (26). Briefly, each bottle was seeded with 8.8 x 106 cells in 100 ml of growth medium. After incubation for 48 h at 37 °C, the culture medium was replaced with growth medium containing 0.04 µg/ml nocodazole. The cells were incubated for 2 h at 37 °C, and prometaphase cells were isolated by shaking. Prometaphase cells (2 x 105 cells) were incubated with 100 µg/ml Ro peptide-streptavidin complex in 1 ml of 0.5% bovine serum albumin-Ham's F-12 medium containing 1% Neutridoma (Roche Applied Science) for various periods at 37 °C. To release the Ro peptidestreptavidin complex from the cell membranes, the peptide-treated cells were washed three times with Ham's F-12 medium containing 10 µM PE liposomes (PE/phosphatidylcholine = 1/1) and 1% Neutridoma and further incubated in the same medium at 37 °C.

Pull-down Assay for GTP-RhoA—The pull-down assay was performed as described previously (10). Briefly, cells were lysed by buffer A (50 mM Tris-HCl (pH7.4), 100 mM NaCl, 1 mM EDTA, 30 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, and protease inhibitor mixture CompleteTM (Roche Applied Science). Cell lysates were cleared by centrifugation at 15,000 x g for 15 min at 4 °C. Supernatants were then incubated with 30 µg of glutathione S-transferase/Rho-binding domain fusion conjugated with glutathione beads for 2 h at 4 °C. The beads were washed with buffer A three times and subjected to SDS-PAGE on a 12.5% gel. Bound RhoA was detected by Western blot using a monoclonal antibody against RhoA (Santa Cruz Biotechnology).

Fluorescence Microscopy—Cells were fixed with either 3.7% (w/v) formaldehyde in PBS for 30 min at room temperature or 10% (w/v) trichloroacetate in PBS for 15 min on ice, followed by a 10-min permeabilization with 0.1% Triton X-100 in PBS. The cells were then blocked with PBS containing 5% (w/v) skimmed milk for 30 min at 25 °C and reacted with the first antibodies for 16 h at 4 °C. Thereafter, the cells were washed with PBS and incubated for 1 h at 37°C with secondary antibodies (Cy3-conjugated goat anti-rat IgG (Amersham Biosciences), Alexa FluorTM488-conjugated goat anti-rabbit IgG, Alexa FluorTM594-conjugated goat anti-rabbit IgG, Alexa FluorTM488-conjugated goat anti-mouse IgG, and Alexa FluorTM594-conjugated goat anti-rabbit IgG (all from Molecular Probes)) diluted to 1/500. For F-actin and DNA staining, cells were incubated with 40 ng/ml TRITC-phalloidin and 0.2 µg/ml DAPI in PBS for 1 h at 25°C. Fluorescence images were obtained using a laser-scanning confocal microscopy LSM 510 (Carl Zeiss).

Plasmid Construction and Expression—PTB701-FLAG and pcDNA3-FLAG were used to construct a mammalian expression vector for N-terminally FLAG epitope-tagged PIP5K{beta} and PIP5K{gamma} as described previously (29). The kinase-deficient PIP5K{beta} mutant (PIP5K{beta}-KD) was prepared by replacing the conserved aspartic acid at position 307 of PIP5K{beta} with alanine as described previously (29). The expression vectors for GFP-PH and GFP-PH(PKB/Akt) were generous gifts of Dr. H. Yagisawa (Himeji Institute of Technology) and Dr. U. Kikkawa (Kobe University), respectively. CHO cells cultured on glass coverslips were transfected with each plasmid using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. After 24 h, cells were fixed and examined by immunofluorescence microscopy. In some experiments, pcDNA3-FLAG-PIP5K{beta}-KD was co-transfected with pGFP-PH into CHO cells (pcDNA3-FLAG-PIP5K{beta}-KD:pGFP-PH = 5:1 (w/w)). For the plasma membrane labeling, the fixed cells were incubated with 5 µg/ml 1,1'-dilinoleyl-3,3,3',3' -tetramethylindocarbocyanine perchlorate (FAST DiITM) (Molecular Probes) for 1 h at 25 °C.

Microinjection—CHO cells grown on CELLocate glass coverslips (Eppendorf) were synchronized at the G1/S boundary by a double thymidine block method (10). In brief, cells were incubated with 2 mM thymidine for 16 h at 37 °C, released from arrest, and then arrested at G1/S again with 2 mM thymidine. The cells were then placed under normal growth conditions (time 0). After 10 h ({approx}1 h before the initiation of mitosis), cells were co-injected with 10 mg/ml Oregon green-conjugated dextran (Moleculer Probe) and 2 mg/ml either control mouse IgG or either of the two distinct anti-PI(4,5)P2 monoclonal antibodies, 2C11 or AM212. Injections were performed with an Eppendorf transinjector (model 5242) and micromanipulator (model 5171) attached to a Zeiss Axiovert 100 microscope. After 24 h, cells were fixed and incubated with 40 ng/ml TRITC-phalloidin and 0.2 µg/ml DAPI in PBS for 1 h at 25 °C for F-actin and DNA staining, respectively.

Flow Cytometry—The pcDNA3 plasmids containing either FLAG-tagged wild-type or mutant PIP5K{beta} were transfected with pEGFP-N1 vector (Clontech) into CHO cells by using Lipofectamine Plus reagents (FLAG-PIP5K{beta}/pEGFP-N1 = 5/1 (w/w)). After 48 h, the cells were trypsinized and fixed in cold 70% ethanol. Cell cycle parameters were determined by propidium iodide labeling of nuclear DNA. The GFP-positive cells were selected and analyzed for DNA contents on a flow cytometer, FACScan (BD Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Accumulation of GTP-bound RhoA at Cytoplasmic Bridge—We have previously shown that a membrane lipid PE is exposed on the cell surface of the cleavage furrow during cytokinesis and that immobilization of the cell surface PE by a PE-binding peptide inhibits disassembly of the contractile ring, resulting in formation of an F-actin-rich cytoplasmic bridge between the daughter cells (26, 27). To further understand the molecular mechanisms underlying this cytokinesis blockade by the peptide, we investigated the distribution and the activity state of small GTPase RhoA, which plays a key role in the contractile ring organization during cytokinesis (4, 5). In normal cell division of CHO cells, RhoA was localized 2 h after the initiation of mitosis only in the middle of the bridge, a region known as the midbody (Fig. 1A). In contrast, RhoA was highly accumulated in the cytoplasmic bridge of the peptide-treated cells and co-localized with F-actin (Fig. 1A). The peptide bound on the surface of the cleavage furrow can be removed by incubating the peptide-treated cells with PE-enriched liposome, resulting in disassembly of the contractile ring and formation of binucleated cells (27). When the peptide-treated cells were incubated with the PE-enriched liposomes, RhoA relocated from the cytoplasmic bridge to the cytoplasm of the binucleated cells (Fig. 1A). These observations suggest that the PE-binding peptide affects the dissociation of RhoA from the cleavage furrow membrane.

To examine whether the accumulated RhoA is the GTP-bound active form or the GDP-bound inactive form, we measured the amounts of cellular GTP-bound RhoA of the peptide-treated cells by a pull-down assay using glutathione S-transferase-fused Rho-binding domain of rhotekin (10, 11). As a control experiment, normally dividing CHO cells collected 0, 45, 90, and 180 min after the release from prometaphase arrest by nocodazole were subjected to the pull-down assay (Fig. 1B). The cellular level of GTP-bound RhoA transiently increased at 45 min in the telophase and then returned to the basal level at 90 min in G1 phase, which is essentially consistent with previous reports (10, 11). Likewise, the GTP-bound RhoA level of the mitotic cells incubated with the PE-binding peptide increased at 45 min; however, the maximum level was maintained up to 180 min after the release from the prometaphase arrest, suggesting that RhoA that accumulated at the cytoplasmic bridge was the GTP-bound active form. Furthermore, when the peptide-treated cells were incubated in the presence of PE-enriched liposomes, the cellular GTP-bound RhoA level gradually decreased and eventually reached the basal level at 180 min after the removal of the PE-binding peptide (Fig. 1B). These results indicate that the trapping of the cell surface-exposed PE by the peptide impairs RhoA inactivation in the late stage of cytokinesis.



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  		FIGURE 1.
The PE-binding peptide inhibits down-regulation of RhoA during late cytokinesis. A, CHO cells synchronized in prometaphase were incubated without (Control) or with (+SA-Ro) 50 µg/ml SA-Ro for 2 h and then fixed and co-stained with anti-RhoA antibody (red, left panels) and anti-actin antibody (green, middle panels). The SA-Ro-treated cells were then incubated in the presence of 10 µM PE liposomes for 3 h (Wash) and fixed and co-stained with anti-RhoA antibody (red, left panels) and anti-actin antibody (green, middle panels). Right panels are merged images. B, the pull-down assay for GTP-RhoA. CHO cells were cultivated in the absence (Control) or presence (+SA-Ro) of 50 µg/ml SA-Ro. Lysates of cells collected 0, 45, 90, and 180 min after the nocodazole release were subjected to the pull-down assay, and precipitated RhoA was detected by Western blot analysis (upper panel of each experiment). The amount of input RhoA was shown in the lower panel. The SA-Ro-treated cells were then incubated in the presence of 10 µM PE liposomes for 3 h (Wash), and the cell lysates of cells collected 0, 60, 120, and 180 min after the initiation of the incubation were subjected to the pull-down assay.

 
PIP5K but Not Other Rho Effectors Colocalized with RhoA at Cytoplasmic Bridge—RhoA performs its cellular functions by binding to downstream effectors (4, 5). Of the identified Rho-binding proteins, citron-kinase (6, 7), ROCK/Rho-associated kinase/Rho-kinase (8), and mammalian homologue of Drosophila diaphanous (9) have been shown to localize at the cytokinetic apparatus and to play essential roles in cytokinesis of mammalian cells. Analyses using specific antibodies revealed that neither citron-kinase (Fig. 2A), ROCK, nor mammalian homologue of Drosophila diaphanous (data not shown) was detected at the cytoplasmic bridge of the peptide-treated cells. Instead, punctate structures were observed in the cytoplasm. In addition, the cytoplasmic bridge formation was not significantly affected by the ROCK inhibitor Y-27632 (data not shown). These data suggest that RhoA stabilizes the contractile ring in the cytoplasmic bridge through a novel signaling pathway.



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  		FIGURE 2.
Co-localization of RhoA and PIP5K{beta} in the division-arrested cells. A, cells treated with SA-Ro for 2 h were fixed and co-stained with citron-kinase (green, upper panel) and RhoA (red) or PIP5K{beta} (green, lower panel) and RhoA (red). B, R-41 mutant cells were cultured in the ethanolamine-deficient medium for 3 days and then fixed and co-stained with citron-kinase (green, upper panel) and RhoA (red) or PIP5K{beta} (green, lower panel) and RhoA (red). C, CHO cells transfected with GFP-PH were treated with 50 µg/ml SA-Ro for 2 h (upper panels). The cells were then fixed and stained with rhodamine-phalloidin. The left and the middle panels represent GFP-PH (green) and F-actin (red), respectively. In the lower panel, CHO cells treated with SA-Ro for 2 h were then fixed and co-stained with lysenine (green) and rhodamine-phalloidin (red). Right panels are merged images.

 
We next investigated other RhoA effectors and found that phosphatidylinositol 4-phosphate 5-kinase {beta} (PIP5K{beta}) was co-localized with RhoA at the cytoplasmic bridge of the peptide-treated cells (Fig. 2A). To define the role of PE movements in PIP5K{beta} translocation from the cleavage furrow, we analyzed its localization in a CHO mutant cell line (R-41) that is defective in PE biosynthesis and exhibits a defect in the contractile ring disassembly, resulting in cytokinesis arrest at late telophase (27, 28). In R-41 cells arrested in cytokinesis, a co-localization of PIP5K{beta} with RhoA, but not other Rho effectors (including citron-kinase), was observed at the cleavage furrow (Fig. 2B). These data demonstrate that PE redistribution is also coupled to the translocation of PIP5K{beta} from the cleavage furrow.

PIP5K converts phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (30-32). Thus, we examined the intracellular distribution of PI(4,5)P2 using the pleckstrin homology (PH) domain of phospholipase C{delta}1, which specifically binds to PI(4,5)P2 on biological membranes (33). Green fluorescent protein-fused PH (GFP-PH) domain displayed a robust accumulation at the bridge membrane in the peptide-treated cells (Fig. 2C), indicating that PI(4,5)P2 is locally produced on the inner leaflet of the bridge membrane. Interestingly, staining with a sphingomyelin-specific binding protein, lysenin (34), revealed that the plasma membrane sphingomyelin, which is distributed on the cell surface uniformly in interphase cells (34), was excluded from the surface of the cytoplasmic bridge (Fig. 2C). Together with our previous findings that the cell surface PE is specifically localized on the cytoplasmic bridge membrane (26), these observations suggest that a particular lipid domain in which PE and PI(4,5)P2 are concentrated on the outer and inner leaflet of bilayer, respectively, is formed at the cytoplasmic bridge. Furthermore, this lipid domain may be involved in the stabilization of the contractile ring, leading to the cytoplasmic bridge formation.

Local Production of PI(4,5)P2 at Cleavage Furrow Membrane—Because this study provides the first evidence suggesting a local accumulation of PIP5K and PI(4,5)P2 in the cleavage furrow of dividing cells, we further examined the role of PI(4,5)P2 in cytokinesis. In normally dividing cells, PI(4,5)P2 localized uniformly in the plasma membrane during metaphase and early anaphase (Fig. 3, A and B); whereas, in the late telophase, a marked accumulation of PI(4,5)P2 to the cleavage furrow was observed (Fig. 3, C and D, arrow). The intense signal at the cleavage furrow did not appear to reflect the concentration of the plasma membrane itself, because a membrane marker DiI was rather evenly distributed on the plasma membranes (Fig. 3E). In contrast, no significant accumulation of the GFP signal was detected either by a mutant PH domain, PH(K40A), which represents much lower binding affinity to PI(4,5)P2 compared with the original PH domain (33) (Fig. 3F), or by the PH domain derived from PKB/Akt, which specifically binds to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) (data not shown). Similar to PI(4,5)P2 localization in dividing cells, although PIP5K{beta} localized uniformly at the plasma membrane during metaphase and early anaphase, in the telophase, PIP5K{beta} became co-localized with RhoA in the cleavage furrow (Fig. 4A). To validate these immunofluorescence data, we next transfected CHO cells with plasmids containing PIP5Ks tagged with FLAG epitope and examined its localization in the dividing cells. Like the endogenous PIP5K{beta}, FLAG-PIP5K{beta} became associated with the cleavage furrow membrane in the telophase of cytokinesis (Fig. 4B), whereas FLAG-PIP5K{gamma}, another isoform of PIP5K, was not significantly concentrated at the furrow (Fig. 4D). Additionally, we detected co-localization of endogenous RhoA with FLAG-PIP5K{beta} in the cleavage furrow (Fig. 4C). Taken together, these observations suggest that local production of PI(4,5)P2 at the furrow membrane is mediated, at least in part, by PIP5K{beta} accumulated at the furrow in the telophase of cytokinesis.



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  		FIGURE 3.
Accumulation of PI(4,5)P2 in the cleavage furrow membrane of dividing cells. CHO cells were transfected with GFP-PH and then cultivated for 24 h. Cells were fixed and stained with anti-tubulin antibody (red, middle panels). Cells in metaphase (A), anaphase (B), telophase (C), and late telophase (D) are shown. E, the GFP-PH-transfected cells at late telophase were fixed and incubated with 5 µg/ml FAST DiITM for 1 h. F, the cells transfected with GFP-PH(R40A) at telophase were fixed and stained with anti-tubulin antibody (red, middle panels). The right panels are merged images. Arrows indicate the cleavage furrow. Scale bar,10 µm.

 
PI(4,5)P2 in Furrow Membrane Is Essential for Cytokinesis—To define the role of PI(4,5)P2 in cytokinesis, we examined the effect of induced expression of either wild-type PIP5K{beta} or a mutant PIP5K{beta} devoid of kinase activity (PIP5K{beta}-KD) on cell division. The cells expressing PIP5K{beta}-KD became multinucleated 36 h after transfection, whereas those expressing either wild-type PIP5K{beta} or mock vector did not exhibit such a phenotype (Fig. 6A). Next, we co-transfected a vector containing EGFP with a vector harboring each wild-type PIP5K{beta} or PIP5K{beta}-KD into CHO cells, and the DNA content of EGFP-positive cells was assessed by flow cytometry 48 h after transfection. As shown in Fig. 6B, overexpression of PIP5K{beta}-KD produced cells with 4 N DNA content more frequently (35.4% of the EGFP-positive cells) compared with those induced by mock vector (9.1% of the EGFP-positive cells) and wild-type PIP5K{beta} (11.7% of the EGFP-positive cells), indicating that overexpression of PIP5K{beta}-KD affects cell division. No significant accumulation of PI(4,5)P2 at the cleavage furrow was observed in ~65% (45/72 cells) of the cells transfected with PIP5K{beta}-KD. In contrast, a robust accumulation of PI(4,5)P2 was apparent in ~75% (41/56 cells) of the mock-transfected cells during late telophase (Fig. 6C, arrow). These results suggest that PIP5K{beta}-KD functions as a dominant negative mutant to inhibit PI(4,5)P2 production at the cleavage furrow during cytokinesis. Because cell division of the PIP5K{beta}-KD-transfected cells proceeds to the stage just before the separation of two daughter cells with single nuclei (Fig. 6C), it is likely that PIP5K{beta} contributes to PI(4,5)P2 production at the cleavage furrow and that PI(4,5)P2 in the furrow membrane is critical in the progression of the telophase of cytokinesis.



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  		FIGURE 4.
PIP5K{beta} co-localizes with RhoA in the cleavage furrow. A, CHO cells were fixed and co-stained with anti-PIP5K{beta} antibody (green) and anti-RhoA antibody (red). B-D, CHO cells transfected with either FLAG-tagged PIP5K{beta} (green in B and C) or FLAG-tagged PIP5K{gamma} (green in D) were fixed and stained with anti-RhoA antibody (red in A, C, and D) or anti-tubulin antibody (red in B). Right panels are merged images. Arrows indicate the cleavage furrow. Scale bar,10 µm.

 
To further confirm the role of PI(4,5)P2 in cytokinesis, we examined the effects of two distinct anti-PI(4,5)P2 monoclonal antibodies, AM212 (35) and 2C11 (36), on cell division. To examine the specific effects of these antibodies on cytokinesis, cells were once synchronized at G1/S phase by the double thymidine block (10) and were then microinjected with either 2C11, AM212, or control IgG ~1 h before the initiation of mitosis. Although cells injected with control mouse IgG divided normally, ~40% of cells injected with either AM212 (38.1 ± 7.5; n = 5) or 2C11 (31.7 ± 4.6; n = 3) became multinucleated 24 h after injection (Fig. 5). These results provide the first direct evidence of PI(4,5)P2 involvement in cytokinesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Blockage of RhoA Down-regulation by PE-binding Peptide—Compared with the recent progression in understanding the molecular mechanisms involved in the formation and constriction of the contractile ring, the mechanisms underlying the disassembly of the contractile ring and the final membrane separation remain largely unknown. In the present study, we found that PE-binding peptide inhibited disassembly of RhoA from the cytoplasmic bridge membrane, to which the peptide bound (Fig. 1A). A pull-down assay for GTP-RhoA revealed that RhoA inactivation during late cytokinesis was prevented by the peptide treatment (Fig. 1B), suggesting that RhoA at the bridge is the GTP-bound active form. In addition, inhibition of RhoA inactivation was reversed by removal of the surface-bound peptide (Fig. 1). These findings suggest that exposure of PE on the cleavage furrow membrane might be involved in the temporospatial down-regulation of RhoA activity during late cytokinesis.

Two proteins, Cyk-4/MgcRacGAP (13, 14) and Nir2 (15), are shown to play an essential role in completion of cytokinesis by down-regulation of RhoA functions. Interestingly, both Cyk-4/MgcRacGAP and Nir2 each contain a putative lipid-binding domain, C1 homologous domain and phosphatidylinositol transfer domain, respectively. Because both lipid-binding domains are closely adjacent to their functional domains (GAP domain of Cyk-4/MgcRacGAP; Rho-inhibitory domain of Nir2), it might be possible that lipids on the furrow membrane may be involved in regulation of their activities. It would be feasible to investigate the effects of lipid molecules on their activity in vitro.



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  		FIGURE 5.
Inhibition of cytokinesis by microinjection of anti-PI(4,5)P2 antibodies. A, two distinct anti-PI(4,5)P2 antibodies, AM212 and 2C11, were co-microinjected with Oregon green-labeled dextran (injection marker) into synchronized cultures ({approx}1 h before the initiation of mitosis) of CHO cells. Injected cells were identified by Oregon green-labeled dextran (left panels). Cells were also co-stained for DNA with DAPI (middle panels) and for F-actin with rhodamine-phalloidin (right panels). Arrows indicate multinucleated cells. Scale bar, 20 µm. B, formation of multinucleated cells by microinjection of the designated antibodies. Oregon green-positive multinucleated cells were counted under a fluorescence microscopy. Data are average of three independent experiments.

 
Local Production of PI(4,5)P2 at Cleavage Furrow—During normal cytokinesis, GFP-PH was found to accumulate at the cleavage furrow membrane (Fig. 3, C and D). Such accumulation was not observed by using GFP-PH (R40A), a mutant GFP-PH with low binding affinity to PI(4,5)P2, or GFP-PH(PKB/Akt), a PI(3,4,5)P3-specific probe (37, 38), suggesting a specific accumulation of PI(4,5)P2 on the furrow membrane. PI(4,5)P2 in the plasma membrane appears to be predominantly produced by phosphorylation of phosphatidylinositol 4-phosphate by PIP5K at the D-5 position of the inositol ring (30, 31, 37). Immunohistochemical studies indicated that PIP5K{beta} (but not PIP5K{gamma}) accumulates at the cleavage furrow during cytokinesis (Fig. 4). In addition, a kinase-deficient PIP5K{beta} mutant prevented PI(4,5)P2 accumulation at the furrow (Fig. 6C), strongly suggesting that PIP5K{beta} contributes to PI(4,5)P2 production at the cleavage furrow of dividing cells. PIP5K has been reported to be physically associated with and activated by small GTPases, such as Arf6 (29), Rac (39), and RhoA (40, 41). During cytokinesis, most of RhoA accumulates in the cleavage furrow (10-12). Cellular level of the GTP-bound active form of RhoA is also transiently increased during cytokinesis (10). In addition, PIP5K{beta} co-localized with RhoA at the furrow during late telophase, as well as at the cytoplasmic bridge of the peptide-treated cells (Fig. 4B). It is therefore likely that PIP5K{beta} is recruited to the furrow and activated by RhoA during cytokinesis, resulting in local production of PI(4,5)P2 at the furrow membrane. A very recent report suggests that phosphatase with tensin homology (PTEN), a PI3 phosphatase, is localized at the cleavage furrow of dividing Dictyostelium discoideum cells to reduce phosphatidylinositol 1,4,5-trisphosphate concentration on the furrow membrane (42). It is thus also possible that PIP5K may cooperate with PTEN to elevate PI(4,5)P2 concentration at the furrow.



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  		FIGURE 6.
Effects of a kinase-deficient mutant of PIP5K{beta} on cytokinesis. A, CHO cells were transfected with either GFP vector alone (Mock), FLAG-tagged PIP5K{beta} (PIP5K{beta}), or a kinase-deficient mutant of PIP5K{beta} (PIP5K{beta}-KD) and cultured for 36 h. Cells expressing wild-type or the mutant PIP5K{beta} are visualized by FLAG staining (green). DNA was stained with DAPI (blue). Scale bar, 20 µm. B, flow cytometric analyses of DNA contents. CHO cells were co-transfected with GFP and either vector alone (Mock), FLAG-tagged PIP5K{beta} (PIP5K{beta}), or a kinase-deficient mutant of PIP5K{beta} (PIP5K{beta}-KD). After 48 h of culture, DNA contents of GFP-positive cells were analyzed. C, effects of a kinase-deficient mutant of PIP5K{beta} on PI(4,5)P2 production in the cleavage furrow. CHO cells co-transfected with GFP-PH (green) and either vector alone (Mock) or a kinase-deficient mutant of PIP5K{beta} (PIP5K{beta}-KD) were cultured for 24 h and stained with anti-tubulin antibody (red). Right panels are merged images. Scale bar, 10 µm.

 
It has been demonstrated that Rho-kinase/ROCK mediates PIP5K activation by RhoA (43, 44). PI(4,5)P2 accumulation both in the cleavage furrow of normal dividing cells and in the cytoplasmic bridge of the peptide-treated cells was not prevented by a Rho-kinase/ROCK-specific inhibitor Y-27632 (data not shown). Thus, multiple different signaling pathways, including the ROCK signaling pathway, might be involved in PIP5K regulation by Rho GTPase during cytokinesis.

Microinjection of either of two distinct anti-PI(4,5)P2 antibodies, 2C11 and AM212, inhibited cytokinesis (Fig. 5A). In addition, overexpression of a kinase-deficient mutant of PIP5K prevented both local production of PI(4,5)P2 at the cleavage furrow (Fig. 6C) and completion of cell division (Fig. 6, A and B). These results strongly suggest that PI(4,5)P2 at the furrow membrane plays an essential role in proper progression of cytokinesis. PI(4,5)P2 might also be involved in regulation of the nucleus structure, because the nucleus morphology appeared abnormal in the injected cells (Fig. 5A).

Neither furrow formation nor early furrow ingression appeared to be affected by overexpression of the kinase-deficient mutant of PIP5K (Fig. 6C). Together with the observation that PI(4,5)P2 accumulation at the furrow membrane became detectable in the telophase (Fig. 3, C and D), these data suggest that local production of PI(4,5)P2 at the furrow membrane is required in late processes of cytokinesis. This possibility is consistent with previous studies in other organisms, such as budding yeast (45), Drosophila spermatocytes (46), and Drosophila cultured cells. A Drosophila mutant defective in PI 4-kinase, which generates PI 4-phosphate, a precursor of PI(4,5)P2 for PIP5K, has a defect in germ line cytokinesis (46). Interestingly, neither formation nor constriction of the contractile ring is impaired in the dividing spermatocytes, but the furrow is unstable, and daughter cells fuse each other. Thus, phosphatidylinositol derivatives including PI(4,5)P2 may play a critical role in stabilizing the interactions between the contractile ring and the plasma membrane in the final stage of cytokinesis, rather than the contractile ring assembly or constriction. This idea is supported by the observation that the contractile ring components, including F-actin, myosin II, ezrin/radixin/moesin proteins, and RhoA, were stabilized in the cytoplasmic bridge of the PE-binding peptide of the treated cells, in which PI(4,5)P2 is highly accumulated (Fig. 2C) (27).

Lipid Domain Formation at Cleavage Furrow and Contractile Ring Rearrangement—In this study, we showed that PI(4,5)P2 is predominantly localized at the furrow membrane during cytokinesis (Fig. 3, C and D). This PI(4,5)P2 production on the inside of the furrow membrane appears to be coupled to the PE exposure on the furrow surface, because PI(4,5)P2 accumulation was induced by immobilization of cell surface PE by a PE-binding peptide (Fig. 2C). These findings raise the possibility that a unique membrane domain with a particular lipid composition is formed at the cleavage furrow membrane during cytokinesis. This possibility is consistent with previous observations that the furrow membrane has a different lipid as well as protein composition from the cortical membrane (23, 24).

Several models could be proposed to explain the role of this particular lipid domain in cytokinesis. One explanation is that PI(4,5)P2 regulates cytoskeletal dynamics during cytokinesis. PI(4,5)P2 binds directly to a variety of actin-regulatory proteins and modulates their functions (31, 32). Of the PI(4,5)P2-binding proteins, ERM proteins (47), profilin (48), and cofilin (49) are implicated in cell division. Alternatively, local accumulation of PI(4,5)P2 may anchor PH domain-containing proteins, such as anillin (50), to the plasma membrane. Mammalian septins, such as Nedd5 (51) and H5 (52), also contain a conserved polybasic domain that binds PI(4,5)P2 and thus may require PI(4,5)P2 for the spatial assembly at the furrow membrane of dividing cells.

An intriguing alternative explanation is that the PE- and PI(4,5)P2-enriched domains may play a role in vesicle fusion during cleavage furrow formation, which provides proteins and lipids to an enlarged plasma membrane surface as cytokinesis progresses (16-19). The role of PI(4,5)P2 in vesicle trafficking and membrane fusion has been demonstrated in a variety of experimental settings (31). PE-rich membranes also tend to form non-bilayer intermediates that have been hypothesized to exist during the membrane fusion process (22). It is thus possible that the PE- and PI(4,5)P2-enriched membrane may provide an active zone to the cleavage furrow for efficient membrane fusion as well as scission. It is of interest to note that fly embryos mutant for either Rab11 or nuclear fallout (Nuf), both involved in trafficking of vesicles through the recycling endosome, display severe defects in actin cytoskeleton remodeling during cellular furrow formation, suggesting that cytoskeletal remodeling during cell division requires membrane delivery to the furrow (1-3). Our studies showed that PE exposure on the furrow surface plays a crucial role in actin reorganization during late cytokinesis as well as membrane separation (27, 28). Thus, the lipid domain formed on furrow membrane may provide a link between membrane separation and actin remodeling through regulating the membrane trafficking and vesicle fusion to the furrow membrane. Further probing the dynamic change of membrane lipid distribution as well as composition in the cleavage furrow will provide new insight into the role of membrane lipid in cytokinesis.


   FOOTNOTES
 
* 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

1 Present address: Howard Hughes Medical Institute, Department of Physiology and Biochemistry, University of California San Francisco, 1550 4th St., San Francisco, CA 94143. Back

2 To whom correspondence should be addressed: Div. of Supramolecular Biology, Institute for Chemical Research, Uji, Kyoto 611-0011, Japan. Tel.: 81-774-38-3250; Fax: 81-774-38-3256; E-mail: umeda{at}scl.kyoto-u.ac.jp.

3 The abbreviations used are: ROCK, Rho-associated coiled coil-forming protein kinase; PE, phosphatidylethanolamine; PIP5K, phosphatidylinositol 4-phosphate 5-kinase; PH, pleckstrin homology; GFP, green fluorescent protein; GFP-PH, green fluorescent protein-tagged pleckstrin homology domain of phospholipase C{delta}1; EGFP, enhanced green fluorescent protein; Ro, Ro09-0198; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; DAPI, 4'6'-diamidino-2'-phenylindole; KD, kinase-deficient; PKB, protein kinase B; GAP, GTPase-activating protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. H. Yagisawa (Himeji Institute of Technology) for expression vector PH-GFP, Dr. U. Kikkawa (Kobe University) for expression vector PH-GFP (PKB/Akt), and Drs. K. Fukami and T. Takenawa (University of Tokyo) for anti-PI(4,5)P2 monoclonal antibody 2C11.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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