The actin-binding and bundling protein, EPLIN, is required for ...

[Cell Cycle 8:5, 757-764; 1 March 2009]; ©2009 Landes Bioscience

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The actin-binding and bundling protein, EPLIN, is required for cytokinesis

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Megan Chircop,1,2 Vanessa Oakes,1 Mark E. Graham,2 Maggie P.C. Ma,2 Charlotte M. Smith,2 Phillip J. Robinson2 and Kum Kum Khanna1 Institute of Medical Research; Post Office Royal Brisbane Hospital; Brisbane, Queensland, Australia; 2Children’s Medical Research Institute; The University of Sydney; Westmead, New South Wales, Australia

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among distant organisms, septins are required for cytokinesis in D. melanogaster,13 C. elegans14 and mammals.7 Septin filaments associate with actin filaments at the cleavage furrow, where they co-operate to achieve membrane ingression.10,15 These two filament systems are thought to be linked indirectly by the actin-binding protein anillin6,8,16 or potentially by other actin-binding proteins and/or directly since Sept2 binds myosin II.17 Several lines of evidence suggest that the actin-binding protein, Epithelial Protein Lost In Neoplasm (EPLIN), may play a role in cytokinesis. EPLIN is preferentially expressed in human epithelial cells.18 Its expression is lost in most epithelial cell-derived human tumors, which may contribute to the motility of invasive tumor cells.19 Two EPLIN isoforms, α and β, are expressed from a single gene,18,20 where the β isoform is 160 amino acids longer at its N-terminus.20 EPLIN contains a centrally located LIM domain and via its N- and C-termini binds actin to promote the parallel formation of actin filament structures by cross-linking and bundling actin filaments.19 This is in an analogous manner to the cleavage furrow protein anillin.21 In this study we show loss of EPLIN expression causes cytokinesis failure. This is associated with mis-localization of key cytokinesis proteins, such as Sept2 and the GTPase, RhoA and Cdc42, to the cleavage furrow. The results suggest a mechanism by which loss of EPLIN may induce aneuploidy, which contributes to genomic instability and cancer.

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Cytokinesis involves two phases: (1) membrane ingression followed by (2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLCS19) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.

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Key words: EPLIN, actin, myosin II, Sept2, cytokinesis, contractile ring, RhoA, Cdc42

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Introduction

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Cytokinesis in mammalian cells begins during anaphase with membrane ingression at the cell equator forming an intracellular bridge between nascent daughter cells. Ingression is driven by a contractile ring, composed of parallel filaments of actin and myosin II.1 Myosin regulatory light chain (MRLC) is a component of the myosin II filaments, and its phosphorylation pn T18 and S19 drives the assembly of the actin-myosin II contractile ring2 and activity of the myosin II motor.3,4 The major phosphorylation site is S19, which allows myosin II to interact with actin to assemble an actinmyosin II complex and initiate contraction.5 A second cytoskeletal regulator of cytokinesis is the septin family of GTP-binding proteins that form filamentous complexes.6 Of the twelve septin genes in mammals, Sept2, Sept4, Sept7 and Sept9, localize to the cell cortex, contractile ring and midbody of mitotic cells.7-12 Cytokinesis is disrupted by micro-injection of anti-Sept2 or anti-Sept9 antibodies and by transfection of siRNAs against Sept2, Sept7 or Sept9.7,9,10 Despite mechanistic differences in cytokinesis *Correspondence to: Megan Chircop; Children’s Medical Research Institute; 214 Hawkesbury Road; Westmead, New South Wales 2145 Australia; Tel.: +61.2.9687.2800; Fax: +61.2.9687.2120; Email: [email protected] Submitted: 01/04/09; Accepted: 01/18/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/7878 www.landesbioscience.com

Results Depletion of EPLIN induces cytokinesis failure. To assess if EPLIN is required for cytokinesis we utilized siRNA to deplete EPLIN from HeLa cells. Two siRNA sequences targeting EPLIN were tested for their knockdown efficiency. At 72 h post-transfection, EPLINα expression was reduced by both siRNA sequences compared to untransfected and GFP siRNA transfected controls (Fig. 1A). EPLIN siRNA sequence 1 was slightly more efficient. A longer exposure of the immunoblot was required to visualize the effect of these siRNAs on the β isoform, which was also reduced. A significant number of EPLIN-depleted cells were multinucleated, indicating failed cytokinesis (Fig. 1B and C). Sequence 1 resulted in a higher incidence of multinucleation than sequence 2 (Fig. 1B). We next determined that EPLIN depletion was directly causing cytokinesis failure and was not an indirect effect that occurred following

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several rounds of division. Following only one round of division, 32.8 ± 7.9% of cells depleted of EPLIN (sequence 1) were multinucleated compared to control cells (8.7 ± 1.2% of GFP siRNA transfected; Fig. 1C). We conclude that EPLIN is required for the successful completion of cytokinesis. EPLIN localizes to the cleavage furrow and associates with myosin II. The requirement of EPLIN for cytokinesis and its previously established association with actin implies that it may associate with the contractile ring. Therefore, we sought to investigate if EPLIN was localized to the cleavage furrow. Since the polyclonal anti-EPLIN antisera does not distinguish between the two EPLIN isoforms, α and β,18 we assessed the localization of each isoform using two different cell lines, HeLa and MCF-7 cells, which predominantly express the α and β isoform, respectively (Fig. 2A). In both cell lines, endogenously expressed EPLIN was highly enriched at the cell cortex during metaphase (Fig. 2B and data not shown). As cells progressed into telophase, EPLIN, like actin, accumulated in the cleavage furrow, with less associated with the cell cortex (Fig. 2B). Pre-immune sera did not decorate these cellular locations in HeLa and MCF-7 cells (data not shown). The myosin II inhibitor, blebbistatin, blocks furrow contraction without disrupting the positioning of the contractile ring.22 Endogenous EPLINα was concentrated at the blebbistatin-arrested furrow in HeLa cells where it co-localized with TRITC-phalloidin (Fig. 2C). This staining is reminiscent to the location of anillin and myosin II in a blebbistatin-arrested cleavage furrow.23 We also provide evidence that EPLIN associates with the contractile ring as myosin II was immunoprecipitated with both Flagtagged EPLIN α and β proteins from asynchronously growing HeLa cells (Fig. 2D) and from HeLa cells synchronised at early mitosis and cytokinesis (Fig. 2E). As expected this complex also contained actin (Fig. 2E). We conclude that EPLIN can associate with components of the contractile ring including actin and myosin IIb. EPLIN contributes to the accumulation of actin and active myosin II at the cleavage furrow. We next asked whether the localization of cleavage furrow components is disrupted in EPLINdepleted cells. EPLIN expression was observed to be homogenously depleted in all cells by >90% at the cell cortex and stress fibres during interphase (Fig. 3A) and at the cell cortex and cleavage furrow during cytokinesis (Fig. 3B). Consistent with the previously described role for EPLIN in actin stabilisation, cortical actin staining was observed to be absent/reduced in interphase cells depleted of EPLIN compared to GFP siRNA cells (Fig. 3C). Similarly, we observed a reduction in actin at the cell cortex and no cleavage furrow accumulation of actin during cytokinesis in EPLIN-depleted cells (Fig. 3D). Interphase microtubules seemed to show increased bundling. However, midzone microtubules established during cytokinesis were not affected (Fig. 3C and D). The actin-binding partner, myosin IIb concentrated at the cleavage furrow from early (anaphase) to completion of ingression (telophase; Fig. 3E), as previously described.22,24 In contrast to actin, its localization was not affected in EPLIN-depleted cells (Fig. 3E). This was confirmed by quantifying the fluorescence intensity of myosin IIb staining in EPLIN-depleted cells compared to GFP siRNA cells (data not shown). We next assessed the activity of the myosin II motor by using an antibody that specifically recognises MRLC phosphorylated at S19. During anaphase, MRLCS19 localized normally to the ingressing furrow in EPLIN-depleted cells (Fig. 3F). However, during the final stages of ingression, MRLCS19 staining

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Figure 1. EPLIN depletion increases binucleated HeLa cells. (A) Lysates prepared from untransfected HeLa cells and HeLa cells transfected with GFP siRNA or EPLIN siRNA 1 or 2 at 60 h post-transfection were immunoblotted with anti-EPLIN antibodies. Short and long exposures of the immunoblot are shown to display expression levels of EPLIN α and β. γ-tubulin levels show equal loading. (B) HeLa cells were transfected as described in A). At 60 h post-transfection, cells were immunostained for α-tubulin. Graph represents percentage of cells with ≥2 nuclei/cell (multinucleation) for each experimental sample (mean ± S.D. from three independent experiments). Per sample >200 cells were scored. (C) HeLa cells were synchronized by double thymidine block and transfected with the indicated siRNA. At 20 h post-release from the second thymidine block, cells were fixed and immunostained for α-tubulin. Per sample >200 cells were scored for multinucleation. Graphs illustrate the mean ± S.D. from two independent experiments.

was reduced at the cleavage furrow of EPLIN-depleted cells (Fig. 3F). The fluorescence intensity ratio of MRLCS19 staining at the cleavage furrow/polar region revealed a 40.7% reduction at the cleavage furrow in EPLIN-depleted cells compared to control GFP siRNA cells (Fig. 3G). A similar result (37.2% reduction) was achieved by quantifying the fluorescence intensity of MRLCS19 staining at the cleavage furrow and normalising this to the amount of myosin II (heavy chain) staining (data not shown). Therefore, EPLIN contributes to the efficient accumulation of actin and active myosin II at the cleavage furrow during the final stages of ingression. EPLIN associates with Sept2 and is required for its cleavage furrow localization. Septin structural filaments co-operate with actin filaments at the cleavage furrow to achieve membrane ingression.6,10,15 However,

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Figure 2. EPLIN locates to the cleavage furrow and associates with myosin IIb during cytokinesis. (A) HeLa and MCF-7 cell lysates immunoblotted with antiEPLIN anti-sera. These cells predominantly express the EPLIN α and β isoforms, respectively. (B) Representative images of asynchronously growing HeLa cells at the indicated mitotic phase are shown displaying localization of EPLIN (red) and α-tubulin (green). DNA shown in blue. (C) HeLa cells expressing endogenous EPLIN were arrested at the onset of cytokinesis by the active myosin II inhibitor, blebbistatin. Representative microscopy images reveal that EPLIN co-localizes with actin filaments (TRITC-phalloidin) at the cleavage furrow. (D) Lysates prepared from asynchronously growing HeLa cells expressing Flag vector, or Flag-EPLIN α or β were immunoprecipitated with an anti-Flag antibody and immunoblotted for myosin IIb. Immunoblots show that myosin IIb associates with both EPLIN isoforms. (E) HeLa cells expressing Flag-EPLINβ were synchronized in early mitosis (prophase) or cytokinesis using nocodazole. Lysates were immunoprecipitated with an anti-Flag antibody and immunoblotted for myosin IIb and actin. Actin and myosin IIb associate with EPLINβ during all stages of mitosis.

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this interaction is not direct and thought to be mediated via an actinbinding protein. Thus, we aimed to determine if EPLIN is required for Sept2 cleavage furrow localization. As previously described,7 we confirm that endogenous Sept2 localizes to the ingressing cleavage furrow and midbody region that contains microtubules (Fig. 4A). We show that Sept2 and GFP-EPLIN α and β co-localize at the intersection where the cleavage furrow meets the midbody microtubules (Fig. 4A). Endogenous EPLIN associated with GST-tagged Sept2 in vitro using a pull-down assay from HeLa cell lysates. GST alone or another septin family member, GST-Sept7 did not bind EPLIN (Fig. 4B). Furthermore, immunoprecipitation assays demonstrated that Flag-EPLINβ and Sept2 associate in HeLa cells during interphase and mitosis (Fig. 4C). Despite the variation in Sept2 present in each of the lysates, densitometry revealed a 2.2 fold increase in Sept2 association with Flag-EPLINβ during mitosis compared to during interphase (data not shown). We conclude that EPLIN can associate with another cleavage furrow component, Sept2.

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Next, we asked whether Sept2 localization at the cleavage furrow is dependent on EPLIN. At the onset of cytokinesis, Sept2 localized normally to the ingressing furrow in EPLIN-depleted cells (Fig. 5A). However, during late stage cytokinesis, Sept2 localization to the cleavage furrow/midbody was either aberrant and/or reduced in approximately 27.2 ± 5.8% of EPLIN-depleted cells (n = 29) compared to 3.5 ± 0.5% of control cells (n = 58; Fig. 5A). This reduction correlates with the number of failed cytokinetic events observed in cells depleted of EPLIN (Fig. 1). The defect in Sept2 localization was observed using either siRNA (data not shown) and was not due to a decrease in Sept2 protein levels (Fig. 5B). Thus, EPLIN contributes to Sept2 cleavage furrow localization during late stages of cytokinesis. EPLIN contributes to accumulation of the GTPases, RhoA and Cdc42, at the cleavage furrow. During cytokinesis, the phosphorylation of MRLC at the cleavage furrow is mediated by Rho-kinase (ROCK) and citron kinase, which are activated by the Rho GTPase,

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Figure 3. EPLIN is required for actin and active myosin cleavage furrow localization. (A-D) Control GFP siRNA and EPLIN-depleted HeLa cells were fixed 72 h post-transfection (A and C) or synchronised in cytokinesis using RO-3306 (B and D) then fixed and stained for EPLIN, α-tubulin and TRITC-phallodin. DNA is shown in blue. (E and F) HeLa cells were transfected with GFP siRNA and EPLIN siRNA and synchronised by double thymidine block as described in Figure legend 1C. At 10.5 h following release from the second thymidine block, cells were fixed and immunostained for myosin II (E) and MRLCS19 (F). DNA is shown in blue. Representative microscopy images of cells in early cytokinesis (anaphase) and late cytokinesis (telophase) are shown. (G) Quantification of MRLCS19 in control and EPLIN-depleted HeLa cells during telophase. Amount of MRLCS19 is expressed as ratio of fluorescence intensity at the ingressing cleavage furrow of the cell over the fluorescence intensity at the polar region. In EPLIN-depleted cells (n = 5), MRLCS19 is significantly reduced at the cleavage furrow compared with control GFP siRNA cells (n = 4).

RhoA.25,26 During late stage cytokinesis, we observed the concentration of RhoA to be significantly reduced at the cleavage furrow in cells depleted of EPLIN compared to control cells (Fig. 5C). Instead, it was dispersed in a diffuse pattern in the vicinity of the cleavage furrow indicating that EPLIN is required for RhoA recruitment to the cleavage furrow during late stage cytokinesis. 760

Cdc42 localizes to the cleavage furrow in mammalian cells and recruits Sept2 for cytokinesis.27 Cdc42 also regulates septin ring formation in budding yeast during mitosis.28 Analysis of Cdc42 localization confirmed that it accumulates at the ingressing cleavage furrow/midbody in control cells (Fig. 5C). In contrast, the concentration of Cdc42 at the ingressing cleavage furrow/midbody was

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Although EPLIN associates with the cleavage furrow throughout the entire duration of ingression it does not appear to be required for the initial assembly of these cytoskeletal systems. Rather, it appears to function during the final stages of ingression. In support of this idea, only in late stage cytokinesis do we observe that depletion of EPLIN affects recruitment of active myosin II to the cleavage furrow to maintain contractile ring activity, and cleavage furrow recruitment of Sept2 and the small GTPases, RhoA and Cdc42, to control myosin activity and Sept2, respectively. Septin filaments have been proposed to act in protein recruitment29 and as diffusion barriers.30,31 Sept2 recruits myosin II to actin filaments during interphase and cytokinesis in mammalian cells. This direct association is important for full activation of myosin II, i.e., MRLC phosphorylation, during cytokinesis.17 In S. cerevisiae, septin rings are located on both sides of the actinomyosin ring to act as barriers, compartmentalizing the cortex around the cleavage site.32 This maintains diffusible cortical factors, such as the exocyst and the polarizome, at the cleavage site for actinomyosin ring function and membrane abscission.32 A similar diffusion barrier exists at the mammalian midzone.33 Therefore, mammalian septins at the ingressing furrow may function as a diffusion barrier to locally concentrate cleavage furrow proteins to a defined area. EPLIN may be essential to maintaining this diffusion barrier by linking the septin filaments to the contractile ring. We propose that this would trap critical regulators such as RhoA and Cdc42 at the cleavage furrow Figure 4. EPLIN associates with Sept2 during cytokinesis. (A) Representative images of HeLa to maintain sufficient local concentrations of active cells ectopically expressing GFP-EPLIN α and β immunostained for Sept2. An enlargement myosin for completion of ingression. of the ingressing furrow shows EPLIN and Sept2 in close proximity at the cleavage furrow/ EPLIN α and β localize to the cleavage furrow and midbody microtubule interface during cytokinesis. (B) GST alone, GST-Sept2 and GST-Sept7 bound to GS beads were incubated with HeLa cell extract. The amount of EPLIN bound to each their depletion leads to cytokinesis failure suggesting was assessed by immunoblotting with anti-EPLIN antibodies. Expression of GST recombinant that they perform similar roles during cytokinesis. protein was determined by coomassie blue staining. (C) Lysates from HeLa cells synchronized Different cell types preferentially express one of the in interphase and mitosis using nocodazole that ectopically expressed Flag-EPLINβ were immunoprecipitated with an anti-Flag antibody. Immunoprecipitated proteins were immunoblotted two isoforms suggesting that they perform specific functions.18 The identification of binding partners for Sept2. that bind to, or post-translational modification(s) that exist within, the additional 160 amino acids at reduced or mis-localized in EPLIN-depleted cells (Fig. 5C). These the N-termini of the β-isoform may reveal potential isoform-specific findings indicate that EPLIN is required for Cdc42 cleavage furrow functions. localization. Cytokinesis failure results in aneuploidy, increasing genomic instability and oncogenic potential. Multinucleated cells are frequently Discussion observed in tumours.34 Given EPLIN is frequently lost in tumors18 In this study, we show that the actin-binding and bundling and that its absence leads to multinucleation and aneuploidy, it is protein, EPLIN, is required for cytokinesis. Its role in this process possible that loss of EPLIN is one of the crucial steps required for appears to be linked to its ability to associate with components of oncogenic progression. Thus, the tumorigenic stage at which EPLIN the two cytoskeletal systems required for membrane ingression and is observed to be lost will be an important finding as this may idencleavage furrow formation: (1) the actin-myosin II contractile ring tify EPLIN as a biomarker that can be used in a diagnostic setting. and (2) the septin filaments. These two filament systems co-operate Materials and Methods to achieve membrane ingression to complete cytokinesis. Thus, Cell culture and transfection. HeLa (human cervical carcinoma EPLIN may act as an adaptor protein, mediating the interface between the contractile ring and septin filaments during ingression. cell line) cells were maintained in RPMI 1640 medium supplewww.landesbioscience.com

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mented with 10% foetal bovine serum (FBS). Cells were grown at 37°C in a humidified 5% CO2 atmosphere and seeded in 9 cm2 dishes and transfected at 50–60% confluence with 7.5 μg of plasmid DNA for immunoblotting. For immunofluorescence studies, cells were seeded onto coverslips and transfected at 50–60% confluence with 1.5 μg of plasmid DNA. In both cases, cells were transfected with Lipofectamine (Invitrogen) according to the Manufacturer’s instructions. Cell synchronization. For mitotic synchronization, cells were treated with 0.5 μg/ml nocodazole for 16 h. Cells arrested in early mitosis (prophase) were collected by “mitotic shake-off ”. These mitotic cells were then released from the nocodazole block by washing thoroughly with PBS and released into medium containing 100 μM Blebbistatin. Cells were seeded onto coverslips then incubated for 3 h at 37°C in a humidified 5% CO2 atmosphere. For immunoprecipitation experiments, cells were synchronised in cytokinesis by allowing prophase cells (collected by “mitotic shake-off ”) to progress through mitosis for 2.5 h at 37°C in a humidified 5% CO2 atmosphere following release from the nocodazole block. Alternatively, cells were synchronized by double thymidine block assay. In brief, cells were treated with 2.5 mM thymidine (Sigma) in media for 18 h, and then released into media containing 2.4 μM thymidine and 2.4 μM deoxycytidine. At the start of this release period, cells were transfected with the indicated siRNA. After 25 h, cells were treated again with 2.5 mM thymidine for 18 h, and then washed three times with warm PBS allowing cells to enter the cell cycle. Cells were incubated in pre-warmed medium for 10.5 h prior to fixation for immunofluorescence microscopy analysis to enrich the cell population in mitosis. Where indicated, HeLa cells were synchronised at the G2/M border by treatment with the selective cdk1 small-molecule inhibitor, RO-3306 (9 μM)35 for at least 18 h. Cells were allowed to progress through mitosis following RO-3306 wash-out and incubation in pre-warmed medium. Cells were fixed for immunofluorescence microscopy after 2 h when the cell population was enriched in cytokinesis. Plasmid construction. Wild-type and deletion constructs of pGFP-EPLINα, pGFP-EPLINβ, pFlag-EPLINα and pFlag-EPLINβ were described previously.19 The construction of GST-Sept2 and GST-Sept7 were described previously.7,10 siRNA to EPLIN and GFP were generated using the DNA-based pSUPER construct.36 The siRNA target sequences in the sense orientation are: EPLIN siRNA 1, 5'-GGT GAA CCA ACT CAA ACT A-3' and EPLIN

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Figure 5. EPLIN functions during late stage of ingression to recruit Sept2 and the regulatory GTPases, RhoA and Cdc42, to the cleavage furrow. (A) Control (GFP siRNA) and EPLIN-depleted HeLa cells were synchronised by double-thymidine block, then stained for Sept2 (red) at 10.5 h post-release. Representative images show that Sept2 midbody localization at completion of ingression (telophase) is aberrant in EPLIN-depleted cells. DNA is shown in blue. The percentage of HeLa cells (mean ± S.D. from two independent experiments) at the final stages of ingression with reduced/mis-localised cleavage furrow localisation of Sept2 for each experimental sample is shown below. For each sample n > 30. (B) Lysates from untransfected HeLa cells and HeLa cells transfected with GFP siRNA or EPLIN siRNA 1 were immunoblotted for EPLIN and Sept2. (C) Representative images of control (GFP siRNA) and EPLIN-depleted HeLa cells at 10.5 h post-release from double thymidine block stained for RhoA and Cdc42 (red). Images show that cleavage furrow localization of these proteins is either reduced or aberrant in EPLIN-depleted cells. DNA is shown in blue.

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siRNA 2; 5'-GTG GAA GGA AGA TCT CTG A-3'. The GFP siRNA construct was described previously.37 Details of primers used are available upon request. All plasmids were confirmed by DNA sequencing. Immunofluorescence microscopy. For RhoA localization experiments, cells were fixed with ice-cold 10% TCA for 15 mins.38 For all other immunostaining experiments, cells were fixed in 4% paraformaldehyde/PBS for 20 mins at room temperature. Following fixation, cells were washed three times with PBS, permeabolized in 0.2% Triton X-100/PBS, then blocked in 3% bovine serum albumin/PBS for 45 mins before the required primary antibody was applied. Antibodies

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Characterization of the mammalian septin H5: distinct patterns of cytoskeletal and membrane association from other septin proteins. Cell Motil. Cytoskeleton 1999; 43:52-62. 12. Zhang J, Kong C, Xie H, Mcpherson PS, Grinstein S, Trimble WS. Phosphatidylinositol polyphosphate binding to the mammalian septin H5 is modulated by GTP. Curr Biol 1999; 9:1458-67. 13. Neufeld TP, Rubin GM. The Drosophila peanut gene is required for cytokinesis and encodes a protein similar to yeast putative bud neck filament proteins. Cell 1994; 77:371-9. 14. Nguyen TQ, Sawa H, Okano H, White JG. The C. elegans septin genes, unc-59 and unc61, are required for normal postembryonic cytokineses and morphogenesis but have no essential function in embryogenesis. J Cell Sci 2000; 113:3825-37. 15. Kinoshita M. The septins. Genome Biol 2003; 4:236. 16. Oegema K, Savoian MS, Mitchison TJ, Field CM. Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis. J Cell Biol 2000; 150:539-52. 17. Joo E, Surka MC, Trimble WS. Mammalian SEPT2 is required for scaffolding nonmuscle myosin II and its kinases. Dev Cell 2007; 13:677-90. 18. Maul RS, Chang DD. EPLIN, epithelial protein lost in neoplasm. Oncogene 1999; 18:7838-41. 19. Maul RS, Song Y, Amann KJ, Gerbin SC, Pollard TD, Chang DD. EPLIN regulates actin dynamics by cross-linking and stabilizing filaments. J Cell Biol 2003; 160:399-407. 20. Chen S, Maul RS, Kim HR, Chang DD. Characterization of the human EPLIN (Epithelial Protein Lost in Neoplasm) gene reveals distinct promoters for the two EPLIN isoforms. Gene 2000; 248:69-76. 21. Field CM, Alberts BM. Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J Cell Biol 1995; 131:165-78. 22. Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, Sellers JR, et al. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 2003; 299:1743-7. 23. Straight AF, Field CM, Mitchison TJ. 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The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast. Mol Biol Cell 2003; 14:4051-66. 29. Hanrahan J, Snyder M. Cytoskeletal activation of a checkpoint kinase. Mol Cell 2003; 12:663-73. 30. Barral Y, Mermall V, Mooseker MS, Snyder M. Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast. Mol Cell 2000; 5:841-51.

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raised against the following proteins were used: EPLIN,18 α-tubulin (clone DM1A; Sigma), TRITC-labelled phalloidin (Sigma), myosin IIb (AbCam), MRLC-S19 (Cell Signalling), RhoA (Santa Cruz), Cdc42 (Santa Cruz) and Sept2.7 Cells were then washed with PBS and incubated with either Alexa 488- or Alexa 546-conjugated secondary antibody (Molecular probes). Cell nuclei were counterstained with the chromosome dye, 4'-6-Diamidino-2-phenylindole (DAPI; Sigma). After extensive washing, coverslips were mounted onto glass microscope slides with Mowiol. Cells were viewed and images were captured with a scanning confocal fluorescence microscope (Leica). Alternatively, images were captured with an Olympus IX80 inverted microscope using a 40x or 100x oil immersion lens and deconvolved using AutoDeblur v9.3 (AutoQuant Imaging, Watervliet, NY). To measure fluorescence intensity, Metamorph software was used. Immunoprecipitation and immunoblotting. Cellular extracts were prepared in lysis buffer B as described previously.37 For FlagEPLIN immunoprecipitation experiments, at 30 h post-transfection, cells were synchronised using nocodazole at the indicated cell cycle stage. The prepared lysates were pre-cleared with protein G-Sepharose beads, then incubated with anti-Flag antibody (M2; Sigma) overnight. Immune complexes were collected with protein G-Sepharose beads, washed twice with lysis buffer then fractionated by SDS-PAGE for immunoblot analysis. Antibodies recognising the following proteins were used for immunoblotting: EPLIN,18 Flag (M2; Sigma), myosin IIb (Abcam), -tubulin (T5192; Sigma), actin (Sigma) and Sept2.7 Primary antibody bound to the indicated protein was detected by incubation with a horseradish peroxidase-conjugated secondary antibody (Sigma). Blotted proteins were visualized using the ECL detection system (Amersham Pharmacia Biotech). GST pull-down assay. GST-alone, GST-Sept2 or GST-Sept7 were expressed in Escherichia coli, then bound to glutathione sepharose (GS) beads according to a previously published method.39 Prepared HeLa cell extract (1 mg) was pre-cleared with GS beads, then incubated with either 5 μg of GST alone, GST-Sept2 or GST-Sept7 bound to GS beads at 4°C with rotation for 3 h. Protein complexes bound to GS beads were then washed 4 times with lysis buffer B.37 Bound proteins were eluted from the beads by boiling in SDS sample buffer for 5 mins, then resolved by SDS-PAGE and transferred to nitrocellulose membrane for immunoblot analysis. Expression of GST-tagged recombinant protein was assessed by SDS-PAGE and Coomassie blue staining.

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We thank members of Khanna laboratory for helpful discussion and Armando van der Horst for critically reading the manuscript. We are extremely grateful to Dr. Raymond S. Maul for providing EPLIN antibodies and constructs. We thank Drs. Kinoshita, Trimble and C. Field for providing the GST-Sept7, GST-Sept2 and GFP-anillin constructs, respectively. This work was supported by grants to Dr. Kum Kum Khanna from Susan G. Komen Breast Cancer Foundation (USA) and National Health and Medical Research Council (NH&MRC) of Australia and the Australian Research Council (ARC). Dr. Megan Chircop (née Fabbro) was supported by a Peter Doherty Fellowship from the NH&MRC of Australia.

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EPLIN is required for cytokinesis

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31. Takizawa PA, DeRisi JL, Wilhelm JE, Vale RD. Plasma membrane compartmentali­zation in yeast by messenger RNA transport and a septin diffusion barrier. Science 2000; 290:3414. 32. Dobbelaere J, Barral Y. Spatial coordination of cytokinetic events by compartmentalization of the cell cortex. Science 2004; 305:393-6. 33. Schmidt K, Nichols BJ. A barrier to lateral diffusion in the cleavage furrow of dividing mammalian cells. Curr Biol 2004; 14:1002-6. 34. Kops GJPL, Weaver BAA, Cleveland DW. On the road to cancer: Aneuploidy and the mitotic checkpoint. Nature Rev Cancer 2005; 5:773-85. 35. Vassilev LT, Tovar C, Chen SQ, Knezevic D, Zhao XL, Sun HM, et al. Selective smallmolecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci USA 2006; 103:10660-5. 36. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296:550-3. 37. Fabbro M, Savage K, Hobson K, Deans AJ, Powell SN, McArthur GA, et al. BRCA1BARD1 complexes are required for p53Ser-15 phosphorylation and a G1/S arrest following ionizing radiation-induced DNA damage. J Biol Chem 2004; 279:31251-8. 38. Hayashi K, Yonemura S, Matsui T, Tsukita S. Immunofluorescence detection of ezrin/ radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues. J Cell Sci 1999; 112:1149-58. 39. Frangioni JV, Neel BG. Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 1993; 210:179-87.

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