A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development

Research Paper

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A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development Andrew Wilkins*, Jonathan R. Chubb*† and Robert H. Insall‡ Background: Dictyostelium possesses a surprisingly large number of Ras proteins and little is known about their activators, the guanine nucleotide exchange factors (GEFs). It is also unclear, in Dictyostelium or in higher eukaryotes, whether Ras pathways are linear, with each Ras controlled by its own GEF, or networked, with multiple GEFs acting on multiple Ras proteins.

Addresses: *MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK. ‡School of Biosciences, Birmingham University, Edgbaston, Birmingham B15 2TT, UK. †Present

Results: We have identified the Dictyostelium gene that encodes RasGEFB, a protein with homology to known RasGEFs such as the Son-of-sevenless (Sos) protein. Dictyostelium cells in which the gene for RasGEFB was disrupted moved unusually rapidly, but lost the ability to perform macropinocytosis and therefore to grow in liquid medium. Crowns, the sites of macropinocytosis, were replaced by polarised lamellipodia. Mutant cells were also profoundly defective in early development, although they eventually formed tiny but normally proportioned fruiting bodies. This defect correlated with loss of discoidin Iγ mRNA, a starvation-induced gene, although other genes required for development were expressed normally or even precociously. RasGEFB was able to rescue a Saccharomyces CDC25 mutant, indicating that it is a genuine GEF for Ras proteins.

address: MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, UK. Correspondence: Robert Insall E-mail [email protected] Received: 13 June 2000 Revised: 11 October 2000 Accepted: 11 October 2000 Published: 1 November 2000 Current Biology 2000, 10:1427–1437 0960-9822/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

Conclusions: RasGEFB appears to be the principal activator of the RasS protein, which regulates macropinocytosis and cell speed, but it also appears to regulate one or more other Ras proteins.

Background The ras subfamily of proto-oncogenes encode membranebound GTPases, which have been implicated in the control of a wide variety of cellular processes [1]. Although initial studies focused on the role of the Ras protein in growth control in mammalian cells [2,3], Ras signalling pathways have now been found in almost all eukaryotes and shown to control processes as diverse as differentiation [4], apoptosis [5], synaptic transmission [6], cell-cycle arrest and senescence in mammalian cells [7], cytoskeletal rearrangements and pinocytosis [8], cytokinesis [9], chemotaxis [10] and yeast pheromone signalling [11]. Ras GTPases exist in two conformations, an active GTP-bound state and an inactive GDP-bound form. The cyclic interconversion between these two states is controlled by GTPase-activating proteins (GAPs), which inactivate Ras by stimulating its weak intrinsic GTPase activity, and guanine nucleotide exchange factors (GEFs), which activate Ras by promoting the exchange of bound GDP for GTP. It is through GAPs and GEFs that extracellular signals are thought to control the activation state of Ras. The first RasGEF to be identified was Cdc25p of Saccharomyces cerevisiae, which is required for Ras-mediated activation of adenylyl cyclase and is thus essential for proliferation and spore germination [12]. In Drosophila

melanogaster, the Son-of-sevenless (Sos) protein functions upstream of Ras in R7 photoreceptor differentiation [13]. In mammals, there are at least six RasGEFs: the closely related Sos1 and Sos2 [14,15], RasGRP [16], the closely related RasGRF1 and RasGRF2 proteins [17,18], and CNRasGEF [19]. The phenotypes produced by deletion or inhibition of RasGEFs have sometimes revealed previously unidentified roles for Ras. For example, mice lacking RasGRF1 are impaired in memory consolidation, implicating RasGRF1-mediated Ras signalling in synaptic events leading to formation of long-term memory [6]. Thus, an understanding of the physiological role of Ras may be aided by an investigation of RasGEF function. In Dictyostelium, at least eight ras subfamily genes are expressed at different times throughout the life cycle, five of which have been published: rasB, rasC, rasD, rasG and rasS. RasD and RasG are most similar to the prototypic human H-Ras, both having only six conservative changes in the first 80 residues relative to H-Ras — the region thought to contain the residues most important for effector interaction. RasD and RasG are 82% identical, with only two conservative changes in the first 80 residues. RasB, RasC and RasS are more distantly related to the prototypic Ras. Interestingly, the effector region of human H-Ras (amino acids 32–40) is identical in all Dictyostelium

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Ras proteins except RasS and RasC. The rasB, rasC and rasS genes are expressed throughout the life cycle of Dictyostelium [20–22]; rasG is expressed maximally during vegetative growth and is undetectable by the onset of aggregation [23] whereas rasD expression is restricted to the multicellular stage of development being first detectable around the tipped mound stage and maximal during the slug stage [23]. We have previously described the phenotypic consequences of single disruptions of the rasG, rasD and rasS genes: rasG– cells have impaired cell movement and are defective in cytokinesis [9]; rasD– cells exhibit impaired phototaxis during the slug stage of development, but no defects in cell-fate specification or pattern formation are evident [24]; rasS– cells are impaired in solid- and fluidphase endocytosis, and yet have an increased cell speed [25]. To date, only a single Dictyostelium RasGEF has been described, encoded by the aimless gene [10]. The phenotype caused by deletion of aimless is different from that of the ras null mutants generated so far — aimless– cells fail to aggregate upon starvation and are unable to synthesise or respond chemotactically to cAMP. Thus, other RasGEFs must exist that regulate the Ras activity required for cellular cytokinesis, endocytosis and for slug phototaxis. Here, we report the identification and characterisation of a second Dictyostelium gene encoding a RasGEF.

Results Identification and cloning of RasGEFB

Using the tBLASTn algorithm to search the Tsukuba Dictyostelium sequencing project databases [26], we identified a partial cDNA (clone SSK260) with strong homology to known GEF domains. This partial cDNA clone was used to probe a random-primed λZAPII cDNA library. Multiple, overlapping, partial cDNA fragments were obtained which,

when assembled, contained a continuous open reading frame. The gene was designated rasgefB. The predicted open reading frame encodes a protein of 1529 amino acids with a mass of 174 kDa (Figure 1a). The sequence is characterised by stretches of repeated glutamine and asparagine residues, a feature of unknown significance seen in many Dictyostelium proteins. The carboxy-terminal region of this protein contains the putative RasGEF catalytic domain comprising the Cdc25 homology domain (Figure 1b; this region is found in the catalytic domains of known GEFs for Ras and Ral subfamily proteins) and the REM box (Figure 1c; this region is found only in Ras-specific GEFs). The Cdc25 homology domain and the REM box share 32% and 40% amino acid identity, respectively, with those of the human Sos1 GEF. Outside the conserved catalytic domain, the remainder of the RasGEFB protein contains no common sequence motifs and has no obvious sequence homology with known proteins. Disruption of rasgefB

A rasGEFB gene disruption vector (see Supplementary material) was transfected into AX3 cells, and transformants were cloned following 7 days of selection in blasticidin S. Of 20 independent clones examined, five were found to contain a simple disruption in rasgefB. All clones were phenotypically similar under all assay conditions tested, and therefore the data for only one rasgefB– clone are presented here. The wild-type rasgefB gene was retransfected back into this clone, under the control of the constitutive act15 promoter, as a control for non-specific effects of transformation. This cell line, which will be referred to as rasgefB–rasgefB, behaved like AX3 in all experiments, confirming that the rasgefB cDNA was fully functional. The rasgefB cDNA was used to probe a northern blot of Dictyostelium total RNA. A single 5 kb mRNA was detected

Figure 1 (a) MVVILWGWNENRFNTLDQVISFDFIEENKKLYLKILENEKQQKIYNISNLIGRRMDNYRDNNNNNGNGSNINNNGIIINS SMGSQYFNLNKGIIIVESDNETFLIEEIDYLGKFPILKQFIKCEEFIGISTLEIISNIEKQLSNIVNLKSNTTEQTDRKY KTNLIDFKESIIQLEKDCKNLLKQSNLINPSIKYSVVKSVKFLLKEIKFEFYHGDINIKRIELFQQLKKFLKIHFWNNPN LILNSGSNIDMTDSNDSISSEEINSPNDLNLSLSSFPSISSPSNSSYNNINTLTFSNNNNNNNNNSNNNNNNNNNNNNNN SIINSNNNNNSNNNSNISNSSNNNNNNSNNSNNISINVSKTGNNSVINSPTQSSSCYIDCDDTMSNYTDCTNTTNTNSTT TTTTTTTTYGNNLNVYNNNNNNNTNNNNNNVPYKKPALKMMKSPSFKIQLIQEVYLEIMDQCKKIQNYLYELLDTYLTDE EYINNNNNNNNKDISILFENNTSYCIAQEIFISLIKNSNDFLYQVQLIDLTDTRKKSKELSEVSNQLFYSTQQLFPSISD HLDFIEMDIFTNNNTTTTTTTTTTTTNTTTNPSNTIKSSHSSLLSSSYSSSKDDDIVLTINKVLSKLDTFSKEYTKLIGN NLSSNYYVDSGNNNESVSSPKCGLFLSHSLSTDSPMTYVKRNTSSGSSASTSSYTTPVSSSASLSFSSSTQTPSSAASQH HIQQILQQQQQQQQQQQQQQQQQQPQQIQSQSQQQHQQQQQQQQQQQQQQQQQQQQQQQQQQQQIQSQSQQQQQLNRKPH YSSISPTSMLVYNATGGKFGVVNQNNNSAESTPTFQLNTMDILNSLMGKEGQNSNTNNANNPNNNNNNNNNNNNNNNNNN NNNNNNENKNENKNETNKSGHNYNNSSTSTLSSSTTIGDLANVSSTNSPNSSTPNLLLPPHQFHNHNREYNHSHHHLLSS SSNNVMINNRSIGKSFSTGNFSQPQPIMPSNGIGGSGGGGLGTTLSVGNGGIVGGLQSRSSSFEENQLQYAIESFMEVYN HLTSKDKVDTSIWLEEEKEDINVYYINNTNIDGKFYRSIKYASLNQLILKLTCESNTELRFTKTFITTYRSFTTGDIFLM KLIQRYFTPNLKNIVPYQFVQKIQIPIQLRVLNVLRLWIEQYPSDFQQPPQITTQSTQPIQNSTTQPQPQPQPQQPQPQL QQSTNLQNSTIQQPSLFKMLTAFLNSTRRNGHGQYSDLILKKFKQQKQKDRLQLPIINNGQGIPKAKIFWMKYSTEFIFS QSSTDIAEQLTLLDFDSYKSIEEIELLNQAWSKPDQKTNTPNIASMVNRFNSFSSFVSWAILRENDIKQRSKMMLKMIKI CYALYKLSNFNGLLAGLSGLMASGVYRLNHTKSLISKQYQKKFDFLCKFLDTKKSHKVYRDIIHSTCPPLIPYLGIYLTD LTFIEDGNQDEIKGLINFKKREMIFNTILEIQQYQQQGYTIKPKPSVLGFLCELPHMSDKKKFEDDTYEQSILLEPKNST KLEVMNAKK

(c) RasGEfB: 1079 IKYASLNQLILKLTCESNTELRFTKTFITTYRSFTTGDIFLMKLIQRY 1127 IK ++ +LI +LT ++ F +TF+TTYRSF + +L LI+R+ hSos : 562 IKGGTVVKLIERLTYHMYADPNFVRTFLTTYRSFCKPQELLSLLIERF 610

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(b) RasGEfB: 1272 KYSTEFIFSQSSTDIAEQLTLLDFDSYKSIEEIELLNQAWSKPDQKTNTPNIASMVNRFN 1331 ++ T +++ +IA QLTLL+ D Y+ ++ EL+ W+K D++ N+PN+ M+ + hSos : 768 QFETFDLMTLDPIEIARQLTLLESDLYRKVQPSELVGSVWTKEDKEINSPNLLKMIRHTT 827 RasGEfB: 1332 SFSSFVSWAILRENDIKQRSKMMLKMIKICYALYKLSNFNGLLAGLSGLMASGVYRLNHT 1391 ++ + I+ +++++R ++ ++I+I + L+NFNG+L +S + + VYRL+HT hSos : 828 NLTLWFEKCIVEAENFEERVAVLSRIIEILQVFQDLNNFNGVLEIVSAVNSVSVYRLDHT 887 RasGEfB: 1392 KSLISKQYQKKFDFLCKFLDTKKSH-KVYRDIIHSTCPPLIPYLGIYLTDLTFIEDGNQD 1451 + ++ +K +D + ++ + H K Y ++S PP +P++GIYLT++ E+GN+D hSos : 888 FEALQERKRKILD---EAVELSQDHFKKYLVKLKSINPPCVPFFGIYLTNILKTEEGNND 944 RasGEfB: 1452 EI----KGLINFKKRE 1463 + K LINF KR hSos : 945 FLKRKGKDLINFSKRR 960

Current Biology

The RasGEFB protein contains a carboxyterminal RasGEF domain. (a) Predicted amino-acid sequence of RasGEFB (GenBank accession number AF275723). The putative RasGEF catalytic domain is shaded. (b,c) Alignment of amino-acid sequences of the (b) Cdc25 homology domain and (c) Rasspecific REM box from the RasGEFB and Sos1 (hSos) catalytic domains. Pluses indicate conservative substitutions. (d) Northern blot. Total RNA (20 µg), extracted from parental AX3 and rasgefB– cells at the indicated times of development (in hours), was separated on a 1% agarose gel and transferred to a nylon membrane. The membrane was cut horizontally and hybridised simultaneously to a radiolabelled fragment of the rasgefB cDNA and an IG7 probe used as a loading control.

Research Paper Dictyostelium RasGEFB regulates endocytosis and cell speed Wilkins et al.

The osmolarity of HL5 axenic medium is fivefold higher than that of the KK2 buffer used for the bacterial suspension experiments [27]. As rasgefB– cells were capable of proliferation in a suspension of bacteria but not when cultured in axenic medium, it was therefore possible that the inability of rasgefB– cells to proliferate was a consequence of the high osmolarity of the axenic medium. To address this, the ability of rasgefB– cells to proliferate in HL5 axenic medium containing a dense suspension of heat-killed bacteria was assessed. As in the previous set of experiments, the rasgefB– cells had a doubling time of 7 hours whereas AX3 cells doubled every 4 hours (Figure 2d). It therefore seems likely that the proliferation defect of rasgefB– cells in axenic medium was not caused by the change in osmolarity experienced by the cells when transferred to axenic medium. As a consequence of the proliferation defect of rasgefB– cells in axenic culture, cells had to be maintained on bacterial lawns or in a shaking bacterial suspension. All the results described below were obtained using cells from bacterial lawns.

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Dictyostelium cells lacking the rasG or rasS genes are unable to proliferate in shaking axenic culture; rasG– cells have a cytokinesis defect and become large and multinucleate [9] whereas rasS– cells are unable to perform fluid-phase endocytosis [25]. To determine whether the rasgefB mutant shared these phenotypes, the proliferation rate in shaking axenic culture of vegetative rasgefB– cells relative to the AX3 parent was assessed (Figure 2a). AX3 cells proliferated with a doubling time of approximately 8 hours whereas, even after 100 hours, the rasgefB– cell lines failed to proliferate significantly. When viewed under a haemocytometer, rasgefB– cells kept in shaking axenic culture for several days did not exhibit the huge increase in cell size seen in shaking, axenic rasG– cells (data not shown), suggesting that the proliferation defect is not due to a failure in cytokinesis. The failure of cells to grow in normal axenic medium could be due to reduced uptake of nutrients. Alternatively, rasgefB– cells could be defective in some form of nutrient sensing, for example, for glucose or amino acids, which is required for axenic growth. We therefore measured growth in MA medium, a richer medium designed for growth of cells that lack axenic mutations. As shown in Figure 2b, rasgefB– cells proliferated in MA medium, albeit slowly. Under these conditions, we were unable to grow NC4, the parent of AX3 (Figure 2b). Similarly, rasgefB– cells could proliferate when shaken in a dense bacterial suspension, albeit at a reduced rate relative to AX3 cells; rasgefB– cells had a doubling time of 7 hours whereas AX3 cells doubled every 4 hours under these conditions (Figure 2c).

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throughout growth and development in AX3 cells, with maximal expression after 8 hours of starvation (Figure 1d). This mRNA species was absent in rasgefB– cells, confirming the functional deletion of the rasgefB gene.

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Proliferation of wild-type and rasgefB– cells in axenic culture and bacterial suspension. Cell proliferation in (a) shaken axenic culture, (b) rich medium, (c) shaken bacterial suspension, and (d) shaken bacterial suspension in axenic medium. (a,c,d) AX3 and rasgefB– cells were seeded at approximately 2 × 105 per ml into (a) flasks of HL5 medium, or into a suspension of (c) live Escherichia coli B/r strain in KK2 medium or (d) heat-killed E. coli B/r in HL5 medium. (b) NC4 and rasgefB– cells were seeded at approximately 2 × 104 per ml into flasks of MA medium. All cultures were shaken at 150 rpm and, at the indicated times, the cell number was determined using a haemacytometer. In all cases, the mean ± SD of three independent cultures is plotted against time.

The rasgefB– cells are impaired in both fluid-phase endocytosis and phagocytosis

Only cells capable of efficient fluid-phase endocytosis can proliferate in liquid medium. A number of Dictyostelium mutants that have impaired axenic growth show a corresponding impairment in fluid-phase endocytosis [25,27]. To investigate the fluid-phase endocytosis of rasgefB– cells, their ability to take up the fluid-phase marker tetramethylrhodamine isothiocyanate (TRITC)–dextran in axenic culture was assessed (Figure 3a). AX3 cells took up TRITC–dextran rapidly, reaching a maximum after approximately 125 minutes, whereas rasgefB– cells showed almost no accumulation of the marker during this time. Thus, it seems that rasgefB– cells are incapable of normal fluidphase endocytosis in axenic medium. This defect is consistent with the impaired proliferation of rasgefB– cells in axenic medium. As rasgefB– cells showed a reduced proliferation rate in bacterial suspension, it was possible that they were also

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(a) Fluid-phase endocytosis, and (b,c) phagocytosis of (b) bacteria and (c) S. cerevisiae by wild-type and rasgefB– cells. AX3 and rasgefB– cells were seeded at (a) 5 × 106 per ml into flasks of HL5 medium containing 2 mg/ml TRITC–dextran, or (b) 1 × 106 per ml into flasks containing a suspension of E. coli B/r in KK2 medium, or (c) 2 × 106 per ml into flasks of KK2 containing a suspension of TRITC–dextranlabelled yeast (109 cells per ml). All cultures were shaken at 150 rpm; 1 ml of each culture was removed at various times and (a) the amount of fluid uptake, or (c) the relative uptake of yeast, determined using a fluorimeter, or (b) the optical density at 600 nm determined using a spectrophotometer. In (a,c), the graphs show

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the data points from three separate experiments, the best-fit curve being fitted to their mean value. In (b), the graph shows the

impaired in phagocytosis. The ability of rasgefB– cells to phagocytose live bacteria was assessed (Figure 3b). The rasgefB– cells reduced the optical density of a bacterial suspension at about a third of the rate of AX3 cells. The rasgefB– cells were also severely impaired in phagocytosis of Saccharomyces cells — AX3 cells took up the yeast cells rapidly, reaching a maximum after approximately 150 minutes, whereas rasgefB– cells showed almost no accumulation during this time (Figure 3c). Therefore, the reduced proliferation rate of rasgefB– cells in bacterial suspension correlates with impairment of phagocytosis. It should be noted that these phagocytosis experiments used bacteria at a tenfold lower density than that used for the proliferation assays described in Figure 2c. There is therefore no inconsistency between the ability of cells to proliferate in bacterial suspension and the phagocytosis defects observed here. Aberrant morphology of rasgefB– cells

Dictyostelium cells lacking RasG or RasS have aberrant cellular morphology when attached to surfaces: rasG– cells appear flattened and non-polar [9] whereas rasS– cells are elongate and highly polarised, with prominent pseudopods [25]. To examine the cellular morphology of the AX3 and rasgefB– cells from axenic culture, scanning electron microscopy (SEM) was performed (Figure 4). AX3 cells had a rounded morphology with some membrane ruffles but few filopods. Circular ruffles or crowns were prominent on the majority of the cells, usually on the dorsal surface (214 crowns were present on 200 AX3 cells). These crowns are the formation sites of macropinosomes, the vesicles responsible for fluid-phase uptake by axenic cells [28]. In contrast, rasgefB– cells were strikingly more flattened, elongate, polarised and dominated by a single large lamella. Although there were an increased number of membrane protrusions in the rasgefB– cells, none of the rasgefB– cells examined possessed the dorsal circular

mean ± SD of three independent cultures; the decrease in the density of the bacterial culture was taken as a measure of phagocytosis.

ruffles typical of normal crowns. The absence of normal crowns correlates well with the severe fluid-phase endocytosis defect of rasgefB– cells. The morphology of rasgefB– cells was similar to that of rasS– cells, although the polarity of rasgefB– cells was if anything more exaggerated. It was also similar to that of normal Dictyostelium cells that have developed to an aggregation competent state [29]. There were also striking differences between rasgefB– and AX3 cells in filamentous actin (F-actin) distribution (Figure 5). In axenic AX3 cells, the majority of F-actin was recruited into the macropinocytic crowns. In rasgefB– cells, which did not have crowns, F-actin was usually localised in a single large lamella at the leading edge of the cell. These F-actin distributions were consistent with the cellular morphologies observed by SEM (Figure 4). Although there were fewer differences between rasgefB– and AX3 cells when they were grown on bacteria (data not shown), the F-actin distribution remained markedly different in the two cell types. In bacterially grown AX3 cells, the majority of F-actin was recruited into several small pseudopods whereas in rasgefB– cells the majority of Factin was concentrated in a single large lamella (Figure 5). The rasgefB– cells move unusually rapidly

Cellular morphology and F-actin distribution were similar in rasgefB– and rasS- cells. In the case of rasS– cells, this correlates with a threefold increase in the speed of movement of vegetative cells [25]. To determine whether this was also true for rasgefB–, cells were transferred to tissue culture plates and time-lapse images collected to allow analysis of cell speed. Bacterially grown AX3 cells moved at a rate of 6 ± 1.6 µm/minute whereas rasgefB– cells moved at 13.3 ± 2.1 µm/minute. In the case of axenic cells, AX3 cells moved at a rate of 1.8 ± 0.3 µm/minute whereas rasgefB– cells moved at 15.2 ± 4.1 µm/minute. Therefore under both sets of conditions, the speed of rasgefB– cells

Research Paper Dictyostelium RasGEFB regulates endocytosis and cell speed Wilkins et al.

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Morphology of wild-type and rasgefB– cells. Vegetative (a,b) AX3 and (c,d) rasgefB– cells were placed in HL5 medium for 24 h, then prepared for SEM as described in the Materials and methods. Arrowheads indicate crowns, the dorsal, circular ruffles that are the sites of macropinosome formation. Although present on the majority of AX3 cells, these structures were never seen on rasgefB– cells.

was significantly greater than that of AX3 cells (two sided t-test, p < 0.001). This increase in cell speed is consistent with the observed F-actin distribution and cellular morphology of rasgefB– cells. This unusually fast movement did not appear to compromise the chemotaxis of rasgefB– cells. When challenged with a cAMP-containing micropipette , they moved up the cAMP gradient with great accuracy (Figure 6). In common with most highly polar cells, they most often moved by turning an established leading edge (Figure 6, first three panels), but would also change cell polarity by advancing new pseudopods up the gradient if stimulated appropriately (Figure 6, last six panels). Wild-type cells were less chemotactic under these conditions (data not shown), either because of their slower movement or because they were better at generating their own, competing, signals. To test the dose-response of chemotaxis, a two-drop assay was performed in which approximately 0.1 µl of a cAMP solution was spotted near a 0.1 µl drop of cells [30]. Under these conditions, rasgefB– cells showed positive chemotaxis to cAMP to the same extent and over the same concentration range as AX3 cells. The weak aggregation of rasgefB– cells (shown below) is therefore not due to a simple defect in chemotaxis. Defects in multicellular development

When spotted onto a lawn of bacteria, wild-type cells grow by phagocytosis, clearing a plaque in the lawn, which increases in size as the cells proliferate. Roughly two

Current Biology

Distinct patterns of F-actin distribution in rasgefB– cells. (a) Parental AX3 and (b) rasgefB– cells were cultured in HL5 medium for 24 h before fixation and staining with Texas red–phalloidin as described in the Materials and methods. In the parent, F-actin accumulated mainly in the crowns, which are the normal site of macropinosome formation. This staining pattern was never seen on rasgefB– cells, in which F-actin accumulated in a single large pseudopod at the leading edge of each cell. Vegetative, bacterially grown (c) AX3 and (d) rasgefB– cells were similarly fixed and stained. There was a concentration of F-actin in a single large pseudopod at the leading edge of rasgefB– cells, which was not seen in AX3 cells. The scale bars represent 5 µm.

zones are visible in the plaque; a zone on the periphery where the bacterial lawn has thinned but is not completely cleared, called the ‘feeding front’, and a central zone in which the bacteria have been cleared and the Dictyostelium cells are in various stages of multicellular development. When a similar amount of rasgefB– cells was spotted onto a bacterial lawn, the plaque formed was strikingly different from that of AX3 cells (Figure 7a). The plaques of rasgefB– cells were two or three times larger than those of AX3 cells. Although the bacterial lawn was thinned, rasgefB– plaques were never fully cleared of bacteria. In addition, although some loose aggregates of cells appeared, the rasgefB– plaques contained relatively few small fruiting bodies. The increased speed of vegetative rasgefB– cells coupled with their reduced phagocytic rate provides an explanation for the large diffuse plaques formed by rasgefB– cells on bacterial lawns. The mutant cells are highly motile, and thus can move faster across the bacterial lawn, causing an increase in colony diameter. Furthermore, the rasgefB– cells only partially cleared the bacterial lawn as they moved out, a phenotype consistent with the impaired phagocytosis observed in the mutant line. The size of plaques formed by rasgefB– cells was strikingly similar to that of plaques formed by rasS– cells [25]; however, the multicellular development of rasS– cells on bacterial lawns is apparently normal.

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Development of wild-type and rasgefB– cells. (a) Vegetative AX3 and rasgefB– cells (103 of each) were spotted onto lawns of Klebsiella aerogenes bacteria on SM agar plates. After 7 days’ incubation at 22°C, the resultant plaques were photographed. (b) Vegetative AX3 and rasgefB– cells were deposited on nitrocellulose filters at a density of 3 × 106 cells/cm2, and photographed after 36 h of development. The scale bars represent 1 mm.

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Chemotaxis of rasgefB– cells. Bacterially grown rasgefB– cells were developed in shaken suspension, in the presence of exogenous cAMP pulses, for 5 h; 5 × 106 cells were allowed to adhere to a 24 mm glass coverslip, then stimulated by an adjacent micropipette containing 10 µM cAMP. The micropipette was moved at 220, 290 and 350 sec.

Development of rasgefB– cells on nitrocellulose filters or on non-nutrient agar was also aberrant (Figure 7b). AX3 cells completed multicellular development within 24 hours, forming spore-containing fruiting bodies. After 24 hours, rasgefB– cells had only begun to form very small aggregates. Over the next 12 hours, these structures proceeded to undergo apparently normal morphogenesis, resulting in minute but morphologically normal fruiting bodies that contained mature, functional spores. Although belatedly able to aggregate, rasgefB– cells never formed streams, which are characteristic of efficient aggregation, when developed on non-nutrient agar (data not shown). The developmental defect of rasgefB– cells was unaffected by

Gene expression is tightly regulated throughout the development of Dictyostelium and certain genes serve as markers of the extent of development. One of the earliest genes expressed after starvation is the gene encoding the lectin discoidin-Iγ; disIγ expression is first detectable after 2 hours of starvation and expression is maximal after approximately 5 hours, thereafter its expression is repressed by the pulses of extracellular cAMP that constitute the aggregation stimulus [31]. The next set of genes to be expressed includes those encoding the aggregationstage cAMP receptor cAR1 and the adenylyl cyclase ACA, which are required to generate the extracellular cAMP that constitutes the aggregation stimulus; cAMP signalling is also required for induction of a number of aggregation stage genes including the cell–cell adhesion molecule csA [32]. To analyse the physiological state of rasgefB– cells during growth and early development, we analysed the expression of these early developmental genes in AX3 and rasgefB– cells upon starvation (Figure 8a–c). In the case of carA and acaA, mRNA levels were measured with or without treatment with exogenous cAMP pulses every 6 minutes to mimic normal signalling. During the first few hours of development, AX3 cells exhibited typical expression patterns for disIγ, carA and acaA mRNA, and csA protein (Figure 8a–c); rasgefB– cells showed similar expression profiles for carA and acaA in the absence of exogenous cAMP, and similar induction of the csA protein. However, disIγ expression was almost entirely absent in rasgefB– cells (Figure 8a), and carA and acaA

Research Paper Dictyostelium RasGEFB regulates endocytosis and cell speed Wilkins et al.

Figure 8 AX3 0 2 4 6 8 10

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(b) csA

AX3 0 2 4 6 8

(c)

rasgefB– 0 2 4 6 8

carA + cAMP acaA carA – cAMP acaA Current Biology

Gene expression during early development of wild-type and rasgefB– cells. (a) Vegetative AX3 and rasgefB– cells were plated at a density of 3 × 106 cells/cm2 on nitrocellulose filters and allowed to develop. Cells were harvested at the indicated times (in hours) and total RNA prepared; 20 µg RNA was separated on a 1% agarose gel, transferred to nylon membrane and hybridised with a radiolabelled disIγ cDNA probe. (b) As in (a), except that harvested cells were dissolved in SDS–PAGE sample buffer, separated on a 10% polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The blot was probed with a monoclonal antibody to csA protein, and visualised using a Pierce Supersignal kit. (c) AX3 and rasgefB– cells were starved in a suspension of KK2 at 5 × 107 cells per ml, with or without addition of 100 nM cAMP every 6 min. Cells were harvested at the indicated hours of development and total RNA prepared; 20 µg RNA was separated on a 1% agarose gel, transferred to a nylon membrane and hybridised with radiolabelled carA and acaA cDNA probes.

were expressed precociously in cAMP-treated cells. These results have a number of important implications for understanding of the phenotype of rasgefB– cells. Firstly, rasgefB– cells had the properties of aggregating Dictyostelium cells: highly polarised morphology, a large number of filopods and a dramatic decrease in the rate of phagocytosis [29,33]; they were also highly motile, moving approximately three times faster than vegetative cells [34]. It therefore seemed possible that the phenotype of rasgefB– cells might reflect premature development to an aggregation-competent state. However, rasgefB– cells did not express any early developmental or aggregation-stage genes during vegetative growth. Thus, the increased cell speed, reduced endocytic rate and polarised morphology of rasgefB– cells is not a simple consequence of the cells being inappropriately advanced into development.

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Secondly, although unable to aggregate, rasgefB– cells were able to induce expression of acaA and carA normally upon starvation. Moreover, the cells would appear to be capable of generating functional pulses of cAMP as the csA protein was induced normally in rasgefB– cells. Thus, the aggregation defect of rasgefB– cells does not appear to be a consequence of the lack of a functional cAMP relay system or, as previously mentioned, an inability to perform normal chemotaxis to cAMP upon starvation. This may indicate that RasGEFB controls some component of an as yet unidentified pathway that is required for aggregation. Thirdly, although carA, acaA and csA expression were normal in rasgefB– cells, there was clearly a defect in early gene expression. The disIγ gene is, in part, induced by factors secreted in response to nutrient depletion and increasing cell density and is usually a good indicator of the onset of starvation [35]. It was therefore surprising that, although rasgefB– cells appeared to enter the developmental programme, they failed to induce disIγ expression. In addition, although able to respond to pulses of exogenous cAMP, the rasgefB– cells seemed to be hypersensitive to cAMP, such that they maximally induced acaA and carA expression several hours earlier than the AX3 parent cells. Taken together, these data suggest that the normal control of early developmental gene expression is lost in rasgefB– cells, implicating RasGEFB in control of the early cellular response to starvation. The cell-type specific genes ecmA and pspA were each expressed at apparently normal levels during multicellular development (see Supplementary material), which indicates that loss of the rasgefB gene does not greatly affect either the differentiation or the proportioning of prestalk and prespore cells. Complementation of a S. cerevisiae CDC25 mutant by rasgefB

Although sequence homology predicts that RasGEFB will function as a GEF for Ras, an inability to produce soluble, recombinant RasGEFB protein precluded a direct demonstration of this in vitro. The temperature-sensitive CDC25-5 S. cerevisiae mutant has previously proved useful for assessing the catalytic activity of putative RasGEFs. Expression of the catalytic domains of the murine RasGEFs, Sos and RasGRF, will complement the phenotype of this yeast mutant, allowing growth at the non-permissive temperature (37°C) [36,37]. However, expression of the S. cerevisiae BUD5 gene, which encodes a GEF for the Rap-related Bud1 protein, is unable to complement the CDC25-5 mutant [38]. To demonstrate that rasgefB encodes a functional RasGEF, DNA encoding the putative catalytic domain (residues 1078–1463) was transfected into CDC25-5 under the control of the strong ADH promoter (see Materials and methods). Expression of the putative catalytic domain of RasGEFB allowed growth of CDC25-5 at the non-permissive temperature in a similar manner to a

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Figure 9 30ºC

37ºC

Vector only

CDC25

rasgefB Current Biology

Complementation of a S. cerevisiae CDC25 mutant by the putative catalytic domain of RasGEFB. The temperature-sensitive CDC25 S. cerevisiae strain LV25-5 was transformed with plasmids expressing either the catalytic domain of Cdc25p or the putative catalytic domain of RasGEFB (residues 1078–1463). An empty pAD4 vector transformation was used as a negative control. See the Materials and methods for details of the rasgefB expression construct. Three clones from each transformation were patched onto two separate Leu– synthetic medium agar plates and incubated at either 30°C or 37°C. Colony growth was assessed after 5 days.

plasmid expressing the Cdc25 catalytic domain [12], whereas transformation with the pAD4 vector alone [39] did not (Figure 9). This strongly supports the idea that RasGEFB functions as a genuine RasGEF in vivo.

Discussion We have identified a novel Dictyostelium RasGEF gene, rasgefB. Dictyostelium cells lacking rasgefB were unable to proliferate in axenic culture and exhibited a reduced rate of proliferation in a bacterial suspension. These proliferation defects correlated with profound defects in fluidphase endocytosis and phagocytosis. In axenic culture, rasgefB– cells had a highly polarised morphology, with an increased number of membrane protrusions and a total absence of crowns, the sites of macropinosome formation. In addition, vegetative rasgefB– cells moved at about twice the speed of the parental AX3 cells. Although capable of cAMP chemotaxis, rasgefB– cells were unable to form normal aggregation streams upon starvation, resulting in the formation of small aggregates that developed into small but morphologically normal fruiting bodies. Activation of RasGEFB

Although the predicted protein sequence of RasGEFB contains a putative RasGEF catalytic domain, the remainder of the sequence has no obvious homology to known proteins. In general, RasGEFs are thought to require recruitment to cellular membranes in order to come into contact with their Ras protein targets [40]. In other RasGEFs, this seems to be mediated via adaptor proteins such as Grb2 [41], which binds to a polyproline region on the Sos RasGEF via its two Src homology 3 (SH3) domains, or by other domains in the molecule such as pleckstrin

homology (PH) domains, which can bind free βγ subunits of heterotrimeric G proteins or to various membrane phospholipids. As RasGEFB contains no obvious means of membrane recruitment, it not clear how it is able to activate its target Ras proteins. Preliminary data from expression of a green fluorescent protein (GFP)–RasGEFB fusion protein suggested that RasGEFB is cytoplasmically localised and not constitutively membrane anchored. It will be interesting to determine whether RasGEFB protein is recruited to the plasma membrane, and if so which cellular signals cause it to relocalize. Signalling pathways downstream of RasGEFB

Cells disrupted in rasS– cells show a strikingly similar phenotype to that of vegetative rasgefB– cells — they are strongly impaired in solid- and fluid-phase endocytosis and have an increased cell speed [25]. The developmental defect of rasgefB– cells is, however, not apparent in any of the known Ras mutants. RasS may therefore be a direct target of RasGEFB in a signalling pathway controlling the balance between endocytosis and motility whereas another, as yet uncharacterised Ras, perhaps RasB or RasC, may mediate the signals from RasGEFB required for normal aggregation. Alternatively, RasS may mediate all signalling from RasGEFB, but other Ras proteins could take over its roles during development. Single and multiple mutants for all the Dictyostelium ras genes will need to be generated to resolve this question. Of the accepted Ras effectors, only the class I phosphoinositide (PI) 3-kinases have been found in Dictyostelium cells so far. Interestingly, cells lacking two class I PI 3kinases (PIK1 and PIK2) show diminished fluid-phase endocytosis and increased motility [42,43]. The endocytosis defect of pik1–/pik2– cells is far less severe than both rasgefB– and rasS– mutants, but this could reflect partial compensation by PIK3. Although a direct link between RasGEFB, RasS and PI 3-kinase has not yet been demonstrated, it seems likely that these three proteins are all members of the same signalling pathway controlling endocytosis and motility Endocytosis and motility defects

The loss of rasgefB resulted in a profound impairment of fluid-phase endocytosis. This correlated with an inability to proliferate in axenic culture. The lack of nutrient availability caused by diminished uptake of medium is a probable reason for the proliferation defect of rasgefB– cells in axenic culture, although it has not been possible to demonstrate this directly. The reduced rate of fluid-phase endocytosis was accompanied by a reduction in the rate of phagocytosis and an increase in cell speed. Together, these additional phenotypes of rasgefB– cells provide an explanation for the enlarged and diffuse colony morphology seen when rasgefB– cells are grown on bacterial lawns.

Research Paper Dictyostelium RasGEFB regulates endocytosis and cell speed Wilkins et al.

The cells were able to move outwards from the centre of the plaque more rapidly, creating a plaque of greater size, while the cells were unable to phagocytose the bacteria as rapidly as wild-type cells, resulting in partial clearance of the bacterial lawn and the characteristically diffuse appearance. This observation also serves to highlight the danger of using plaque size as a measure of cell proliferation rate; rasgefB– cells would appear to be growing faster than wild-type cells as the rasgefB– plaque size increased at a faster rate, but examination of the proliferation rate of rasgefB– cells in shaking bacterial suspension showed that the cells proliferate more slowly. A body of evidence suggests that the processes of endocytosis and cell migration are in constant competition with one another. Competition arises between pseudopods and the membrane protrusions which mediate phagocytosis and macropinocytosis, as they are projected. It appears that the two structures can only coexist in the same cell for a short time [44]. Leading edges have been observed to recruit the actin-binding protein coronin from regressing phagocytic cups, and extending cups can sequester coronin from retracting pseudopods [44]. It therefore appears that competition for cytoskeletal components limits the extent to which a cell can perform phagocytosis and migrate at the same time. These data suggest an inverse correlation between cell speed and the intrinsic rate of endocytosis in the Dictyostelium cell. It may be that the function of the RasGEFB/RasS signalling pathway is to regulate this balance between endocytosis and motility, favouring endocytic feeding during growth and motility while cells are aggregating. Developmental defects

The rasgefB– cells were both delayed in multicellular development and unable to aggregate normally. Although able to initiate expression of the aggregation-stage cAMP receptor and adenylyl cyclase, rasgefB– cells failed to induce expression of discoidin upon starvation. Surprisingly, rasgefB– cells were able to perform normal chemotaxis to cAMP and seemed capable of producing pulses of cAMP as the csA protein was induced normally. Some Dictyostelium mutants are unable to aggregate normally by chemotaxis and do so by random motion and adhesion [45,46]. In most cases, although the resultant aggregates are smaller then normal, the time-scale of development is similar to that of the parental cells. The delay in multicellular development in rasgefB– cells suggests that there is a partial block in development, in addition to the defect in aggregation. The lack of disIγ induction is unlikely to be the cause of the developmental delay or the aggregation defect as cells lacking discoidin-I are able to aggregate normally upon starvation [47]. However, the absence of disIγ may indicate that the developmental delay is caused by a lack of induction of a subset of genes necessary for aggregation. Alternatively, all the necessary

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cellular components required for normal development may be present but signalling through RasGEFB may be required to attenuate a signal inhibitory to aggregation. Although required for normal aggregation, it does not appear that RasGEFB is required for cell-fate specification during later development as both prestalk and prespore cell mRNAs were expressed. Physiological function of rasgefB

In several respects, vegetative rasgefB– cells behaved like aggregation-competent amoebae [4]. They were highly polarised, highly motile, and they had a reduced rate of endocytosis. It was therefore surprising to find that the mutant did not express early developmental or aggregation-stage marker genes during growth. A possible explanation for this phenotype that fits the available data is that there are independent, parallel, rather than sequential, developmental signalling pathways required for the progression of cells to an aggregation-competent state. Loss of one of these signalling pathways, perhaps involving RasGEFB, might give rise to cells that manifest a specific behaviour of starved amoebae, such as rapid migration, without any of the others. The physiological function of RasGEFB may therefore be to control the balance between endocytosis and motility in the context of the early developmental program, favouring endocytic feeding during vegetative growth and motility during early development. In addition, signalling through RasGEFB may be required to allow aggregation, either by inducing the expression of genes required for aggregation or attenuating an aggregation-blocking signal. The loss of RasGEFB would therefore result in an inappropriate increase in motility and concomitant decrease in endocytosis in the absence of starvation and prevent aggregation.

Materials and methods Unless otherwise stated, all chemicals were obtained from Sigma and all restriction and DNA modifying enzymes were from New England Biolabs.

Cloning of rasgefB cDNA and vector construction The Tsukuba Dictyostelium cDNA sequencing project databases [26] were searched using the tBLASTn search engine for clones with homology to the catalytic domain of the putative RasGEF encoded by aimless [10]. One clone, SSK260, was identified. This partial cDNA clone was obtained from T. Morio and the full DNA sequence determined. To obtain a full-length rasgefB cDNA, a λZAPII cDNA library (Stratagene) was screened using the entire cDNA insert of clone SSK260, radiolabelled by the random primer method using [α-32P]dATP (Amersham). Several partial cDNA clones were isolated, which when assembled, encompassed the entire rasgefB open reading frame. The rasgefB gene disruption vector was constructed by inserting the blasticidin resistance gene (bsr) from pBsr∆Bam [9,48] into the single Bst Z117 site of a 1.6 kb, Spe I–Not I fragment of 3′ rasgefB cDNA cloned into the EcoRI and Not I sites of pBluescript KS+. From the resulting vector (pATW71), the 3.9 kb rasgefB/bsr fragment was excised using EcoRI and Not I and used to transform Dictyostelium strain AX3 as described above. A rasgefB expression vector was constructed by inserting the entire rasgefB cDNA into the Hind III and XbaI sites of plasmid pDXA [49] to create plasmid pATW118. This vector expresses rasgefB from the strong constitutive act15 promoter and was used to reintroduce the rasgefB cDNA into

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rasgefB– cells. The rasgefB gene disruption vector (pATW71) and the rasgefB cDNA expression vector (pATW118) were transfected into cells by electroporation as previously described [9]. To assemble the yeast complementation vector, pATW99, containing the 3′ half of the rasgefB cDNA, was digested with BstB I and Not I and a doublestranded oligonucleotide inserted (5′-GGCCTGCAGATGTT-3′ and 5′-CGAACATCTGCA-3′) to create pATW123. This resulted in a Pst I and in-frame ATG translational initiation codon being added before the putative catalytic domain (residues 1078–1463) of rasgefB. The modified GEF domain was excised from pATW123 by Pst I/Sal I digestion and inserted into the Pst I/XhoI sites of pAD4 to create pATW124. This vector expresses the transgene using the strong constitutive ADH1 promoter.

Endocytosis assays Fluid-phase endocytosis was determined by a modification of previous methods [27,44]. Briefly, bacterially grown cells were pelleted at 2000 × g for 2 min, washed free of bacteria with KK2 and plated at 5 × 106 cells per plate in HL5 on 90 mm tissue culture plates for 24 h. Cells were harvested by trituration, pelleted, then resuspended at 5 × 106 cells/ml in 10 ml HL5 containing 2 mg/ml TRITC–dextran (MW 70000) and shaken at 150 rpm; 1 ml of culture was removed at regular intervals and 100 µl trypan blue added to quench extracellular fluorescence. Cells were then washed once, resuspended in 1 ml KK2 and intracellular fluorescence measured by Fluorimeter (Kontron SFM25). Excitation was at 544 nm and emission read at 574 nm. For each cell line, the assay was performed on three separate occasions using the same batch of TRITC–dextran. Results are presented in terms of relative fluorescence with the data points from each separate experiment and their mean value displayed on the same graph. Phagocytosis of bacteria was performed as described previously [50]. Dictyostelium cells were inoculated at a density of 106 cells per ml into a 20 ml suspension of E. coli strain B/r at 109 cells per ml in KK2; 1 ml culture was removed at regular intervals and the optical density at 600 nm measured. Each assay was performed in triplicate, the mean and SD plotted against time and an estimate of phagocytic rate calculated from the best-fit straight line. Phagocytosis of TRITC-labelled yeast was performed as described [44]. The assay was performed exactly as the fluid-phase endocytosis assay above except that vegetative cells were assayed at 2 × 106 in 10 ml KK2 containing TRITC– dextran-labelled yeast at 109 cells/ml (a kind gift from M. Maniak).

Microscopy Visualisation of F-actin in vegetative or axenic cells was performed exactly as described [9]. SEM was performed as follows. Cells were fixed on glass coverslips using 1% osmium tetroxide in KK2 for 5 sec followed by 2% glutaraldehyde in KK2 for 2 h. The fix was removed and the samples were progressively dehydrated through an ethanol series of 30–100% ethanol, then taken into hexamethyl disilane. After air-drying, the cells were sputter-coated in gold, and viewed on a Jeol JSM5410 scanning electron microscope. The mean speed of bacterially grown or axenic cells was estimated using time-lapse recordings of digital phase-contrast images of the cells. Bacterially grown cells were washed free of bacteria, plated in KK2 onto 90 mm tissue culture plates and allowed to adhere for 15 min. Axenic cells were assayed in HL5 medium in 90 mm tissue culture plates; 40 frames were captured at 20 sec intervals. Cell centroids were determined and the mean centroid displacement between each frame was calculated by the software.

Yeast strains, growth and transformation S. cerevisiae strain LV25-5 [12] (a strain carrying the temperature-sensitive CDC25-5 allele) was maintained at 30°C in YPD medium or on YPD agar plates. Yeast transformations were performed according to the TRAFO protocol [51] and transformants selected on Leu– drop-out media agar plates (Bio101). Transformants were patched onto Leu– plates and incubated at 30°C (permissive temperature) or 37°C (nonpermissive temperature) to assess suppression of the temperature sensitive growth defect.

Supplementary material Supplementary material including further methodological detail, two figures showing the strategy used to disrupt the rasgefB gene, and the expression of the ecmA and pspA genes during multicellular development in rasgefB– cells, is available at http://current-biology.com/supmat/ supmatin.htm.

Acknowledgements We gratefully acknowledge Y. Tanaka and the D. discoideum cDNA project in Tsukuba, Japan for the initial 3′ rasgefB clone. We thank Adrian Harwood for advice and assistance, Laura Machesky and two referees for constructive comments on the manuscript, Markus Maniak and Carole Parent for help with experimental methods, and Jan Faix and Günther Gerisch for the monoclonal antibody against csA. R.H.I. was supported by Wellcome Trust Career Development Fellowship 043754 and by an MRC Senior Fellowship.

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