702 Research Article

IntroductionDuring chemotaxis, cells polarize and coordinately movetoward an extracellular signal. To effectively move, cells mustdetect shallow gradients of extracellular signal and translatethis signal into changes in cell shape and cell adhesion (Parent,2004). Particularly important for cell motility is the properextension and retraction of actin-filled pseudopodia (VanHaastert and Devreotes, 2004; Williams and Harwood, 2003).Thus actin polymerization, depolymerization, and thebranching and crosslinking of actin filaments must be undertight spatial and temporal control for effective cell movement(Iijima et al., 2002). To date, an increasing number of actin andactin-associated proteins have been identified that couldregulate the organization of the actin cytoskeleton duringdirected chemotactic migration.

Dictyostelium discoideum is an excellent model to studychemotaxis and cell motility. When starved, Dictyosteliumamoebae secrete pulses of extracellular cAMP and use this cueto stream together to form a multicellular aggregate. As theychemotax toward the cAMP signal, Dictyostelium amoebaerapidly extend and retract pseudopodia enriched in both actinand many actin-associated proteins. How these proteinscoordinately regulate pseudopodium formation is notcompletely understood, but at least part of pseudopodiumbehavior is accomplished by myosins. Two myosin I proteins,MyoA and MyoB, accumulate at the leading edge of streamingcells to regulate retraction of pseudopodia and to suppress non-

productive pseudopodium formation (Novak et al., 1995;Wessels et al., 1996). The conventional myosin, myosin II,concentrates at the trailing edge of chemotaxing cells, andfunctions to suppress lateral pseudopodia (Wessels et al.,1988). In addition to these myosins, actin-associated proteinsalso regulate actin dynamics within pseudopodia. The Arp2/3complex, as well as its activators such as the WASP/SCARfamily of adaptor proteins, functions in actin networkdynamics at the leading edge (Machesky and Insall, 1998;Mullins et al., 1998; Zalevsky et al., 2001). PIR21, acomponent of the SCAR complex, regulates actinpolymerization and affects pseudopodium formation (Bear etal., 1998; Blagg et al., 2003). While some of the importantregulators of pseudopodium formation are known, how theirdiverse activities are coordinated is not well understood.

One important regulator of the actin cytoskeleton ineukaryotic cells is the protein Abp1. Abp1 was one of the firstactin binding proteins identified in the yeast S. cerevisiae(Drubin et al., 1988). Overexpression of Abp1 in yeast altersactin organization and affects polarized cell growth (Drubin etal., 1990; Fazi et al., 2002). The mammalian Abp1 homologuefunctions in receptor-mediated endocytosis (Kessels et al.,2000; Kessels et al., 2001). Both yeast Abp1 and mammalianAbp1 contain an ADFH domain at their amino termini thatbinds to actin. Both yeast and mammalian Abp1 also have anSH3 domain at their carboxyl termini that binds ligands thatfunction in endocytosis. This domain structure supports a role

When starved, Dictyostelium cells respond to extracellularsignals, polarize, and move with strong persistence intoaggregation centers. Actin and actin-associated proteinsplay key roles in regulating both the morphology anddirected movements of cells during chemotacticaggregation. Recently, we identified an ortholog of Abp1 inDictyostelium (Dabp1). The first actin binding proteinidentified in yeast, Abp1 functions in actin-basedendocytosis in yeast and in receptor-mediated endocytosisin mammalian cells. To explore the functions for Abp1 inDictyostelium, we examined the phenotypes of cells thatoverexpressed the Dabp1 protein and cells that eliminatedDabp1 expression. In these mutants, most actin-basedprocesses were intact. However, cell motility was alteredduring early development. During chemotactic streaming,more than 90% of wild-type cells had a single leading

pseudopodium and a single uropodium, whereas more than27% of Dabp1 null cells projected multiple pseuodpodia.Similarly, ~90% of cells that overexpressed Dabp1projected multiple pseudopodia during chemotacticstreaming, and displayed reduced rates of cell movement.Expression of the SH3 domain of Dabp1 showed thisdomain to be an important determinant in regulatingpseudopodium number. These results suggest that Abp1controls pseudopodium number and motility in early stagesof chemotactic aggregation in Dictyostelium.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/119/4/710/DC1

Key words: Actin cytoskeleton, Cell motility, Chemotacticaggregation, Pseudopodium

Summary

Abp1 regulates pseudopodium number inchemotaxing Dictyostelium cellsYanqin Wang and Theresa J. O’Halloran*Department of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin,TX 78712, USA*Author for correspondence (e-mail: t.ohalloran@mail.utexas.edu)

Accepted 17 October 2005Journal of Cell Science 119, 702-710 Published by The Company of Biologists 2006doi:10.1242/jcs.02742

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for Abp1 as a functional link between the actin cytoskeletonand other proteins (Qualmann and Kessels, 2002).

Although Abp1 is found in a diverse array of organisms, thecontribution of Abp1 activity to cellular functions remains anopen question. Here we report on the contribution of Abp1 tocellular function in Dictyostelium. We find that Dabp1(Dictyostelium actin binding protein 1) is concentrated indynamic regions of the cell cortex rich in F-actin. Moreover,Dictyostelium cells engineered to either abolish expression orto increase expression of the Dabp1 protein displayed a distinctphenotype. Altering levels of Dabp1 in cells profoundlyimpaired the ability of cells to limit pseudopodium number inearly development, and limited rates of directed cellmovement. Consequently, cells overexpressing Dabp1 formedsmaller aggregation centers and small fruiting bodies duringdevelopment. These results reveal a specific and critical rolefor Dabp1 in regulation of pseudopodium number duringdirected cell migration.

ResultsDabp1 is a member of the Abp1 familySearching the Dictyostelium gene database(www.dictybase.org) revealed a single gene for Abp1,(accession number: AY437927, Dictyostelium abpE) onchromosome 2. The predicted reading frame for the Dabp1gene, abpE, encoded a protein of 481 amino acids with amolecular mass of 59 kDa. Similar to Abp1 proteins from otherspecies, the amino acid sequence of the Dabp1 protein includedan ADFH (actin depolymerizing factor homology) domain atthe amino terminus (amino acid 1-130), and an SH3 (Srchomology 3) domain at the carboxyl terminus (amino acid 422-481). Relative to the rest of the protein, these domains shareda high degree of amino acid identity with other abp1 proteins.Across its entire length, Dabp1 shared 21% identity withmouse Abp1 and 18% identity with Abp1 in Saccharomycescerevisiae. The ADFH domain in Dabp1 shared 28.5% identitywith mouse Abp1 and 19.2% identity with the S. cerevisiaeAbp1. The SH3 domain was more conserved among Abp1members. The SH3 domain in Dabp1 shared 37% identity withmouse Abp1 and 34% identity with Abp1 in S. cerevisiae (Fig.1). Using a PCR-based approach, we cloned a cDNA for thegene and generated an antibody against the gene product.Western blots probed with affinity-purified antibodies raisedagainst the E. coli-expressed fusion protein revealed that theDabp1 protein migrated anomalously on SDS gels with a

molecular mass of 70±3 kDa (Fig. 2). Whereas cellsoverexpressing Dabp1 tagged with GFP showed multiplebands in western blots, cells that overexpressed Dabp1 withoutthe GFP tag did not display multiple bands (data not shown).

Dabp1 is enriched in the cell cortex and the leadingedgeTo determine the intracellular location of Dabp1, we stainedDictyostelium cells with affinity-purified anti-Dabp1antibodies. Inspection of growing cells by fluorescencemicroscopy revealed an extensive association of Dabp1 withthe cell cortex (Fig. 3, top panels). In polarized cellsundergoing directed migration during early development,Dabp1 was especially concentrated at the leading edge (Fig. 3,bottom panels). To investigate a possible association of Dabp1with the actin cytoskeleton, we stained cells simultaneouslywith phalloidin to label F-actin and with antibodies againstDabp1. In growing cells, Dabp1 colocalized with F-actin atsome, but not all, regions of the cortex (Fig. 3, top panel). Instreaming cells, Dabp1 colocalized with F-actin to a muchhigher extent and completely overlapped with actin at theleading edge (Fig. 3, bottom panel). We observed that theamount of Dabp1 varied in growing cells. One possible reasonis that Dabp1 associates only with portions of the actincytoskeleton that are particularly dynamic, a state that mightdiffer between cells. By contrast, Dabp1 consistentlyassociated with the leading edge of migrating cells, a region ofactive actin remodeling, suggesting that Dabp1 could associatepreferentially with areas of dynamic F-actin in Dictyostelium.

An intact actin cytoskeleton is required for the corticalresidence of Dabp1The localization of Dabp1 at the cell cortex suggested that theactin cytoskeleton could be required for the corticallocalization of Dabp1. To test this idea, we disrupted thefilament-rich actin cortex in wild-type cells with cytochalasinA, a drug that depolymerizes F-actin. Subsequently, stainingF-actin cells with Texas Red (TXRED)-labeled phalloidinconfirmed that cytochalasin dramatically altered cortical actin.Labeling the cytochalasin-treated cells with an antibody

Fig. 1. Domain structures of Abp1 orthologs in different species.M.m., Mus musculus; S.c., Saccharomyces cerevisiae; D.d.,Dictyostelium discoideum; ADFH, actin depolymerizing factorhomology domain; PPP, proline-rich region; SH3, Src homology 3domain. Numbers indicate amino acid identity shared betweendomains of abp1 orthologs.

ADFH

ADFH

ADFH

SH3

SH3

SH3

PPP

PPP

PPP

Abp1/M.m.

Abp1/D.d.

Abp1/S.c.

28.5%

19.2%

37%

34%

Fig. 2. Knockout and overexpression of Dabp1. A western blot ofwhole cell lysates from 2.5�105 wild-type cells (WT), Dabp1 nullcells (Dabp1–) and cells overexpressing Dabp1 (Dabp1+) was stainedwith affinity-purified anti-Dabp1 antibodies.

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against Dabp1 showed that the association of Dabp1 with thecell cortex was abolished concurrent with the loss of corticalactin (Fig. 4A). These results indicated that cortical actin wasrequired for the cortical localization of Dabp1.

Deletion or overexpression of Dabp1 has no effect onthe cortical localization of actinTo examine a possible requirement of Dabp1 for the cortical

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localization of actin, we generated cell lines with alteredexpression of the Dabp1 protein. Using homologousrecombination, we deleted the abpE gene to generate Dabp1null mutants (Dabp1– cells). Cell lines with increased levels ofDabp1 (Dabp1+ cells) were also made by transforming both awild-type strain and the Dabp1 null mutants with anextrachromosomal plasmid that overexpressed the GFP-Dabp1fusion protein. Western blots probed with an anti-Dabp1 serumconfirmed the absence of the Dabp1 protein in Dabp1– cells,and increased levels of Dabp1 protein in Dabp1+ cells (Fig. 2).Initial phenotypic analysis of these strains showed that theabsence of Dabp1 did not influence cell growth, while theoverexpression of Dabp1 retarded cell growth slightly.

To examine the influence of Dabp1 on cortical actin, westained Dabp1– cells and Dabp1+ cells with TXRED-labeledphalloidin. Inspection of growing cells by fluorescencemicroscopy revealed that neither the absence nor increasedlevels of Dabp1 protein altered the cortical organization of theactin cytoskeleton (Fig. 4B). Moreover, after treatment withcytochalasin and subsequent washout, Dabp1– and Dabp1+

cells reestablished localization of actin to the cortex withkinetics similar to wild-type cells (data not shown).

Overexpression of Dabp1 impedes aggregation ofstreaming cellsThe association of Dabp1 with the leading edge of developingcells suggested that Dabp1 could function in areas containingdynamic actin. Active remodeling of the actin cytoskeleton isparticularly important when Dictyostelium amoebae developinto multicellular fruiting bodies in response to starvation.

Fig. 3. Dabp1 is enriched in the cortex andthe leading edge of chemotaxing cells.Growing cells (top panels) and developingcells (bottom panels) were fixed and stainedwith affinity-purified anti-Dabp1 antibodiesfollowed by BODIPY-FL-labeledsecondary antibodies (to detect Dabp1) andTXRED-labeled phalloidin (to detect actin).Arrows and arrowhead show areas whereDabp1 and actin colocalize (in the mergedimages). Bar, 10 �m.

Fig. 4. (A) The cortical localization of Dabp1 requiresan organized actin cytoskeleton. Wild-type cells weretreated with 10 �M cytochalasin A for 40 minutes andthen stained with affinity-purified anti-Dabp1antibodies (Dabp1) and TXRED-labeled phalloidin(actin). (B) Neither the absence of, nor an increase in,Dabp1 protein influences the actin cortex. Wild-typecells (WT), Dabp1 null cells (Dabp1–) and cellsoverexpressing Dabp1 (Dabp1+) were fixed andstained with TXRED-labeled phalloidin. Bar, 10 �m.

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During early development, Dictyostelium cells respond toextracellular cAMP signals and coordinately stream intoaggregation centers (Gerisch, 1987). Actin and actin-associated proteins form a dynamic gel organized into adominant pseudopodium at the leading edge of polarized cells(Gerisch et al., 1993). We therefore tested a possiblecontribution of Dabp1 to the ability of cells to organize theiractin into pseudopodia, adopt a polarized morphology, andmove efficiently during development. Wild-type cells, Dabp1–

cells, and Dabp1+ cells were placed under starvation buffer toinduce chemotaxis into aggregation centers.

Under these conditions, wild-type cells readily adopted apolarized shape (Fig. 5, left panels). By 12-13 hours, streamsof wild-type cells moving into an aggregation center werereadily apparent. By 18 hours, most wild-type cells wereintegrated into either streams or large aggregation centers.Dabp1– mutants followed a similar developmental pattern: by8 hours, cells were elongated, and by 13-18 hours Dabp1– cellswere incorporated into streams centered around anaggregation center (Fig. 5, middle panels). By contrast,Dabp1+ cells were dramatically delayed in early development.Dabp1+ cells were unable to adopt a polarized morphologyefficiently; elongated cells were not seen until 13 hours understarvation buffer (Fig. 5, right panels). Small aggregationcenters surrounded by broken streams of cells were onlyapparent after 18-19 hours.

To examine later developmental stages, we placed equalnumbers of wild-type cells, Dabp1– cells and Dabp1+ cells on

nonnutrient agar plates, a surface on which Dictyostelium cellscomplete development to form multicellular fruiting bodiesconsisting of a spore-filled sorus on top of an elongated stalk.After 14 hours on nonnutrient plates, wild-type cells andDabp1– cells had formed multicellular migrating slugs. At 14hours, most Dabp1+ cells had only aggregated into mounds, anearlier stage of development (Fig. 6). By 28 hours, both wild-type cells and Dabp1– cells were fully developed into robustfruiting bodies consisting of a stalk topped with a sorus full ofspores. By contrast, Dabp1+ cells took about 12 hours longerto develop and formed much smaller fruiting bodies (Fig. 6).Thus overexpression of Dabp1 resulted in delayed aggregationof streaming cells, formation of smaller aggregation centersand smaller fruiting bodies.

Dabp1 influences polarity and pseudopodium number instreaming cells during chemotaxisLight microscopy was used to monitor wild-type cells, Dabp1–

cells and Dabp1+ cells during chemotaxis (Fig. 7). As theystreamed together, wild-type cells adopted a highly elongatedand polarized morphology. The average length of polarizedwild-type cells was 28.1±3.1 �m (n=30). Dabp1– cells werealso elongated in early development, and similar in averagelength to wild-type cells (26.2±3.3 �m; n=12). HoweverDabp1+ cells failed to adopt an elongated shape during earlydevelopment. The average length of the streaming Dabp1+ cellswas 18.9±2.0 �m (n=37), only 67% of the length of streamingwild-type cells.

In addition to decreased cell length,cells that overexpressed Dabp1 alsodisplayed an increase in pseudopodiumnumber. As they chemotaxed, wild-typecells generally formed two protrusionsfrom the cell body, a dominantpseudopodium at the leading edge anda trailing uropodium at the rear.Approximately 92% wild-type cellsexhibited this morphology (Table 1). Ofthe remaining 7.5% of wild-type cells,none had more than five cellularprotrusions. The number ofpseudopodia increased somewhat inthree independent Dabp1– cell lines:26.5% of the null mutants projectedmultiple pseudopodia; none had morethan five pseudopodia. By contrast,Dabp1+ cells displayed a dramaticincrease in pseudopodium number. Themajority of cells (~90%)overexpressing Dabp1 had multiplepseudopodia (Table 1), with some cellsexhibiting as many as 10 pseudopodia.The stellate shape of Dabp1+ cells madeit difficult to discern a distinct leadingpseudopodium and/or a trailinguropodium. Imaging chemotaxing cellsshowed that wild-type cells generallyextended a single pseudopodiumpersistently in the direction ofmovement. Most Dabp1+ cells,however, extended pseudopodia in

Fig. 5. Overexpression of Dabp1 delays early development. Wild-type cells (WT), Dabp1 nullcells (Dabp1–) and cells overexpressing Dabp1 (Dabp1+) were submerged under starvationbuffer to induce chemotaxis, and photographed at various times as indicated. Bar, 100 �m.

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multiple directions. Moreover, we also noticed that theextension and retraction of pseudopodia was slower in theDabp1+ cells, resulting in slower turnover of pseudopodiarelative to wild-type cells (supplementary material Movie 1).Increased numbers of pseudopodia extending in multipledirections were also observed in cells overexpressing Dabp1without the GFP tag, indicating that the phenotype was not dueto the addition of the GFP tag to abp1 (supplementary materialMovie 2). The impaired ability to extend a dominantpseudopodium in a single direction may explain why Dabp1+

cells take longer to stream into aggregation centers.The pseudopodia formed by wild-type cells are enriched in

filamentous actin. The deletion of abp1 had a small effect onpseudopodium number whereas overexpression of abp1resulted in a significantly increased number of pseudopodia inchemotaxing cells. To determine whether these pseudopodiawere also enriched in F-actin, we stained cells with TXRED-

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labeled phalloidin (Fig. 8). Chemotaxing wild-type cellsformed pseudopodia at their leading edges enriched in F-actin.Although Dabp1– and Dabp1+ cells formed multiplepseudopodia, all had normally enriched F-actin.

Dabp1 impedes cell motilityThe delayed aggregation and altered morphology exhibited byDabp1+ cells prompted us to examine whether these cells alsodisplayed defects in motility. To initiate chemotaxis, wesubmerged wild-type cells, Dabp1– cells and Dabp1+ cellsunder starvation buffer. When the cells were activelystreaming, we imaged independent cells moving towardaggregation centers. Wild-type cells moved persistently in onedirection (Fig. 9A; supplementary material Movie 3).Approximately 90% of movements were greater than 8�m/minute, with an average velocity of 12.5±6 �m/minute(n=159, 15 independent cells) (Fig. 9B). Dabp1– cells also

moved persistently in one direction (Fig.9A; supplementary material Movie 4),with around 80% moving at greater than8 �m/minute, and an average velocityof 11.1±6 �m/minute (n=129, 11independent cells) (Fig. 9B). Incomparison, Dabp1+ cells frequentlystalled in one location withoutproductive translocation (Fig. 9A;supplementary material Movie 1). WhenDabp1+ cells translocated, only 34% oftheir movements were greater than 8�m/minute, and their average velocitywas only half that of wild-type cells(6.8±4 �m/minute; n=166, 12independent cells) (Fig. 9B).

The SH3 domain is important for themorphology of streaming cellsduring aggregationThe Dabp1 protein has an ADFHdomain at its amino terminus and an SH3domain at its carboxyl terminus. To testthe functional contribution of thesedomains to Dabp1, we designed twoplasmids to express either the ADFHdomain or the SH3 domain (Fig. 10A).

Fig. 6. Overexpression of Dabp1 affectsearly development. Wild-type cells (WT),Dabp1 null cells (Dabp1–) and cellsoverexpressing Dabp1 (Dabp1+) were placedon nonnutrient agar plates. Images weretaken of cells after the aggregation stage(Aggregates) and after the final fruiting bodystage (Fruiting bodies). Bar, 100 �m.

Fig. 7. Overexpression of Dabp1 causes morphological changes during chemotaxis. Wild-type cells (WT), Dabp1 null cells (Dabp1–) and cells overexpressing Dabp1 (Dabp1+) weresubmerged under starvation buffer until they were actively streaming and photographed tostudy the morphology. Bar, 10 �m.

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Each domain was expressed as a fusion protein with GFP atthe amino terminus. The expression plasmids were introducedinto wild-type cells. Western blots probed with anti-GFPantibodies showed high expression levels for the two GFP-tagged proteins (data not shown).

To examine the influence of these domains on cellphenotype, cells expressing the ADFH domain (ADFH+ cells)and cells expressing the SH3 domain (SH3+ cells) wereinduced to begin chemotaxis by starvation. ADFH+ and SH3+

cells formed normal fruiting bodies when plated on non-nutrient agar. Nonetheless, overexpression of the SH3 domaindramatically altered the morphology of cells during streamingin early development (Fig. 10B). In contrast to ADFH+ cells,which appeared similar in morphology to wild-type cells, moststreaming SH3+ cells exhibited multiple pseudopodia (Table1). Cells overexpressing the SH3 domain exhibited up to 7pseudopodia during chemotaxis. Thus the SH3 domainappeared to be a key functional determinant in controllingpseudopodium number during the aggregation phase of earlydevelopment.

DiscussionIn early development, chemotaxing cells rapidly rearrangetheir actin cytoskeleton in order to adopt an effective shape forcell movement (Pollard and Borisy, 2003; Williams andHarwood, 2003). Remodeling the actin cytoskeleton results inextension of a dominant pseudopodium at the leading edge andretraction of the uropodium at the trailing edge, coordinatedshape changes that allow cells to move in response to aextracellular chemical gradient (Varnum-Finney et al., 1987).Key to productive motility is the tight control of pseudopodiumnumber for cells moving toward an aggregation center (Chungand Firtel, 2002). Here we have shown that overexpression ofDabp1 resulted in formation of multiple pseudopodia andimpaired cell motility during early development. These defectsprobably account for the delay in aggregation center formationin cells overexpressing Dabp1. Because of their inability toform a single dominant pseudopod, Dabp1+ cells frequentlystalled while extending pseudopodia in multiple directions,displayed reduced cell velocities and failed to movepersistently to an aggregation center. As a consequence ofimpaired motility, cells overexpressing Dabp1 formed smalleraggregation centers on starvation plates and made smallfruiting bodies.

A concern with interpreting overexpression experiments isthat phenotypes resulting from overexpression of a protein maynot reflect the normal function of the protein. Nonetheless, thesimilarity in phenotypes exhibited by Dabp1+ cells and Dabp1–

cells supports the interpretation that the phenotypes associatedwith overexpression of Dabp1 reveal its physiological role.Both Dabp1+ cells and three independent Dabp1– cell linesprojected excess pseudopodia during chemotaxis.Overexpression of Dabp1 yielded stronger phenotypes: relativeto Dabp1– cells, Dabp1+ cells exhibited an increased number ofpseudopodia, and also displayed a dramatic reduction in cellmotility. The milder phenotype associated with Dabp1– cellscould reflect redundancy in proteins that regulate the actin

cytoskeleton: when Dabp1 is depleted,other proteins could partially substitute forits function. Functional redundancy duringdevelopment has been noted previously forother proteins associated with the actincytoskeleton (Witke et al., 1992).

Formation of extra pseudopodia waslimited to developing Dabp1+ cells;growing cells that overexpressed Dabp1were normal in appearance. Thisintracellular role for Dabp1 was alsospecific for pseudopodium formation andnot general for other actin-basedprocesses. Most other actin-basedprocesses were intact in the Dabp1 nullmutants, including cytokinesis,pinocytosis and phagocytosis (data notshown). Pseudopodia in streaming cellscontain abundant amounts of F-actin andthe pseudopodia in Dabp1+ cells weresimilarly rich in F-actin. Thus Dabp1+

cells were able to construct pseudopodiathat appeared normal, but were defectivein limiting the number of pseudopodiaduring development.

Table 1. Dabp1 regulates the number of pseudopodiaduring chemotactic aggregation

% cells having more than Cell line two cellular protrusions

WT 7.5±1.0%Dabp1+ 89.0±3.6%Dabp1– 26.5±4.0%SH3+ 87.0±1.2%ADFH+ 18.4±2.0%

Wild-type cells, Dabp1+ cells, SH3+ cells, ADFH+ cells and threeindependent Dabp1– cell lines were used. Cells were submerged understarvation buffer until they were actively streaming, and then photographedusing DIC microscopy. For each cell line, pseudopodia were quantified usingcells imaged in three different experiments.

Fig. 8. Actin distribution in supernumerary pseudopodia in Dabp1+ cells is normal. Wild-type cells (WT), Dabp1 null cells (Dabp1–) and cells overexpressing Dabp1 (Dabp1+) weregrown in medium (Growing cells) or submerged under starvation buffer until they wereactively streaming (Developing cells), and then fixed and stained with TXRED-labeledphalloidin to image the actin cytoskeleton. Bar, 10 �m.

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A repertoire of proteins important for regulation ofpseudopodium number is emerging. One class of proteinsknown to regulate pseudopodium number in Dictyostelium isunconventional myosins. MyoA and MyoB localize to theleading edge of streaming cells and play critical rolesin regulating where and when a cell forms pseudopodia(Morita et al., 1996; Titus et al., 1993; Wessels et al.,1996). These two myosin Is play independent roles insuppressing lateral pseudopodium formation duringchemotaxis (Falk et al., 2003). Null mutants for either MyoAor MyoB extend a greater number of pseudopodia.Overexpression of MyoB inhibits pseudopodium formationand impairs cell motility (Novak and Titus, 1997). Likemyosin Is, Dabp1 was enriched in pseudopodia, andconceivably could influence pseudopodium number byparticipating in a regulatory pathway involving MyoA orMyoB. Dabp1 could also contribute to other pathways thatregulate polymerization of actin in pseudopodia. Forexample, Dabp1 could influence the activity of a protein thatpromotes actin polymerization, such as Arp2/3, also localizedin pseudopodia.

How might Dabp1 influence the activity of anotherregulatory protein? The phenotype resulting fromoverexpression of domains of Dabp1 supports the ideathat Dabp1 binds a regulatory protein via the carboxyl-terminal SH3 domain. Overexpressing the ADFH domainof Dabp1 had little effect on the morphology ofchemotaxing cells. By contrast, overexpression of the SH3domain resulted in an increased number of pseudopodia inchemotaxing cells, similar to the defect seen in cellsoverexpressing full-length Dabp1. This dominant-negativephenotype suggests that the Dabp1 protein influencespseudopodium number through binding partners for the SH3domain. Binding partners for the SH3 domain of Abp1homologues in other species have also been found to beimportant for its function. The SH3 domain of mammalianAbp1 binds to the GTPase dynamin, and overexpression ofthis SH3 domain in cultured cells causes defects in receptor-mediated endocytosis (Kessels et al., 2001). The SH3 of yeastAbp1 binds to six ligands, all proteins involved in endocytosis(Fazi et al., 2002). Conceivably, overexpression of the SH3domain of Abp1 could sequester partners for all of the SH3domains involved in multiple pathways. However,overexpression of the SH3 domain of Abp1 probablysequesters a limited set of discrete ligands. Different SH3domains are sufficiently specific to distinguish subtledifferences in the primary structure of potential ligands(Rickles et al., 1995; Sparks et al., 1996). Thus, it seems morelikely that the dominant negative effect of overexpression ofthe SH3 domain is due to binding a proline-rich ligandimportant for limiting pseudopodium number in chemotaxingcells.

Fig. 9. Dabp1 affects cell motility during chemotaxis in earlydevelopment. (A) Wild-type cells (WT), Dabp1 null cells (Dabp1–)and cells overexpressing Dabp1 (Dabp1+) were submerged understarvation buffer until the cells were actively streaming. Cells werephotographed every 12 seconds using DIC optics. Their positions at15 time points are shown for a typical cell from each cell line.(B) Histogram showing velocities of wild-type cells (WT), Dabp1null cells (Dabp1–) and cells overexpressing Dabp1 (Dabp1+) duringchemotaxis in early development.

Fig. 10. The SH3 domain is important for the function ofDabp1. (A) Diagram of constructs for ADFH and SH3domain of Dabp1. (B) Morphology of wild-type cells (WT)and cells overexpressing the ADFH domain (ADFH+) and theSH3 domain (SH3+). (B) Cells were placed in the starvationbuffer until they were actively streaming (about 9 hours) andthen imaged under DIC optics. Bars, 10 �m.

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Materials and MethodsStrain and cell cultureThe wild-type strain Ax2, Dabp1– (Dabp1 null) cells and Dabp1+ cells (cellsoverexpressing Dabp1) of Dictyostelium discoideum were used. Cells were culturedin HL-5 medium on Petri dishes at 18°C. Dabp1+ cells were maintained in HL-5with 10 �g/ml G418 (geneticin; Gibco-BRL); Dabp1– cells were cultured in HL-5with 5 �g/ml blasticidin (ICN, Biomedicals).

cDNA cloning and sequence analysisThe DNA sequence of yeast Abp1 (accession number X51780) was used to searchthe Dictyostelium genome database (http://www.dictybase.org) for the best matchusing reciprocal BLAST. A single gene ortholog was obtained, Dictyostelium abpE.A second database, InParanoid (http://inparanoid.cgb.ki.se) searched with BLASTalso identified the abpE gene as the Dictyostelium abp1 ortholog. A complete cDNAclone was obtained from a cDNA library using a polymerase chain reaction (PCR)-based strategy using primers 5�CCGGATCCATGGCATCATTAGATATTAGTGA-TCCAGATATTAC3� and 5�CCGAATTCCTCGAGTTACAATTGTTGTACGAAA-TTAGATGGGAAG3�. Predicted protein sequences were analyzed using theMegalign program (DNAStar, Inc., Madison, WI) with the default ClustalVparameters.

Generation of antibodies to Dabp1A cDNA for Dabp1 was cloned into the BamHI and EcoRI sites of the plasmidPGEX-2T. In this plasmid, the cDNA for Dabp1 was inserted downstream of theglutathione S-transferase (GST) gene, resulting in expression of a GST-Dabp1fusion protein. This plasmid was transformed into Escherichia coli DH5� for large-scale protein purification. To purify the GST-Dabp1 fusion protein, cell cultures andcell lysates were prepared as described previously (Vithalani et al., 1998). Thepurified GST-Dabp1 protein was used to generate polyclonal anti-Dabp1 antibodiesin rabbits (Cocalico Biologicals, Reamstown, PA). The polyclonal anti-Dabp1antibodies were affinity-purified on a GST column using a GST orientation kit(Pierce, Rockford, IL).

Replacement of the abpE gene in DictyosteliumThe vector used to generate Dabp1– cells was constructed by first using PCR toamplify sequences corresponding to regions that flanked the 5� and the 3� ends ofthe entire coding sequence for the abpE gene. The primers 5�-GAGCTCTT-GTAGTTCCCCTTACCAAATCATTGTG-3� and 5�-GGATCCGTTTTGGTTCAA-AGAATAATATTTGTTGG-3� were used to amplify a 5� fragment flanked by SacIand BamHI sties. The primers 5� AAGCTTAACTATTTCCATTTGTTTTCCTTAT-TTATCC 3� and 5�-CTCGAGAGGGGTGTCTCTGGCTGTGTCG-3� were used toamplify a 3� fragment flanked by HindIII and XhoI sites. Both fragments weresubsequently cloned into the plasmid pSP72-BSR (Wang et al., 2002) so that theblasticidin gene was flanked by these two fragments. A linear DNA fragmentcontaining this abpE gene replacement cassette was excised using the flanking SacIand XhoI restriction sites. After treating with phosphatase, 5-10 �g of this linearDNA fragment was used to transform Ax2 wild-type cells by electroporation. Cellswere diluted into 96-well plates, and cultured with blasticidin to generate clonaltransformants. Clonal transformants in which the entire coding region of Dabp1 wasreplaced by integration of the blasticidin marker were identified using PCR analysis.The absence of Dabp1 expression was confirmed in these cells by western blotanalysis with anti-Dabp1 antibodies.

Expression of the Dabp1 protein, the SH3 domain and theADFH domainTo make a construct for Dabp1 expression, a cDNA for Dabp1 expression was PCR-amplified from a Dictyostelium cDNA library with primers 3�-CCGAATTCC-TCGAGTTACAATTGTTGTACGAAATTAGATGGGAAG-5� and 5�-GGATCCG-CAGCAGCAGCAGCAATGGCATCATTAGATATTAGTGATCCAGATATTAC-3�.This fragment was cloned into the BamHI and XhoI sites of the plasmid pTX-GFP(Levi et al., 2000), placing green fluorescent protein (GFP) at the amino terminusof the Dabp1 protein. A linker containing five alanines was included between GFPand Dabp1.

To make an expression vector for Dabp1 without GFP tag, we modified the pTX-GFP-Dabp1 plasmid by removing the GFP cassette at the BamHI and HindIII sites,blunting and re-ligating the plasmid.

The ADFH and SH3 domains of Dabp1 were identified by comparing thesequence of Dabp1 with conserved domains using NCBI software(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). To make an expressionvector for the ADFH domain, the first 130 amino acids of Dabp1 were cloned byPCR from a Dictyostelium cDNA library. This sequence was cloned into the pTX-GFP plasmid with GFP placed at the amino terminus of the ADFH domain. A linkerof five alanines separated the GFP and the ADFH domain.

To make an expression vector for the SH3 domain, the carboxyl-terminal 69amino acids of Dabp1 (the 59 amino acids of the entire SH3 domain and tenadditional amino acids amino-terminal to the SH3 domain) were amplified by PCRfrom a Dictyostelium cDNA library. This DNA fragment was cloned into the pTX-GFP plasmid with GFP fused at the amino terminus of the SH3 domain.

Immunofluorescence microscopyAx2, Dabp1– and Dabp1+ cells were harvested at mid-log phase, adjusted to 2�106

cells/ml and allowed to settle on glass coverslips for 20 minutes at 18°C. Cells werefixed for 5 minutes at –20°C by incubation with methanol containing 1%formaldehyde. To improve imaging, some cells were gently flattened with a squareof 2% agar NA (Amersham Biosciences, Uppsala, Sweden) before fixing for 5minutes in methanol containing 1% formaldehyde at –20°C (Fukui et al., 1987).Subsequently, fixed cells were processed for immunostaining.

For immunostaining, fixed cells on coverslips were incubated with affinity-purified anti-Dabp1 antibodies (15 �g/ml) at 37°C for 40 minutes. After washingfour times with phosphate-buffed saline (PBS), the coverslips were incubated withBODIPY FL-conjugated goat-anti-rabbit IgG (30 �g/ml; Molecular Probes,Eugene, OR) at 37°C for 40 minutes. TXRED-labeled phalloidin (0.3 unit/ml;Molecular Probes) was then used to detect actin in fixed cells.

For cytochalasin treatment, cells adherent to coverslips were incubated with 10�M cytochalasin A at room temperature. After 30-60 minutes, cells were flattenedand fixed for 5 minutes in methanol with 1% formaldehyde at –20°C. For double-labeling of both Dabp1 and actin, immunostaining with anti-Dabp1 antibodies wasfollowed with TXRED-labeled phalloidin. Images were taken using an invertedNikon microscope TE200 (Nikon Instruments, Dallas, TX) with a 100� 1.4 NAPlanFluor objective and a Quantix 57 camera (Roper Scientific, AZ) controlled byMetamorph software (Universal Image, PA), and then processed using AdobePhotoshop software.

Light microscopyFor studying the morphology of streaming Ax2, Dabp1– and Dabp1+ cells, the cellswere imaged on an inverted Nikon TE200 microscope with either the 20� objectiveor the 100� objective. For studying development, images were captured on a ZeissSemi SR microscope with 0.8� or 1.2� objectives controlled by NIH imagesoftware.

Velocity measurement assaysTo study the motility of streaming, Ax2, Dabp1– and Dabp1+ cells were harvestedat late-log phase, washed once with the starvation buffer, PDF (2 mM KCl, 1.1 mMK2HPO4, 1.32 mM KH2PO4, 0.1 mM CaCl2, 0.25 mM MgSO4, pH 6.7), and thenresuspended into the same PDF buffer at a density of 2�106 cells/ml. 400 �l of thecell suspension were added to a one-well coverslip-chamber (Nulge-Nunc Int.,Naperville, IL) and incubated at 18°C until cells were actively streaming. Imagesof cells moving toward aggregation centers were recorded at 6-second intervalsusing an inverted Nikon TE200 microscope (Nikon Instruments) with a 60� 1.4NA PlanFluor objective and a Quantix 57 camera (Roper Scientific, AZ) controlledby Metamorph (Universal Image Corp., PA).

The velocity of streaming cells was measured by studying the movement of asingle cell outside of the aggregation center in the images of moving cells. Thelongest extension of the uropodium of the streaming cell was used as a startingpoint. The x and y position of the uropodium in each frame was noted. The distancemoved by the tail at a given time was calculated from the z value derived from thex and y values (z2=x2+y2) and converted from pixels to �m (1 �m=4.52 pixels).Velocity was calculated from the z value and time (v=z/t).

Streaming and development assaysTo study the localization of Dabp1 or actin in the starved cells undergoingchemotactic aggregation, cells were harvested at late log phase, washed once withPDF, and then resuspended at a density of 2�106 cells/ml in PDF buffer. Cellsuspension (200 �l) was spotted on a coverslip and incubated in a humidifiedchamber at 18°C until cells were actively streaming. Cells were then gently flattenedwith an agarose square, and fixed and stained with affinity-purified anti-Dabp1antibodies or TXRED-labeled phalloidin as described above.

To study the development of starved cells on agar plates, 1�108 Ax2, Dabp1–

or Dabp1+ cells were harvested at late log phase. Cells were washed once with PDFand resuspended into 3 ml PDF. The cell suspension was spread on a PDF agarplate (20 g agar/l PDF, 30 ml/plate, prepared the day before use) and allowed tosettle for 40 minutes at room temperature. After aspirating excess liquid, cells wereallowed to develop at 18°C for 30 hours. Images were taken with a Zeiss Semi SRmicroscope with a 0.8� or 1.2� objectives controlled by NIH image software.Western blot analysis demonstrated an equivalent amount of Dabp1 proteinthroughout development in wild-type and in Dabp1+ cells (data not shown).

Quantification of pseudopodium numberDeveloping Ax2, SH3+, ADFH+, Dabp1– and Dabp1+ cells were prepared andimaged as described for the velocity measurement assays. For Dabp1– cells, threeindependent cell lines were examined. For each cell line, approximately 80-100 cellswere selected and an outline of the cell perimeter was drawn. Protrusions extendingfrom the cell body were counted as pseudopodia.

We would like to thank Arturo De Lozanne for helpful commentson the manuscript and Tom Egelhoff for the gift of the pTX-GFP. Thiswork was supported by NIH RO1 GM048625 to T.J.O.

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