Small GTPases in Dictyostelium: lessons from a social amoeba

The Ras superfamily of small GTPases is foundthroughout all eukaryotes and controls a wide varietyof cellular processes including proliferation,differentiation, cell motility, cell polarity andtrafficking of vesicles and macromolecules. Likeheterotrimeric G proteins, they are simple molecularswitches; when bound to GTP they are turned on, butwhen the GTP is hydrolyzed to GDP the switch isturned off. Small GTPases are regulated by two setsof proteins (Fig. 1). Guanine-nucleotide-exchangefactors (GEFs) cause activation by catalysing theexchange of bound GDP with GTP, and GTPase-activating proteins (GAPs) inactivate them byincreasing the rate of GTP hydrolysis. This system ofGTPases, GEFs and GAPs allows great versatility inthe construction of signalling pathways. Signals canbe amplified (one GEF activates several GTPases),integrated (several pathways activate each GEF andGAP, and the behaviour of one GTPase depends onthe total effect of all its GEFs and GAPs) or split (oneGTPase activates any of several effectors). Thisversatility allows small GTPases to mediate a widerange of different biological functions. Mosteukaryotic cells contain several members of each offive broad subfamilies – Ras, Rho, Ran, Rab and Arf –that are grouped by sequence homology and sharesimilar roles. A detailed description of the Rassuperfamily is beyond the scope of this article,however, many recent reviews providecomprehensive coverage1–6.

Investigations of small GTPases have typicallyfavoured the use of mammalian cells. However, alleukaryotes contain representatives of eachsubfamily, and comparisons of their roles in differentorganisms provide a broader understanding of thisubiquitous family. One excellent example is thesocial amoeba, Dictyostelium discoideum.Dictyostelium is widely studied, mainly because of

three experimental advantages. First, genedisruption is fast and straightforward. Second, cellsmove rapidly, in a manner which closely resemblesmammalian cells and that uses similar proteins7.Third, when amoebae start to starve, they aggregatetogether by chemotaxis and form multicellularstructures. Again, this process uses mechanisms andproteins that are closely related to those used inmammalian signalling and development. Thecombination of easy gene disruption and simple,accessible movement, signalling and developmentmakes Dictyostelium a potent experimental tool. Inaddition, it has an unusually large complement ofRas-superfamily GTPases relative to the complexityof its genome, which makes it an ideal organism forthe molecular genetic study of small GTPases. Thisreview highlights some of the emerging similaritiesand apparent differences between the functions ofsmall GTPases in Dictyostelium and otherorganisms.

Revelations from the genome

Raw sequence data from the Dictyostelium genomeand cDNA sequencing projects (Box 1) has alreadyrevealed several surprises about the smallGTPases.

An unexpected profusion of Ras genesSix members of the Ras subfamily have been knownfor several years8, including a Rap that is remarkablysimilar to mammalian Rap1 (Table 1). Genes for atleast four other Ras proteins appear in theDictyostelium sequence databases (Box 1). This is asurprisingly large number for a relatively simpleorganism with a genome of 35 Mb. The more complexgenomes of Caenorhabditis elegans (80 Mb) andDrosophila melanogaster (125 Mb) contain eight andten Ras genes, respectively.

All the Dictyostelium Ras proteins are closerelatives of the mammalian H-,N- and Ki-Rasproteins – no homologues of the more divergent Ral orR-Ras have been identified. Why Dictyostelium needsmore than ten Ras-subfamily genes is not clear, and isdiscussed below.

Numerous Rac genes but apparently no Rho or Cdc42In animal cells, the Rho GTPase subfamily issubdivided further into three subgroups calledCdc42, Rac and, confusingly, Rho. Cdc42 is believed

Although the process of sequencing the Dictyostelium genome is not

complete, it is already producing surprises, including an unexpectedly large

number of Ras- and Rho-subfamily GTPases. Members of these families control

a wide variety of cellular processes in eukaryotes, including proliferation,

differentiation, cell motility and cell polarity. Comparison of small GTPases

from Dictyostelium with those from higher eukaryotes provides an intriguing

view of their cellular and evolutionary roles. In particular, although mammalian

Ras proteins interact with several signalling pathways, the Dictyostelium

pathways appear more linear, with each Ras apparently performing a specific

cellular function.

Small GTPases in Dictyostelium:

lessons from a social amoeba

Andrew Wilkins and Robert H. Insall

TRENDS in Genetics Vol.17 No.1 January 2001

http://tig.trends.com 0168–9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(00)02181-8

41Review

A.Wilkins

MRC Laboratory ofMolecular Biology, Hills Road, Cambridge, UK CB2 2QH.

R. H. Insall*

School of Biosciences,University of Birmingham,Edgbaston, Birmingham,UK B15 2TT.*e-mail:[email protected]

to control cell polarity and the formation of filopodia;Rac is associated with the formation of ruffles andlamellipodia; and Rho is involved in contractilityand the assembly of stress fibres. In Dictyostelium,14 Rac homologues9 have been identified, and morepresumably await discovery as the genome issequenced. However, despite extensive searching,both in vitro and in silico, no homologues of Rho orCdc42 have been discovered. A similar situationexists in plants, where 13 distinct Rac-like proteinshave been identified but there are apparently noRho or Cdc42 homologues10. Because both plant andDictyostelium cells can show a high degree ofpolarity, this implies that the association of Cdc42with cell polarization has appeared more recently inevolution than might have been expected. Also,Dictyostelium makes many filopodia, which aresimilar in size, shape and molecular composition tothose of mammalian cells. Therefore, it appears thatCdc42 is not necessary for the formation of filopodia,as some authors have supposed. Similarly,Dictyostelium, like most other crawling cells, movesusing alternating cycles of expansion andcontraction. Something must therefore be taking thenormal role of Rho in contraction.

Many of the Dictyostelium Racs are very divergentfrom prototypical human Racs, and might thereforeperform the same functions as the Rho and Cdc42proteins from animal and fungal cells. In any case, thenumber of Racs (like the number of Ras genes) seemslarge for such a small genome.

These Ras and Rac proteins, together with 18Rabs, two Rans and several Arfs brings the number ofsmall GTPases identified so far in Dictyostelium toover 40, more than in Drosophila and C. elegans.

Multiple GEFs but no receptor tyrosine kinasesThis impressive small GTPase complement ismatched by an equally unexpected number of GEFsfor the Ras and Rac proteins. Although only twoRasGEFs, Aimless (Ref. 11) and RasGEFB (Ref. 12),are functionally characterized, ~20 others exist inthe cDNA and genome sequence databases. It is asthough there is a RasGEF for every conceivablefunction of Ras. The sequences of Aimless andRasGEFB contain no recognizable signallingdomains outside the RasGEF domain. This is verydifferent from the RasGEFs of yeast and mammals,which contain multiple features including PH (seeGLOSSARY) and SH3 DOMAINS, DAG- and cAMP-bindingsites, and in several cases DH DOMAINS, whichresemble the catalytic domains of GEFs for the Rhosubfamily. This could mean that each DictyosteliumGEF responds to relatively few stimulatory signals,whereas mammalian RasGEFs are morepromiscuous. Only one RacGEF homologue has beenpublished, in an interesting chimaera with anapparent RacGAP (Ref. 13), but the sequencedatabases clearly contain several others.

For the Ras and Rac families, many of the majormammalian effectors are conserved in Dictyostelium(Fig. 2). The ubiquitous MAP KINASE pathway is


http://tig.trends.com

42 Review

Downstream effectors

GEFs

GAPs

GTP-bound(active)Stimulus

GDP-bound(inactive)

TRENDS in Genetics

Fig. 1. Activation of smallGTPases. The GDP-boundform (red) is inactive anddoes not interact withdownstream effectors.GEFs activate smallGTPases by allowing GDPto be replaced by GTP. The GTP-bound form caninteract with downstreameffectors. Hydrolysis ofGTP to GDP returns thesmall GTPase to theinactive state; the rate ofhydrolysis is greatlystimulated by the actionof GAPs. Stimuli cantherefore activate smallGTPases either byenhancing the activity ofGEFs or by inhibitingGAPs.

The Dictyostelium Genome Sequencing Projectinvolves collaborating groups from Köln and Jena(Germany), Houston (USA) and Cambridge (UK).Thesix chromosomes are separated by pulse-field gelelectrophoresis (PFGE), then purified and sequencedusing a whole-chromosome shotgun approach. Morethan threefold coverage of the 34-Mb genome hasalready been obtained, providing at least someinformation on >90% of all genes. Assembly offragments has been underway for some months, witha predicted completion date of 2002. Additionalinformation is obtained from a high-resolutionYAC-based map and HAPPY mapping, in which PCR usingfractions of a single genome is used to correlate thedistance between markers. In addition, a large-scalecDNA sequencing project underway inTsukuba(Japan) has yielded information and clones of a largeproportion of all expressed genes. Proteome andgene-chip studies are under way in several labs, and a

variety of other postgenomic studies are currentlybeing tested.The combination of whole-genometechniques with the straightforward gene knockoutsand biological assays possible in Dictyostelium willallow rapid progress in the next few years.

URLs:

BCI Köln: http://www.uni-koeln.de/dictyostelium/GSC Jena: http://genome.imb-jena.de/dictyostelium/Baylor: http://dictygenome.bcm.tmc.edu/Sanger centre: http://www.sanger.ac.uk/Projects/D_discoideum/HAPPY mapping: http://www.mrc-lmb.cam.ac.uk/happy/happy-home-page.htmlJapanese cDNA projects:http://csmnetj.biol.tsukuba.ac.jp/cDNAproject.htmlDictyosteliumWWW server:http://dicty.cmb.nwu.edu/dicty/dicty.html

Box 1. Dictyostelium genome projects

present and genetic evidence indicates that theremight be at least three separate kinase cascades14–16.However, so far no Raf homologue has beenidentified. Of the other direct Ras effectors, threeclass I PI 3-KINASE homologues have beencharacterized17, and an additional PI 3-kinase and aSUR-8 homologue are present in the sequencedatabases. It should be noted that unlikeSaccharomyces cerevisiae, the known adenylylcyclases from Dictyostelium do not appear to bedirectly activated by Ras. Homologues of the Rho-subfamily effectors IQGAP (Refs 18, 19) and PAK

(Ref. 20) have also been characterized and

homologues of p140Dia, WASP and PIP 5-KINASE exist inthe sequence databases. Scar1, a relative of WASPthat is thought to control the actin cytoskeleton inmany cell types, was originally discovered inDictyostelium21, although it is not yet clear whetherit is an effector for small GTPases, heterotrimericG proteins or some other signalling pathway.

To date, no receptor tyrosine kinases have beenidentified. This strongly indicates that the pathwaysused by signalling molecules such as PDGF, which aretransduced by pathways based on Ras proteins,evolved relatively recently, after Dictyosteliumdiverged from higher animals and fungi.



43Review

Table 1. Members of the Ras and Rac signalling pathways characterized in Dictyostelium

Dictyostelium Closest homologues Suggested roles in mammals Suggested roles in Dictyostelium Ref.

gene

Ras subfamily

rasG Ha-Ras, Ki-Ras, N-Ras (human) Proliferation, differentiation, survival, motility Cell polarity, cytokinesis 25rasD Ha-Ras, Ki-Ras, N-Ras (human) Proliferation, differentiation, survival, motility Slug phototaxis 50rasS Ha-Ras, Ki-Ras, N-Ras (human) Proliferation, differentiation, survival, motility Control of endocytosis and cell speed 37rasB Ha-Ras, Ki-Ras, N-Ras (human) Proliferation, differentiation, survival, motility Not yet known 51rasC Ha-Ras, Ki-Ras, N-Ras (human) Proliferation, differentiation, survival, motility Not yet known 52rap1 Rap1A (human) Phagocytosis, proliferation? differentiation? Control of cytoskeleton? 53

aimless Son of sevenless (Drosophila); Activate Ras proteins in response to multiple Control of adenylyl cyclase and 11CDC25 (Saccharomyces); Sos, signals chemotaxisRasGRF1,RasGRP (mammals)

rasGEFB Son of sevenless (Drosophila); Activate Ras proteins in response to multiple Control of early development, 12CDC25 (Saccharomyces); Sos, signals endocytosis and cell speed RasGRF1, RasGRP (mammals)

PIK1, PIK2, PIK3 Class I PI 3-kinases (all higher Synthesises PI(3,4,5)P3 from PI(4,5)P2; roles Control of endocytosis and differentiation 17,33eukaryotes) in differentiation, survival, motility

pkbA Protein Kinase B/Akt Activated by PI(3,4,5)P3 roles in differentiation, Control of chemotaxis and differentiation 32survival, mobility

rip3 Human ORF JC310 Suppressor of activated Ras Control of adenylyl cyclase and 29(accession number C38677) chemotaxis (cf. Aimless)

MEKKα Raf (all animals) Proliferation, differentiation Differentiation 16DdMEK1 MEK/MAPKK (all eukaryotes) Proliferation, differentiation Chemotaxis 15erk1, erk2 MAP kinases (all eukaryotes) Proliferation, differentiation Chemotaxis, differentiation 27,28,54

Rac subfamily

rac1A, rac1B, Mammalian Rac1 Control of actin cytoskeleton Control of actin cytoskeleton 34,45rac1C (also phagocytosis, proliferation etc.)

racA, racB, Mammalian Rac1 Control of actin cytoskeleton Not yet known 55racC

racC Mammalian Rac1 Control of actin cytoskeleton Control of actin cytoskeleton 46racE Mammalian Rac1 Control of actin cytoskeleton Cortical tension, cytokinesis 56,43racF1, racF2, racG, Mammalian Rac1 Control of actin cytoskeleton Not yet known 9racH, racI, racJ

myoM Bifunctional – RacGEF and RacGEFs activate Rac; unconventional Control of pseudopodium extension? 46unconventional Myosin Myosins are multifunctional motors

racGAP Bifunctional – RacGEF and RacGEFs activate Rac; RacGAPs Regulates actin cytoskeleton 57,34RacGAP domains inactivate Rac

MIHCK Saccharomyces STE20, Ste20 essential for polarization and interacts Controls activity of Myosin I family 58mammalian PAK with heterotrimericG-proteins and Cdc42;

PAKs connect Cdc42 and Rac to multiple cytoskeletal proteins

darlin/darA smgGDS Activates various small GTPases? Required for cellular aggregation 59pakA Saccharomyces STE20, Activates various small GTPases? Regulates Myosin II assembly? 20

mammalian PAKDGAP1 IQGAPs Regulates actin in response to Rac, Negative regulator of actin levels and 18

cadherin, Ca2+ pseudopodia? Required for cytokinesisgapA IQGAPs Regulates actin in response to Rac, Required for cytokinesis 19

cadherin, Ca2+

Polarity, motility and chemotaxis

In addition to their role in formation of filopodia,Cdc42 proteins are implicated in cell polarity inepithelial cells and macrophages in S. cerevisiae andSchizosaccharomyces pombe4,22,23. AlthoughDictyostelium does not appear to possess a Cdc42,amoebae exhibit a distinct polarity – an obvious front,where pseudopods are extended, and a rear, which isretracted as the cell moves. However, it seems likelythat a small GTPase capable of performing the samefunction as Cdc42 is present. This is indicated by thefact that human Cdc42 has the same ability topolymerize actin in lysates of Dictyostelium as it doesin human peripheral blood mononuclear cells andthat it appears to act through endogenous Rac- orCdc42-effector molecules in both cases24. One GTPasedirectly implicated in control of cell polarity inDictyostelium is RasG. Loss of RasG results in a near-total loss of cell polarity and consequent impairmentof cell movement in growing cells25. This is similar tothe situation in S. pombe, where Ras1p is required formaintenance of cell shape and the morphologicalresponse to mating pheromone23. In yeast, Cdc42pcontrols cell polarity downstream of Ras1p (Ref. 23)and it is therefore plausible that a Cdc42-likemolecule operates downstream of RasG to control cellpolarity in Dictyostelium. By contrast, a role for Rasin cell polarity has not been demonstrated inmammalian cells and it will therefore be interestingto determine the effect of ablation of differentmammalian Ras proteins on the morphology ofpolarized or motile cells.

Small GTPases are also implicated in control ofcell motility and chemotaxis. Many similaritiesbetween the role of small GTPases in Dictyosteliumand mammalian cell motility have already emerged.In mammalian fibroblasts, Ras-dependent MAPKactivation is required for chemotaxis to PDGF, butnot for random migration26. A similar situation occursin Dictyostelium where an ERK2 homologue isactivated in response to chemoattractant and isessential for chemotaxis, but not general motility14,27.Another Ras pathway involving the RasGEF Aimless

seems to downregulate the chemoattractant-inducedERK2 activity28. Aimless is also essential forchemotaxis, but not for random cell movement. Inaddition, a Ras effector, RIP3, is required forchemotaxis in Dictyostelium, although itsrelationship to Aimless and ERK2 is unclear29.Interestingly, RIP3 contains a region of homology to ahuman open reading frame, JC310, that suppressesthe effects of activating mutations in S. cerevisiaeRAS2. We speculate that this human protein mighthave a similar role in chemotaxis of mammalian cells.

In neutrophils, PI 3-kinase signalling pathwaysare implicated in the control of cell motility. The Ras-regulated PI 3-kinase p110γ is required for efficientneutrophil chemotaxis30, whereas PKB, a downstreameffector of PI 3-kinase, becomes localized to theleading edge of cells in response to chemoattractant31.Similarly, the Dictyostelium homologue of PKB isrequired for normal motility and chemotaxis and isrecruited to the leading edge of cells in response tochemoattractant32. However, at present, the role of PI3-kinases in Dictyostelium motility and chemotaxisremains unclear because, although Dictyosteliumcells lacking two class I PI 3-kinases are defective inactivation of PKB in response to cAMP (Ref. 32), theydo not display any defects in chemotaxis33.

In Dictyostelium as in mammalian cells, Rho-subfamily GTPases seem to be intimately involved inmediating cell motility. Expression of activatedversions of Rac1B (Ref. 34) or the Rac-activated PAKa(Ref. 20) causes actin polymerization and membraneruffling reminiscent of the effects of activated Racproteins in mammalian cells2. Just asdominant–negative Rac1 inhibits macrophagechemotaxis, so a dominant–negative Rac1B impairschemotactic migration and motility ofDictyostelium22,34. Intriguingly, a different PAKprotein is a major kinase controlling the activity ofmyosin I (Ref. 35). This suggests that Racs canmodulate the cytoskeleton through unconventionalmyosins as well as through the better-understoodmyosin II. It is, however, unclear whethermammalian cells can utilize both pathways.

These emerging similarities betweenDictyostelium and motile mammalian cells in thecontrol of cell motility by small GTPases demonstratethat Dictyostelium is an excellent system for studyingthe role of small GTPases in cell motility.

Eat or run?

GTPases of the Rho subfamily are involved in the controlof endocytosis in mammalian cells4. Althoughexpression of activated Ras causes an increase inmacropinocytosis (the uptake of fluids into vesicles) infibroblasts, the physiological function of Ras inendocytosis remains unclear36. Consequently the findingthat rasS-null Dictyostelium cells are severely impairedin both solid- and fluid-phase endocytosis is of greatinterest37. Furthermore, these mutant cells migratethree times faster than normal. This unusual phenotype



44 Review

PH domain: Pleckstrin-homology domainSH3 domain: Src-homology domain 3DAG: DiacylglycerolDH domain: Dbl-homology domainIQGAP: Rho-subfamily effector with homology to GAPs, capableof binding actin and calcium/calmodulinMAP kinase (MAPK): Mitogen-activated protein kinase PAK: p21-activated kinasePDGF: Platelet-derived growth factorPI 3-kinase: Kinase that phosphorylates phosphatidylinositidesat the 3-position of the inositol ringPIP 5-kinase: Kinase that phosphorylates phosphatidylinositol(4)-phosphate at the 5-position of the inositol ringPKB: Protein kinase BRIP3: Ras-interacting protein-3, interacts specifically with DdRasGSur8: Ras-binding protein with leucine-rich repeatsWASP: Wiskott–Aldrich syndrome protein

Glossary

is also observed in Dictyostelium cells lacking RasGEFB(Ref. 12). Thus, there seems to be a fairly linear Raspathway controlling cell speed and endocytosis.

A body of evidence suggests that the processes ofendocytosis and cell migration are in constantcompetition with one another in Dictyostelium cells.As they are projected, competition arises betweenpseudopods and membrane protrusions called ‘cups’that mediate phagocytosis and macropinocytosis. Itappears that the two structures can only co-exist inthe same cell for a short time38. Leading edges ofpseudopods recruit the actin-binding protein coroninfrom regressing phagocytic cups, and extending cupscan sequester coronin from retracting pseudopods38.It therefore appears that competition for cytoskeletalcomponents limits the extent to which a cell canperform phagocytosis and migrate at the same time.The corollary suggests that any treatment thatcauses a cell to become more motile wouldconcomitantly inhibit endocytosis. This phenomenonhas been observed – phagocytic cups are almostcompletely absent in rapidly moving, aggregatingcells and this correlates with a reduced rate ofphagocytosis39.However,growing cells transferredinto liquid culture medium exhibit a huge increase in

fluid-phase endocytosis and a decrease in cell speed40.Together, these data are indicative of an inversecorrelation between cell speed and the intrinsic rateof endocytosis in the Dictyostelium cell. It might bethat the function of the RasS signalling pathway is tocontrol this balance between endocytosis andmotility, favouring endocytic feeding during growthand motility while cells are aggregating.

It will be interesting to determine whether Rasproteins act together with the Rho subfamily in thecontrol of endocytosis in higher eukaryotes, andwhether any of the Dictyostelium Rac proteinsoperate downstream (or even upstream) of RasS.Perhaps more importantly, is there a similar balancebetween rates of endocytosis and motility in otherorganisms?

Cytokinesis

The involvement of small GTPases in regulatingcytokinesis, the division of the cytoplasm followingmitosis, has been demonstrated in several organisms(reviewed in Ref. 41). Rho-subfamily GTPases areinvolved in all cases – in both Drosophila andC. elegans the RhoI protein is required, whereas inXenopus and humans both Rho and Cdc42 areimplicated.

One great advantage of Dictyostelium for this fieldis that the phenotypes can be conditional. Cytokinesismutants become multinucleate when grown insuspension but adhesion to a surface often allowspartial recovery of normal cell division (see Ref. 42 fora review of cytokinesis mutants). The role for Rho-subfamily members seems to be conserved inDictyostelium, although Racs take the place of Cdc42and Rho. Loss of one Rac, RacE, causes failure in thefinal steps of cytokinesis, apparently because of areduction in the cortical tension of the cells43 (Fig. 3).This seems to be a relatively specific defect as noobvious impairment in other actin-dependentprocesses such as cell motility or endocytosis areevident. Overexpression of activated alleles ofDictyostelium Rac1 proteins34, or ablation of PAKa(Ref. 20) or either of the two IQGAP homologuesGAPA and DGAP1 (Refs 18,19) also interferes withcytokinesis. Neither PAKa nor DGAP1 appears to bea direct effector of RacE, preferentially interactingwith activated Rac1 proteins instead18,20. Theinvolvement of the Dictyostelium IQGAP homologuesin cytokinesis is particularly interesting, because noclear physiological function has been ascribed to theseRac- and Cdc42-binding proteins in mammalian cells,although they are known to bind F-actin.

Ras proteins are usually associated withproliferation and cell-cycle control in higherorganisms, implicating them rather than cytokinesis.It was therefore unexpected to find that loss ofDictyostelium RasG, the closest cellular homologue ofmammalian H-Ras, resulted in a strong cytokinesisdefect without any apparent effect on the rate ofnuclear division25. As with RasS in endocytosis, it will



45Review

Fig. 2. Ras and Racsignalling pathways inDictyostelium and othereukaryotes. Ras (a) andRac (b) signallingpathways are shown. Theprecise inputs andoutputs for Rac, as well asCdc42 and Rho, are thesubject of much debate,so they have not beenshown (reviewed in Ref.60). Similarly, onlyexamples of the widerange of mammalianRacGEFs are shown; thereare many others. Green,pathway memberspublished in Dictyosteliumand other organisms;amber, pathwaymembers that have beenobserved in theDictyostelium cDNA andgenome projects but notyet published; red,present in otherorganisms but not yetfound in Dictyostelium;blue, published forDictyostelium but notother organisms.

WASPMLK3

Synaptojanin2p67phox IQGAP

PIP5K

PAK

POR1

PKNdiaphanousRhotekinRhophilin

citronROK

Ras, R-Ras

SUR-8RasGAP

Nore1Rin1AF6

RIP3

RalGDS

Ral

PI 3-kinase

PKB

Raf/MEKK

MEK

ERK

RasGRF1/2 CNRasGEF RasGRPSos1/2 Cdc25

Growth factorsRTK ligands

Aimless,RasGEFB

Inputs:

GEFs:

Effectors:

Roles: ProliferationDifferentiation

Cell survivalMovement and polarity

DifferentiationRacGEF activation

Motility? Various

RacRho Cdc42

GEFs:

Effectors:

p150RhoGEF Fgd1Tiam-1

DdRacGAP1Vav

(a)

(b)

TRENDS in Genetics

Ca2+cAMP,cGMP

DAGCa2+ ?

be interesting to determine how the Rac proteinscooperate with Ras in control of cytokinesis. It willalso be intriguing to discover whether thisinvolvement of Ras in Dictyostelium cytokinesis isindirect, caused by a general defect in the actincytoskeleton, or whether Ras directly regulates someaspect of cytokinesis – and if so, whether Ras-familymembers in mammalian cells have a similar role.

Redundancy or necessity

In several systems a high level of apparentfunctional redundancy exists within the GTPasefamilies. In mammalian cells the four prototypicalRas proteins elicit indistinguishable cellular effectswhen overexpressed in tissue culture cells and nodifferential effector binding has beendemonstrated3. Moreover, H-Ras and N-Rasknockout mice have no obvious phenotype3.Similarly, in S. cerevisiae the Ras1 and Ras2proteins appear to be largely redundant5.Considering the number of GTPases present inDictyostelium, genetic overlap might be expected toobscure functional delineation of these proteins. It istherefore surprising that those small GTPasescharacterized so far, using loss-of-functionmutagenesis, appear to have highly specific cellularroles. The loss-of-function Ras mutants are a goodexample; rasG − cells have impaired cell polarity andmovement and are defective in cytokinesis25,

whereas rasD − cells exhibit impaired phototaxisduring the ‘slug’ stage of development50 and rasS −

cells are impaired in solid- and fluid-phaseendocytosis yet have an increased cell speed37.

Further evidence for limited functional overlapcomes from the observation that expression of RasD inrasG-null cells using the rasGpromoter can rescue thecytokinesis, but not the motility, defect of rasG-nullcells44. This is somewhat surprising given that RasD andRasG are 82% identical, with only two conservativechanges in the first 80 residues, the region thought tocontain all residues important in effector interaction.Futhermore, yeast two-hybrid analysis of DictyosteliumRas–effector interactions show that RasD and RasGbind to a different subset of effectors, whereas human H-Ras can bind to all the Dictyostelium Ras effectors29.These data hint at two further possibilities. First, theremight be more structural determinants of Ras–effectorbinding specificity than previously thought. Second, andmore speculatively, the functions performed by a singleRas in mammals might be split between several Rasproteins in Dictyostelium. If this were true it couldexplain why there are so many Ras proteins inDictyosteliumand why their functions are easier todefine than in higher eukaryotes. It would beinformative to determine which, if any, of the Rasproteins from other species are able to complement fullythe DictyosteliumRas-null cells. It could be that theostensibly identical mammalian Ras proteins have



46 Review

Fig. 3. Phenotypes of mutants in Rac signalling pathway components. (a–c) Filamentous actin in parental cell (a), multiple filopodia in a mutantlacking the IQGAP-related protein DGAP1 (b) and cuplike structures in a cell overexpressing constitutively active Rac1A (c). Multiple confocalsections of TRITC-phalloidin fluorescence were recorded at intervals of 0.2 µm and processed into a three-dimensional image using a ray-tracingalgorithm. The images are colour-coded according to the emitted fluorescence intensity: blue, areas with low F-actin concentrations; red, with highF-actin concentrations. (d) Multiple nuclei (stained with DAPI) in a racE-null cell grown in suspension, revealing a defect in cytokinesis. (e)Numerous large pseudopodia (‘ramopodia’) in a cell overexpressing the RacGEF domain from MyoM, after hypo-osmotic shock. Stainingrepresents F-actin (green) and coronin (red), projected as a side view from multiple confocal sections. (f) Scanning electron micrograph of cellsexpressing 2–4 times normal levels of RacC. Multiple membrane blebs (e.g. arrowhead) are seen. Photographs were reproduced, with permission,from the following references: (a,c) Ref 45; (b) Ref. 18; (e) Ref. 47; (f) Ref. 46. Photograph (d) is courtesy of A. De Lozanne.

differential properties when expressed in theDictyosteliumRas-null mutants. It might also bepossible to define functional groups of Ras proteins onthe basis of their function in Dictyostelium. The converseof this approach would be to determine whichDictyosteliumRas protein (or combination of Rasproteins) is able to complement loss-of-functionmutations in other systems such as DrosophilaandC.elegans.

Initial evidence suggests that the DictyosteliumRac proteins might also have nonoverlappingfunctions (Fig. 3). For example, the racE-nullmutant (with the specific defect in cytokinesis),cannot be rescued by expressing other familymembers43. Equally, the actin-rich membranestructures formed from constitutive activation ofthe Rac1A (Ref. 45), Rac1B (Ref. 34) and RacC(Ref. 46) proteins are highly distinct, butexpression of activating mutations of RacE does notinduce any obvious actin structures. Moreover,overexpression of the RacGEF domain from theunconventional myosin MyoM results in theformation of completely different membranestructures in response to osmotic shock47. Thissuggests that different Rac pathways have clearlydefined roles, a hypothesis that is supported byrecent work using knockout mice48.

Perspectives

Why are there so many Ras superfamily GTPases inDictyostelium? The initial evidence, discussedabove, indicates that this is not a result ofunnecessary gene duplication producing a surplus offunctionally redundant genes. A more speculativeexplanation is that the signalling networks that usesmall GTPases have evolved in a different directionfrom other eukaryotes. The lack of certain types ofsignalling components, such as tyrosine kinasereceptors, might have necessitated an expansionand specialization of small GTPases to obtain therequired signalling complexity. Also, in higherorganisms there is evidence of extensive crosstalkbetween signalling components and multiple inputsimpinging on single proteins. However, inDictyostelium, at least for Ras, the nonoverlappingcellular functions and large number of RasGEFsimply that a more linear, parallel approach tosignalling has evolved, with each RasGEF receivinga single input and transducing a signal in a fairlylinear fashion through a limited subset of signallingmolecules.

One pattern to emerge from the study of theDictyostelium small GTPases is that Ras proteinsseem to control processes in Dictyostelium that arenormally associated with Rho-subfamily proteinsin higher eukaryotes. Although it has not beendemonstrated that Racs operate downstream ofRas proteins in Dictyostelium cells, it wouldprovide a consistent explanation for much of thedata. This is certainly the case in yeast, and it isclear that Rho- and Rab-subfamily GTPasesoperate downstream of Ras to mediate cellulartransformation in some mammalian tissue culturecells3. Mammalian Ras proteins might well operateupstream of the Rho subfamily in a large number ofcellular processes but, owing to experimentallimitations, their role could have gone undetectedin higher organisms. For example, if a Ras wereinvolved in regulation of cytokinesis in mammaliancells, it might be impossible to detect becausedominant alleles of Ras block cell-cycle progressionand cause apoptosis.

One general question that needs to be addressedby the field is how the data obtained from the use ofactivated or dominant–negative mutant GTPasescompare with data from loss-of-function mutants.Many authors have presumed that expression ofdominant–negative Ras mutants effectively equatesto a loss of Ras function, but several importantresults (e.g. Ref. 49) show that this is anoversimplification. An answer to this question in adefined system would undoubtedly aid us in both ourcomparative interpretation of previous data and indesigning more incisive experiments. Dictyosteliumis a highly suitable system to address this problemrapidly, the sheer number of small GTPasesallowing careful testing of the emerging principles.

The study of small GTPases in Dictyostelium hasalready yielded some extremely interesting data.The presence of filopodia without Cdc42, andcontraction without Rho, indicate that thesepathways evolved more recently than expected. Theimportance to cytokinesis of Racs and theirassociates, and especially Ras proteins, was asurprise. Finally, the apparent specificity ofRasGEFs for single pathways is in stark contrast tothe presumed promiscuity of mammalian RasGEFs.As more information comes to light, it will continueto be fascinating to compare this simple eukaryoticsystem with higher organisms, and determine justhow far small GTPase signalling has been conservedthrough evolution.



47Review

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Acknowledgements

We thank all the teamsinvolved in theDictyostelium cDNA andgenome sequencingprojects; J. Faix, J.Cardelli, A. De Lozanneand T. Soldati for kindlyproviding thephotographs used inFig. 3; and L. Macheskyand D. Knecht for theircritical reading of themanuscript.



48 Review

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Small GTPases in Dictyostelium: lessons from a social amoeba

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