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Regulation of a LATS-homolog by Ras GTPases is important for the control of cell division Müller-Taubenberger, Annette; Kastner, Peter M; Schleicher, Michael; Bolourani, Parvin; Weeks, Gerald Jul 1, 2014

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Regulation of a LATS-homolog by Ras GTPases isimportant for the control of cell divisionMüller-Taubenberger et al.Müller-Taubenberger et al. BMC Cell Biology 2014, 15:25http://www.biomedcentral.com/1471-2121/15/25RESEARCH ARTICLERegulation of a LATS-homae(Nth,polsingdtissue growth in flies and mammals [4-8]. Loss of NDR/ that LATS kinases are involved in the density-dependentMüller-Taubenberger et al. BMC Cell Biology 2014, 15:25http://www.biomedcentral.com/1471-2121/15/25and evolutionary functions of these important regulators. Inparticular Dictyostelium is an easily accessible eukaryoticSchillerstr. 42, 80336 Munich, GermanyFull list of author information is available at the end of the articleLATS kinase activity has been related to the developmentof various human malignancies [1,4,9].The Hippo pathway was identified first by geneticscreens in Drosophila melanogaster. The Ste20 kinasecontrol of cell proliferation through a cell morphology-based mechanism which is mediated by stress fibers andcooperates with a cell adhesion-based mechanism [10-12].Homologs of the Hippo pathway components havebeen shown to be present in yeast [5,13], Dictyosteliumdiscoideum [14], and Capsaspora owczarzaki [15], thus pro-viding an opportunity for the genetic analysis of the essential* Correspondence: amueller@lrz.uni-muenchen.de†Equal contributors1Anatomy III - Cell Biology, Ludwig Maximilian University of Munich,shown to have a tumor suppressor function and to controlResults: In this study we used the model organism Dictyostelium discoideum to analyze the functions of NdrC, ahomolog of the mammalian LATS2 protein, and present a novel regulatory mechanism for this kinase. Deletion of thendrC gene caused impaired cell division and loss of centrosome integrity. A yeast two-hybrid analysis, using activatedRas proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain. Further in vitro pull-downassays showed that NdrC binds RasG and RasB, and to a lesser extent RasC and Rap1. In cells lacking NdrC, the levels ofactivated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC.Conclusions: Dictyostelium discoideum NdrC is a LATS2-homologous kinase that is important for the regulation of celldivision. NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG. Changed levels of both,RasB or RasG, have been shown previously to interfere with cell division. Since a defect in cell division is exhibited byNdrC-null cells, RasG-null cells, and cells overexpressing activated RasB, we propose a model for the regulation ofcytokinesis by NdrC that involves the antagonistic control by RasB and RasG.Keywords: Cell division, Dictyostelium discoideum, Ras GTPase, LATS kinaseBackgroundNuclear Dbf-related/large tumor suppressor (NDR/LATS)kinases have been shown recently to control pathwaysregulating mitotic exit, cytokinesis, cell proliferation, mor-phological changes and apoptosis [1-3]. Work over thepast decade has revealed that LATS kinases are core com-ponents of the Hippo signaling pathway that has beenHippo (MST1/2 in mammals) and the NDR family kinaseWarts (LATS1/2 in mammals) constitute the core of thesignaling cascade. The elucidation of the mechanisms thatregulate the Hippo pathway and the identification of inter-actors contributes to our understanding how growth andorgan size in flies and mammals are controlled and whymisregulation leads to the formation of cancer. Recentstudies in Drosophila and mammalian cells have suggestedimportant for the controlAnnette Müller-Taubenberger1*†, Peter M Kastner1†, MichAbstractBackground: Nuclear Dbf-related/large tumor suppressorpathways that regulate mitotic exit, cytokinesis, cell growcore components of the Hippo signaling cascade and imand organ size in flies and mammals, and homologs are aproto-oncogens regulate many biological functions, includof LATS kinases or Ras GTPases have been implicated in the© 2014 Müller-Taubenberger et al.; licensee Bithe Creative Commons Attribution License (htdistribution, and reproduction in any mediumDomain Dedication waiver (http://creativecomarticle, unless otherwise stated.Open Accessolog by Ras GTPases isof cell divisionl Schleicher1, Parvin Bolourani2 and Gerald Weeks2DR/LATS) kinases have been shown recently to controlmorphological changes and apoptosis. LATS kinases arertant tumor suppressors controlling cell proliferationo present in yeast and Dictyostelium discoideum. Rasdifferentiation, proliferation and apoptosis. Dysfunctionsevelopment of a variety of cancers in humans.oMed Central Ltd. This is an Open Access article distributed under the terms oftp://creativecommons.org/licenses/by/2.0), which permits unrestricted use,, provided the original work is properly credited. The Creative Commons Publicmons.org/publicdomain/zero/1.0/) applies to the data made available in thismodel system to gain insights into a variety of basic cellularprocesses, including the regulatory machinery controllingcell division [16,17]. The LATS/NDR family of Dictyosteliumconsists of two LATS-related kinases, NdrC and NdrD, aswell as two NDR-related kinases, NdrA and NdrB [18,19].In the present study, we have explored the function ofNdrC, and provide evidence that NdrC plays an importantrole in cell division. Based on the data presented, we proposethat its activity is antagonistically controlled by RasB andRasG, two members of the Ras subfamily of GTPases.Results and discussionIdentification of NdrC as a Ras GTPase interacting proteinDictyostelium discoideum NdrC (DDB0349842) belongsto the LATS/NDR kinase family, which constitutes asubgroup of AGC (protein kinase A/G/C-related) kinases[18,20]. The Dictyostelium LATS/NDR family consistsof four kinases, two ‘shorter’ NDR kinases (NdrA/B),and two ‘larger’ LATS/NDR-related kinases (NdrC/D)that are characterized by an extended N-terminus [19].Similarly, the mammalian LATS/NDR kinase family iserimain(hdespinran-RMüller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 2 of 10http://www.biomedcentral.com/1471-2121/15/25Figure 1 NdrC interacts with Ras proteins. A. In yeast two-hybrid expas a strong interactor of RasG and Rap1. Domain organization of NdrC, andregulatory domain, aa 650–710), phosphorylation site (T703), catalytic dom(activation segment, aa 914–928; regulatory phosphorylation site S917), HMof GST-NdrC-RBD to Ras proteins in vivo. The RBD of NdrC was used toThe bound material was analyzed by Western blotting using antibodiestested in all pull-down experiments, and consistently there was no bindstaining of a duplicate gel with Coomassie Blue (lower panel); only theshowing the pull-down of His-tagged Ras proteins by GST-tagged NdrCGDP-bound) were allowed to bind in vitro to GST-tagged bacterially expreMethods. The amount of bound Ras proteins was detected by Western bloments with various activated Ras proteins as bait, NdrC was revealedapping of the Ras binding domain (RBD, aa 107–284), NTR (N-terminal(kinase domain aa 718–1019, subdomains I-X), I (insert, aa 867–913), ASydrophobic motif aa 1092–1099; phosphorylation site T1095). B. Bindingtect Ras proteins in Dictyostelium cell lysates as described in Methods.ecific to RasB, RasG, RasC and Rap1 (upper panel). GST-only wasg (here shown only for RasB). Equal sample loading was verified byge of the strongest band is shown. C. Representative Western blotBD. Recombinant His-tagged Ras proteins (constitutively GTP- orssed NdrC-RBD. For details of the quantitative assay please seetting using an anti-His Tag antibody.Müller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 3 of 10http://www.biomedcentral.com/1471-2121/15/25subdivided into two ‘larger’ LATS kinases (LATS1/2)and two ‘shorter’ NDR kinases (NDR1/2) (Additionalfile 1: Figure S1A). The Dictyostelium NdrC kinase ismade up of 1,312 amino acids (147 kDa), and its proteinsequence comprises the general features described forother LATS/NDR kinases, which include an N-terminalregulatory domain (NTR), an insert sequence (I) be-tween subdomain VII and VIII of the catalytic domain,an activation segment (AS) as well as a conservedhydrophobic motif (HM) (Figure 1A, Additional file 1:Figure S1B).When the Dictyostelium GTPases RasG, RasC andRap1 were employed as bait in a yeast two-hybridscreen, NdrC was identified as a novel interacting pro-tein that exhibited strong positive interactions with allthree proteins. This screen also revealed that RasG andRap1 exhibited strong positive interactions with the pre-viously described Ras-binding domain (RBD) containingproteins PL3K and Rip3. In contrast, NdrC was the onlyprotein that bound RasC in the yeast two-hybrid screen.The RBD of NdrC was localized between amino acid resi-dues 107 and 284, the sequence that interacted with allthree Ras proteins in the yeast two-hybrid assays (Figure 1Aand data not shown). The minimal Ras-binding domainof NdrC was not defined further by additional experi-ments. A bioinformatics analysis did not reveal evidenceof a RBD sequence in this region, but this is not surprisingsince the RBD sequences of a number of other RBD pro-teins were not detected by bioinformatic analyses [21].Ras protein binding to NdrC was confirmed by pull-down assays using the identified RBD of NdrC tagged toGST and Dictyostelium lysates (Figure 1B). Bound Rasproteins were detected by Western blot analysis using spe-cific anti-Ras polyclonal antibodies. The experiment re-vealed that the NdrC-RBD interacted not only with RasG,RasC and Rap1, but also with RasB (Figure 1B). This resultwas confirmed and extended by pull-down experimentsusing GST-tagged NdrC-RBD in combination withHis-tagged Ras subfamily proteins that allowed a morequantifiable comparison of the binding. These inter-action studies showed that NdrC bound to the acti-vated Ras proteins but not to the wild type (Figure 1C),although the wild-type proteins did bind in the pres-ence of GTPγS (data not shown). NdrC bound best toactivated RasG and RasB, with lower levels of bindingto activated Rap1 and RasC (Figure 1C). NdrC did notinteract with either RasD or RasS (Figure 1C).ndrC-null cells exhibit a severe defect in cytokinesis andaberrant numbers of centrosomesTo investigate the cellular function of NdrC, a knockoutstrain of the ndrC gene was generated by homologous re-combination using a disruption construct carrying aninverted blasticidin-S resistance cassette within the partialndrC gene sequence (Figure 2A). The most striking pheno-type of the ndrC-null cells when compared to the wild typewas that the majority of mutant cells were larger and mul-tinucleated (Figure 2B). When wild-type and ndrC-nullcells were fixed and stained with TO-PRO-3 to visualizethe nuclei, and counterstained for actin using Alexa Fluor488-coupled phalloidin, the increased number of nuclei inthe mutant was clearly revealed (Figure 2C, D), and this in-crease was quantified by nuclei counts of fixed, DAPI-stained cell preparations (Figure 2E). The percentage ofmono-nucleated cells in wild type was 95% compared toonly 29% in ndrC-null cells (Figure 2E). The number ofnuclei per cell in the ndrC-nulls peaked at 1, 2, 4 and 8 in-dicating concerted mitosis (Figure 2E).An analysis of living cells lacking NdrC revealed thatthey were able to perform mitosis, but an impairment ofcell division resulted in the formation of multinuclearcells. Frequently, putative daughter cells were still con-nected by thin cellular bridges suggesting division by cyto-fission rather than mitosis-coupled cytokinesis (Figure 2B,right). The generation times, measured for ndrC-null andwild-type cells were almost identical, regardless of whethercells were grown in shaking culture in rich axenic mediumor on a solid substrate with bacterial lawns (Additional file2: Figure S2). Since ndrC-null cells are considerably largerthan wild-type cells, their cell masses increase at consider-ably faster rates indicating an additional defect in the con-trol of cell growth.Inspection of multinucleated ndrC-null cells, fixed andstained with TO-PRO-3 for visualization of nuclei andimmunostained with anti-α-tubulin antibodies to visualizemicrotubules and centrosomes, revealed a high percentageof mutant cells carrying increased numbers of centro-somes (Figure 3). Frequently, the supernumerous centro-somes were not connected to the nuclei (Figure 3B). Toquantify the disruption of centrosome integrity, the ratioof centrosomes visualized by anti-α-tubulin staining to nu-clei visualized by DAPI staining, was determined for wild-type and ndrC-null cells (Figure 3D). About one third(31%) of the ndrC-null cells had more than one centro-some per nucleus, compared to only 0.6% in wild-typecells. ndrC-null cells with evenly distributed centrosomesunderwent mitosis in a synchronized manner (concertedmitosis) (Figure 3E), but although the increased ratio ofcentrosomes per nuclei did not affect the ability of cellsto perform concerted mitosis, the unattached surpluscentrosomes were not capable of forming mitotic spin-dles (Figure 3F).Aberrant numbers of centrosomes have also been ob-served for knockout strains of mouse LATS2 [22,23] andhuman NDR1/2 [3,24]. The cell division defect in mam-malian cells has been attributed to a disruption of thesignaling cascade controlling cytokinesis on the onehand [25], and a control of nuclear division on the otherMüller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 4 of 10http://www.biomedcentral.com/1471-2121/15/25[26]. Human LATS1 was also reported to act as mitoticexit network kinase [27], responsible for regulation ofthe G2/M-arrest [28]. In addition, the Drosophila LATS-homolog Warts regulates mitotic progression [29]. How-ever, despite these clear indications for a role of NDR/LATS kinases in regulation of the cell cycle and/or celldivision, the underlying mechanisms are largely unknown.Subcellular localization of NdrCGene expression profiling data indicated that NdrC isexpressed at very low levels throughout the developmentalFigure 2 NdrC-null cells are impaired in cell division. A. Gene replacemand P1-P3 indicate the primer combinations that were used initially to idenand ndrC-null cells. ndrC-null cells are much larger than wild-type cells, andimage). C. Fixed wild-type cells stained with TRITC-phalloidin for actin andfor actin and TO-PRO-3 for DNA. E. Histogram showing the percentage of cndrC-null mutants. Cells were grown in Petri dishes, fixed, stained, and for erepeated in an independent experiment with almost identical results. Bars,cycle of Dictyostelium (http://dictyexpress.biolab.si) [30].In order to specify the cellular localization of NdrC, poly-clonal antibodies were raised against NdrC. Immunolabel-ing experiments with fixed Dictyostelium cells indicatedthat NdrC is preferentially enriched at the centrosome(Figure 4A). This localization was confirmed by co-stainingwith a monoclonal antibody directed against a genuinecentrosomal component, the corona protein CP224[31]. The centrosomal localization of NdrC was detect-able only in a subfraction of cells, suggesting a cellcycle-dependent enrichment. Expression of full-lengthent of ndrC (DDB0219984) by a blasticidin-S resistance cassette. P1-P2tify gene knockouts by PCR. B. Live-cell microscopy of wild-type (left)often divide by traction-mediated cytofission (as shown in the rightTO-PRO-3 for DNA. D. Fixed ndrC-null cell stained with TRITC-phalloidinells carrying the indicated numbers of nuclei in wild-type andach strain the nuclei of >500 cells were counted. The counting was10 μm.Müller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 5 of 10http://www.biomedcentral.com/1471-2121/15/25NdrC with a N-terminal GFP-tag confirmed the localizationat the centrosome. NdrC had not been detected in aprevious proteome screen of the Dictyostelium centro-some [32], but was found to co-purify with centrosomesthat were isolated from Dictyostelium wild-type and cellsexpressing GFP-NdrC (Additional file 3: Figure S3).Over-expression of a fusion protein comprising GFP andthe N-terminal 300 amino acids of NdrC containing theRBD domain, showed a very similar localization as that re-ported previously for the Ras-binding domain of humanRaf1 that relocates upon stimulation to active zones of thecell cortex [33,34] (Figure 4B). This finding indicates a pref-erential binding of RBD of NdrC to membrane-associatedRas proteins. A GFP-fusion protein comprising the cata-lytic domain and the C-terminus of NdrC (amino acid res-idues 435 to 1312) localized to the cytoplasm and showedno specific enrichment (Additional file 4: Figure S4).Figure 3 Cells lacking NdrC show centrosomal aberrations. A. In compsupernumerous centrosomes. D. Histogram depicting the percentage of nocentrosomes in wild-type and ndrC-null cells. E, F. Visualization of mitotic santibodies, and staining of DNA with TO-PRO-3. Spindle formation occurs swith supernumerous centrosomes (F). Bars 5 μm in (A), and, 10 μm in (B)Putative control of NdrC by Ras GTPase family membersWe have shown previously that rasG-null cells or cellsoverexpressing RasB-(G12T) exhibit a multinucleated cellphenotype that is similar to the one exhibited by the ndrC-null cells [35,36]. In addition, severe defects in cytokinesisin cells overexpressing the exchange factor RasGEF-Q,which acts specifically upstream of RasB have been de-scribed [37]. Given that NdrC contains a RBD, it is conceiv-able that RasB or RasG exert regulatory functions on NdrC.To further investigate the possible roles of RasG orRasB in NdrC function, we determined the subcellularlocalization of RasG and RasB. Wild-type cells overex-pressing GFP-tagged RasB or RasG were normal in re-spect to cell size and nuclei number, and the GFP-taggedproteins predominately localized to the cortex (Figure 4C,D). The GFP-tagged activated forms of the proteins, RasB-(G12T) and RasG-(G12T), also localized predominately toarison to wild-type, B, C multinucleated ndrC-null cells frequently showrmal (centrosomes/nuclei = 1) and aberrant (centrosomes/nuclei > 1)pindles in fixed ndrC-null cells by immunolabeling with anti-α-tubulinynchronously in multinucleate cells (E), as well as in multinucleate cells, (C), (E), and (F).Müller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 6 of 10http://www.biomedcentral.com/1471-2121/15/25the cortex (Figure 4E-G). However, whereas RasG-(G12T)overexpression did not affect the cell phenotype (Figure 4E),overexpression of RasB-(G12T) caused disturbed cyto-kinesis characterized by an impairment of cells to severthe connection between daughter cells (Figure 4F). Thisresulted in enlarged, multinucleated cells (Figure 4F), adefect similar to that described previously for cells ex-pressing an untagged RasB-(G12T) [35].Both, GFP-tagged RasB and GFP-RasG bound to theGST-NdrC-RBD, as shown by in vitro pull-down assays ofFigure 4 Localization and interaction of NdrC with Ras proteins. A. Ndimmunolabeled with centrosome-specific monoclonal anti-CP224 antibodiedetected with anti-mouse Alexa Fluor-568 and anti-rabbit Alexa Fluor-488 adetected. Bar, 10 μm. B. Live-cell imaging of a Dictyostelium cell expressingBar, 10 μm. C. Live-cell imaging of GFP-RasB expressing wild-type cells showto the cortex of wild-type cells, shown by live cell microscopy. E. GFP-RasG(G1of GFP-RasB(G12T) expressing wild-type cell. G. GFP-RasB(G12T) expressed in won the right), and results in enlarged multinucleate cells. Bars in (C)-(G), 5 μmand thereby activity of the GFP-tagged Ras-GTPase. GFP-tagged RasG overexpbound RasG were detected by Western blotting using anti-GFP antibodies. I.Total cell extracts from wild-type or ndrC-null cells were bound to GST-Byr2-Rdown or of total Ras protein in the lysate was determined by Western blottindata shown is for a single experiment, but similar results were obtained in twwild-type AX2 cells expressing GFP-tagged Ras proteins(Figure 4H). Although NdrC also binds RasC and Rap1,cell division is not affected by either the loss or up-regulation of Rap1 [38], or by the loss or up-regulationof RasC [39]. Thus the significance of the binding ofRap1 and RasC to NdrC remains to be established.Further evidence for a possible functional connectionbetween the Ras-GTPases and NdrC was obtained bymeasuring the levels of activated Ras protein in thendrC-null cells by Western blot analysis of GST-Byr2-rC localizes to the centrosome. Fixed Dictyostelium cells weres, and polyclonal anti-NdrC antibodies. Primary antibodies werentibodies. In NdrC-null cells, no staining with anti-NdrC antibodies wasGFP-NdrC-RBD. Left image, GFP-NdrC-RBD signal; right image, merge.s localization of GFP-RasB to the cell cortex. D. GFP-RasG localization2T) localizes to the cortex and filopodia of wild-type cells. F. Cytofissionild-type cells localizes to the cortex and filopodia (image enlargement. H. GST-NdrC-RBD pull-down of RasB tagged to GFP shows interactionressed in wild-type cells interacts with the GST-NdrC-RBD. The levels ofLevels of activated Ras proteins in wild-type compared to ndrC-null cells.BD as described in Methods. The amount of activated Ras proteins pulledg using specific polyclonal antibodies against RasB, RasG, and Rap1. Theo other experiments.RBD pull-downs using specific antibodies for the Rasfamily members. Levels of activated RasB and RasGwere increased in the absence of NdrC compared towild-type cells, indicating that NdrC acts as a negativeregulator for activation of RasG and RasB (Figure 4I). Incontrast, the levels of activated Rap1 and the total levelsof all the Ras GTPases were found to be identical in ly-sates of wild-type and NdrC-null cells (Figure 4I). Themechanism involved in the feedback inhibition of RasGand RasB activation by NdrC, presumably at the level ofRasGEF activity, is not known at this time, but it appearsto be a general mechanism for downstream Ras effectorsin Dictyostelium, since similar results have been observedfor pik null and ripA null cells.The findings that an absence of RasG and the overex-pression of activated RasB both result in a multinucleatephenotype suggest the possibility that RasB and RasGplay antagonistic roles in regulating NdrC during cytokin-esis (Figure 5). The multinucleate phenotype of rasG-nullcells [36] is consistent with a role for RasG in activatingNdrC, whereas the multinucleate phenotype generated byover-expression of either activated RasB [35], or the RasB-specific exchange factor GefQ [37], indicates that RasB isa negative regulator of cytokinesis and cell division.Müller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 7 of 10http://www.biomedcentral.com/1471-2121/15/25Figure 5 Model suggesting the regulation of NdrC by RasGTPases as explained in the text.The regulation of NdrC by RasG and RasB implies thatthese Ras proteins would also be localized at the centro-some or in the nucleus. When cells were labeled withGFP-tagged Ras proteins, we find wild-type RasB andRasG, as well as constitutively active forms of the two pro-teins, are localized predominantly at the plasma mem-brane. This result contrasts somewhat with a previousstudy, using antibody staining, that revealed RasB clearlylocalized at the nucleus [35]. When we stained cells withan antibody highly specific to RasG, the majority of thestain was associated with the cell cortex, but some stainwas also detected in the cytoplasm and in the nucleus(Additional file 5: Figure S5). These results imply that onlya small proportion of the cellular RasG and RasB is local-ized in the nucleus, and are consistent with RasG andRasB both having other functions in the cell. In fact, RasGis not only involved in cytokinesis, but also in folatechemotaxis, in maintenance of cell shape, and in cell mo-tility [40]. In addition, it has been shown recently thatRasG, acting through PI3K, is important for pinocytosis[41]. Evidence for other specific functions for RasB is lessdirect, but gefQ-null cells exhibit myosin-II over-assemblyand defects in polarity [37], implying a possible role forRasB in these functions, and rasB appears to be an essen-tial gene [35], suggesting an additional vital function forRasB.ConclusionsWe present evidence for a novel mechanism for the regu-lation of cell division in Dictyostelium, involving theLATS2-homolog NdrC and members of the Ras subfamilyof GTPases, RasB and RasG. We propose an antagonisticregulation, whereby the NdrC is activated by RasG andinactivated by RasB. The previously described interactionbetween the Schizosaccharomyces pombe Cdc42, a smallGTPase, and Orb6, a member of the LATS/NDR groupof kinases [42] suggests the possible generality of thistype of mechanism, and it will be important to investi-gate whether the mammalian LATS kinases are also reg-ulated by small GTPases during cell division.MethodsCell culture, gene replacement and transformation ofDictyosteliumCells of the Dictyostelium discoideum wild-type strainAX2-214, or mutant cells derived from it, were cultivatedat 21°C in nutrient HL5 medium (Formedium), either inshaken culture at 150 rpm or in Petri dishes without shak-ing. To induce starvation, cells were washed twice in17 mM Soerensen’s phosphate buffer (PB), pH 6.0, andshaken at a density of 107 cells per ml in the buffer.To generate ndrC-null mutants, wild-type AX2 cellswere transformed with the gene replacement construct(Figure 2A) by electroporation using a Bio-Rad gene pulserdescribed previously [41]. In brief, 400 μg of purifiedMüller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 8 of 10http://www.biomedcentral.com/1471-2121/15/25at 0.8-0.9 kV and 3 μF, and 4-mm cuvettes. Independenttransformants were selected by addition of 7.5 μg/ml ofBlasticidin-S (ICN Biomedicals Inc.). Transformants werecloned by spreader dilutions on lawns of non-pathogenicKlebsiella aerogenes. Three independent NdrC-knockoutclones were identified by testing genomic DNA for inser-tion of the resistance cassette into the ndrC gene by PCR.For expression of fluorescently tagged Ras GTPases,AX2 wild-type and NdrC-null cells were transformed byelectroporation with pDEX derived plasmids enabling theexpression of GFP-RasB, GFP-RasB-G12T, GFP-RasG andGFP-RasG-G12T. Transformants were selected by addition of10 μg/ml of G418 (Sigma-Aldrich) or 7.5 μg/ml Blasticidin-S(ICN Biomedicals Inc.), and cloned.Live-cell microscopyCells were transferred into an open chamber and washedtwice with Soerensen phosphate buffer. Confocal imageswere taken using an inverted LSM 510 Meta confocalmicroscope (Zeiss) equipped with a 63× Neofluar 1.4 ora 100× Neofluar 1.3 oil immersion objective. For excita-tion, the 488-nm argon ion laser line and the 543-nm aswell as the 633-nm helium neon laser lines were used,and emission was collected using 505–530 nm, 585–615 nmband-pass or a 650 nm long-pass filter.ImmunofluorescenceFor immunolabeling, AX2 wild-type or ndrC-null cells,settled onto glass coverslips, were fixed with a mixtureof 15% (v/v) of saturated picric acid and 2% (w/v) para-formaldehyde in 10 mM Pipes-HCl, pH 6.0, at roomtemperature for 20 min, and post-fixed with 70% ethanolfor 10 min. α-tubulin was detected using monoclonal ratantibodies (YL1/2) [43] and Alexa Fluor 568-conjugatedgoat anti-rat IgG (Invitrogen). For visualization of fila-mentous actin, cells were stained with Alexa Fluor 488-phalloidin (Molecular Probes). DNA was visualized eitherwith TO-PRO-3 or with DAPI.Polyclonal antibodies were generated against GST-taggedNdrC-RBD. The sequence encoding amino acid residues 1to 299 of NdrC was cloned into the bacterial expressionvector pGEX-6P1 (GE Healthcare). Purification of the GST-fusion protein was performed using standard procedures,and the recombinant protein was used to immunize a fe-male white New Zealand rabbit together with the adjuvantGerbu100 (Gerbu Biochemicals).GST-RBD pull-down assay of endogenous Ras proteinsThe binding of endogenous activated Ras proteins to theGST-Ras Binding Domain (GST-RBD) of DictyosteliumNdrC was determined as described previously [39]. 350 μlof Dictyostelium wild-type AX2 or ndrC-null cell suspen-sion (5 × 107 cells/ml) were lysed by mixing with an equalvolume of 2× lysis buffer. 400 μg of protein lysate wereHis-tagged Ras protein were incubated with 100 μg ofGST-RBD on glutathione sepharose beads and tumbledin binding buffer at 4°C overnight. Beads were washedthree times and analyzed by SDS-PAGE, and Westernblots were probed with anti-His tag monoclonal antibody(Santa Cruz Biotechnology).Additional filesAdditional file 1: Figure S1. Comparison of NdrC to other LATS/NDRkinases and LATS/NDR kinase sequence signatures of DictyosteliumNdrC. A. Sequence identities of the catalytic domain of D. discoideumNdrC with the catalytic domains of D. discoideum NdrD, NdrA and NdrB,in comparison to Homo sapiens NDR1 and 2, and LATS1 and 2, and theDrosophila melanogaster LATS-homolog Warts. Numbers indicate per centidentity within the catalytic domains compared to NdrC catalytic domaindetermined by BLASTp. B. Sequence signatures of Dictyostelium NdrC.The NTR region (amino acid residues 650 to 710) carries a conservedphosphorylation site at threonine 703. The catalytic domain (subdomainsI-X; amino acid residues 718 to 1019) contains an AGC-kinase specific insert(I; amino acid residues 867 to 913) as well as an adjacent activation segment(AS; amino acid residues 914 to 928) containing a conserved regulatoryphosphorylation site at serine 917. The conserved hydrophobic motif(HM; amino acid residues 1091 to 1099) corresponds to the consensussequence F_X_X_Y/F_T_Y/F_K/R carrying a putative phosphorylationsite at threonine 1095 [1].Additional file 2: Figure S2. Growth rates of ndrC-null cells compared towild-type. A. Growth rates of ndrC-null cells compared to wild-type cells in richmedium under shaking conditions. B. Growth of wild-type and ndrC-null cellson bacterial lawns of non-pathogenic K. aerogenes on agar plates.Additional file 3: Figure S3. NdrC co-purifies with centrosomes.Centrosomes were isolated from cells expressing GFP-NdrC by purificationof nuclei followed by pyrophosphate treatment and sucrose densitycentrifugation. The nuclei fraction with the associated centrosomes wasdisintegrated by pyrophosphate and passage through a 5-μm meshincubated with 100 μg of GST-RBD and glutathione seph-arose beads (GE Healthcare) at 4°C for 1 hour. 50 μl of 1×SDS gel loading buffer was added to the pelleted beads,and the suspension was boiled for 5 min. Samples weresubjected to SDS-PAGE, and Western blots were probedwith polyclonal antibodies directed against RasB, RasG,RasC, or Rap1. Equal sample loading was verified by stain-ing of a duplicate gel with Coomassie Blue.Interaction of His-tagged Ras and GST-NdrCTo produce 6xHis-tagged Ras-GTPases, wild-type RasG,RasC , RasB, RasS, Rap1 and RasD, and the constitutivelyactivated forms of RasG (G12T), RasC (G13T), RasB(G15T), RasS (G12T), Rap1 (Q65E) and RasD (G12T)were cloned into pET-21a (Novagen) [41]. To expressGST-NdrC, a fragment encoding amino acid residues1–299 of NdrC was cloned into pGEX6P-1. Recombinantproteins were expressed in Escherichia coli BL21 (DE3)codonplus-RIL (Stratagene), and protein concentrationswere determined using the DC Protein Assay (Bio-Rad).His-Ras/RBD-NdrC-GST interaction was examined aspolycarbonate filter. Centrosomes were isolated via two consecutive sucrosestep gradients of 80% and 50%, followed by 80%, 70%, 55% and 50%Müller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 9 of 10http://www.biomedcentral.com/1471-2121/15/25steps in SW-40 tubes (Beckman) centrifuged at 55,000 × g for 1 h at 4°C.Immunostaining of methanol-fixed centrosomes was performed with monoclonalanti-CP224 antibodies [31]. The primary antibodies were visualized withAlexa Fluor-568 anti-mouse IgG (Invitrogen). Centrosomes labeled byanti-CP224 antibodies are red, those containing GFP-NdrC are green, andthose containing both labels are yellow. Very similar results were obtainedwith centrosomes isolated from wild-type cells and immunostaining withpolyclonal anti-NdrC-RBD antibodies and Alexa Fluor-488 anti-rabbit IgG.Additional file 4: Figure S4. Localization of GFP-NdrC(435–1312).A. Scheme of the GFP-tagged NdrC (435-1312) construct. B. Live-cell imagingof a Dictyostelium wild-type cell expressing GFP-NdrC(435–1312). Bar, 5 μm.Additional file 5: Figure S5. Immunolocalization of RasG in wild-typecells. Wild-type cells were fixed and immunostained with polyclonalantibodies directed specifically against RasG. Primary antibodies weredetected with Alexa Fluor-488 anti-rabbit IgG (green). Nuclei werevisualized by staining with DAPI (blue). Bar, 5 μm.Abbreviationsaa: Amino acids; GST: Glutathione-S-transferase.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAMT and GW designed and supervised research; AMT, PMK and PBperformed research; MS advised experiments and contributed financially;AMT, PMK, PB and GW analyzed data; AMT and GW wrote the manuscript.All authors read and approved the final manuscript.AcknowledgementsThe excellent technical assistance of Thi-Hieu Ho, Marlis Fürbringer andGudrun Trommler is gratefully acknowledged. We thank Ralph Gräf (Potsdam)for the anti-CP224 antibodies, and Arjan Kortholt (Groningen) for providing thefull-length GFP-NdrC construct. This work was supported by grants from theDeutsche Forschungsgemeinschaft to AMT and MS (SFB 914, TP7), the CanadianInstitute of Health Research to GW, and the Elitenetzwerk Bayern and UniversitätBayern e.V. to PMK.Author details1Anatomy III - Cell Biology, Ludwig Maximilian University of Munich,Schillerstr. 42, 80336 Munich, Germany. 2Department of Microbiology andImmunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.Received: 31 January 2014 Accepted: 25 June 2014Published: 1 July 2014References1. Hergovich A, Stegert MR, Schmitz D, Hemmings BA: NDR kinases regulateessential cell processes from yeast to humans. Nat Rev Mol Cell Biol 2006,7(4):253–264.2. Hergovich A: Regulation and functions of mammalian LATS/NDR kinases:looking beyond canonical Hippo signalling. Cell Biosci 2013, 3(1):32.3. Hergovich A, Cornils H, Hemmings BA: Mammalian NDR protein kinases:from regulation to a role in centrosome duplication. Biochim Biophys Acta2008, 1784(1):3–15.4. 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Wehland J, Willingham MC: A rat monoclonal antibody reactingSubmit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionMüller-Taubenberger et al. BMC Cell Biology 2014, 15:25 Page 10 of 10http://www.biomedcentral.com/1471-2121/15/25specifically with the tyrosylated form of alpha-tubulin. II. Effects on cellmovement, organization of microtubules, and intermediate filaments,and arrangement of Golgi elements. J Cell Biol 1983, 97(5 Pt 1):1476–1490.doi:10.1186/1471-2121-15-25Cite this article as: Müller-Taubenberger et al.: Regulation of a LATS-homologby Ras GTPases is important for the control of cell division. BMC Cell Biology2014 15:25.Submit your manuscript at www.biomedcentral.com/submit


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