Despite their lower threshold formoving brood, low-temperature-incubated ants selected warmerparts of their artificial nest for broodplacement. Weidenmuller et al. [3]speculate that this behaviour relates tothe best strategies for incubating broodin spring and summer. They suggestthat in spring, heat is a limitingresource. Thus, colonies place broodin the warmest part of the nest, butstand ready to move it away rapidlyshould the temperature becomesuper-optimal. In summer, heat isthe enemy, and it is probably best ifbrood is kept in parts of the nestwhere high temperatures are neverexperienced, thus saving the labourof frequently moving brood around,and reducing the risk that it will everexperience lethal temperatures.Hence, a rapid response to risingtemperature is not required, for thebrood is located in a cool partof the nest. This argument is a bitcounter-intuitive, and I am notentirely convinced by it; however,the speculation is supported by thefact that high-temperature-rearedworkers moved brood tocooler parts of the artificial nestthan low-temperature-rearedworkers.

Finally, Weidenmuller et al. [3]found that adult ants that wereexposed to increasing temperatures

repeatedly developed lower taskthresholds and thus started to movepupae at lower temperatures thanthey had done previously. Thissupports the idea that task thresholdcan be reinforced by experience [7],increasing the variance in taskthresholds among workers ina colony. Interestingly, althoughexperience decreased the thresholdfor carrying out brood, it did notchange the threshold for the firstresponse; that is, picking the broodup. This suggests that experiencedworkers do not become moresensitive to increasing temperature,but become more efficient in theirresponse to it.

The Weidenmuller et al. [3] studyis important because it demonstratesanother way by which non-geneticmechanisms can result in significantinter-individual behaviour withincolonies of insects. The work focussedon brood incubation behaviour, butit is not unreasonable to suspectthat thresholds of other behaviourscould also be affected by a worker’sexperience as a pupa or larva. Forexample, in the honey bee, larval diethas a profound effect on morphology,and likely the behaviour of thesubsequent adults [8]. Thus, larvalfeeding, pupal incubation, age andgenetics may all interact to produceworkers with different task thresholds,

and thus colonies that are morehomeostatic and efficient.

References1. Bonabeau, E., Theraulaz, G., and

Deneubourg, J.-L. (1996). Quantitative study ofthe fixed threshold model for the regulation ofdivision of labour in insect societies. Proc. R.Soc. Lond. B 263, 1565–1569.

2. Bonabeau, E., Theraulaz, G., andDeneubourg, J.L. (1998). Fixed responsethresholds and the regulation of division of laborIn insect societies. Bull. Math. Biol. 60, 753–807.

3. Weidenmuller, A., Mayer, C., Kleineidam, C.J.,and Roces, F. (2009). Preimaginal and adultexperience modulates the thermal responsebehavior of ants. Curr. Biol. 19, 1897–1902.

4. Myerscough, M., and Oldroyd, B.P. (2004).Simulation models of the role of geneticvariability in social insect task allocation.Insectes Sociaux 51, 146–152.

5. Jones, J., Myerscough, M., Graham, S., andOldroyd, B.P. (2004). Honey bee nestthermoregulation: diversity promotes stability.Science 305, 402–404.

6. Oldroyd, B.P., and Fewell, J.H. (2007). Geneticdiversity promotes homeostasis in insectcolonies. Trends Ecol. Evol. 22, 408–413.

7. Weidenmuller, A. (2004). The control of nestclimate in bumblebee (Bombus terrestris)colonies: interindividual variability and selfreinforcement in fanning response. Behav.Ecol. 15, 120–128.

8. Allsopp, M.H., Calis, J.N.M., and Boot, W.J.(2003). Differential feeding of worker larvaeaffects caste characters in the Cape honeybee,Apis mellifera capensis. Behav. Ecol. Sociobiol.54, 555–561.

Behaviour and Genetics of Social InsectsLaboratory, School of Biological SciencesA12, University of Sydney, NSW 2006,Australia.E-mail: b.oldroyd@usyd.edu.au

DOI: 10.1016/j.cub.2009.09.038

Current Biology Vol 19 No 22R1036

Metastasis: Wherefore Arf Thou?

The small GTP-binding protein Arf6 is known to be an important regulator of theactin cytoskeleton and of cell motility associated with metastasis. A recentstudy identifies yet another role for Arf6 in metastasis — as a regulator ofplasma-membrane-derived microvesicle release.

Richard T. Premont*and Robert Schmalzigaug

Perhaps the most perilous moment incarcinogenesis is when transformedcells break free from their originalmicroenvironment, seeking to flourishelsewhere. As tumor cells migrate awayfrom their site of origin, they can eludethe remaining normal spatial controlsthat might have kept the tumor fromexpanding explosively and can go onto find new environments where theycan thrive, forming new and oftenmore aggressive tumors.

Established anti-cancer therapiestarget cell proliferation, as transformedcells divide relentlessly in the absenceof feedback controls that normally limitthe growth of non-transformed cells.An alternative therapeutic approachwould be to prevent tumor cells frommigrating away from their site of origin;much effort has therefore beenexpended in developing a molecularunderstanding of cell migration innormal and tumor cells. The Arf6protein is one important mediator oftumor-cell migration and invasivenessduring metastasis, suggesting that

Arf6-regulated pathways may providevaluable targets for therapeuticintervention to reduce metastaticpotential.

Arf6 is a member of theADP-ribosylation factor (Arf) family ofsmall GTP-binding proteins [1]. Likeall GTP-binding regulatory proteins,Arf6 cycles between an inactiveGDP-bound form and an activeGTP-bound form. Activity is controlledby guanine nucleotide exchangefactors (GEFs) that promote GDPrelease and GTP binding, andGTPase-activating proteins (GAPs)that promote hydrolysis of bound GTPto GDP [1]. Unlike other Arf familymembers, which function primarilyto coordinate intracellular vesicletrafficking events in the Golgi andendoplasmic reticulum, Arf6 functionsprimarily at the plasma membrane [1].The traditional view of Arf6 is that it

DispatchR1037

does have a role in coordinating vesicletrafficking, but that it specificallyregulates traffic to and from theplasma membrane [1]. As such, Arf6is an important regulator of cellularsignaling by affecting the ability ofreceptor proteins to make their wayto and from the cell surface. Arf6 alsohas an important role in coordinatingcell shape and motility by regulatingthe actin cytoskeleton, especiallyin tandem with the Rho-familymember Rac1 [2–4]. Arf6 functionsas a signaling intermediate, linkingcell-surface receptors to theactivation of several enzymesinvolved in plasma membrane lipidmetabolism and lipid-messengergeneration [1]. Given the diversityand complexity of Arf6 functions,and the apparently limited capacityof other Arf isoforms to perform thesespecific tasks, it is not surprisingthen that mice lacking Arf6 dieduring embryogenesis [5].

Arf6-regulated pathways havebeen studied extensively in the contextof cancer, especially in tumor-cellmetastasis. In previous work, Sabeand co-workers [6,7] exploredArf6-dependent migration and invasionin breast cancer-derived cell lines.Breast tumor cells with a highlyinvasive phenotype had increasedexpression of Arf6 compared with lessinvasive cells [8], and siRNA-mediatedknockdown of Arf6 expression reducedcell migration and invasiveness [6].Active Arf6 promoted the formation oftumor cell invadopodia — membraneprotrusions that project into and digestthe extracellular matrix [6]. Arf6localized to invadopodia, and aninactive Arf6 mutant preventedinvadopodia formation and matrixdigestion [9]. In a series of recentstudies (reviewed in [7]), Sabe andco-workers delineated a pathwaypromoting metastatic potential inbreast cancer cells that began withstimulation of the EGF receptor, toactivation of the Arf GEF GEP100/BRAG2, of Arf6, and of the ArfGAPprotein ASAP1/AMAP1, which actsas an Arf6 effector to recruit paxillinand cortactin to invadopodia.D’Souza-Schorey and colleagues [9,10]performed similar studies with the LOXinvasive melanoma cell line. LOXmelanoma cells stably expressingmutant forms of Arf6 that were eitherlocked in the active, GTP-bound stateor the inactive, GDP-bound state werecompared with wild-type LOX cells

SOS

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Current Biology

Cytoskeletal and focal adhesion regulation

Microparticleshedding

EGFreceptor

Figure 1. Arf6 signaling pathways in the regulation of tumor-cell migration and metastasis.

In the well-known mitogen-activated protein kinase pathway, the EGF-bound EGF receptorrecruits the Ras GEF Sos to activate the small GTP-binding protein Ras, which initiates theRaf–MEK–Erk kinase effector cascade to increase cell proliferation. Arf6 is similarly activateddownstream of extracellular receptors, such as the EGF receptor as shown here, as well as byintracellular signals that activate Arf GEF proteins such as ARNO or GEP100. For many Arf6pathways, the specific GEF remains to be identified (shown as ‘ArfGEF?’). Activated Arf6 bindsto effector proteins (such as phospholipase D (PLD), and the Rac1 GEFs Dock180 and kalirin),some of which also function as regulatory ArfGAPs (ASAP1, ASAP3). These effectors initiatefurther signaling pathways, through Rac1 or Erk, that promote further cellular events, ormay directly recruit proteins that alter cellular function (such as paxillin and cortactin ininvadopodia).

(expressing only endogenous Arf6)in a xenograft model using athymicnude mice [10]. Mice injectedsubcutaneously with LOXARF6-GTP cellsdeveloped primary tumors that weremuch smaller and grew more slowlythan those in mice injected with LOXcells or LOXARF6-GDP cells [10].However, despite this slower growth,LOXARF6-GTP cells were much moreinvasive of immediate surroundingtissue. Nevertheless, in a directmetastasis establishment assay, inwhich tumor cells are injected into thetail vein, LOXARF6-GDP cells showeda small reduction in the number andsize of lung metastases compared withLOX cells, whereas LOXARF6-GTP cellsfailed to induce lung metastases. Thesefindings indicate that Arf6 functions incontrolling both cell proliferation andcell migration ability during metastasis.In glioma cells, Arf6 has been shownto promote invasion through activationof Rac1 [11] and also to mediateEGF-stimulated proliferation [12]. In

various other cell types, the Arf GEFsARNO/cytohesin-2 and EFA6 [13]have been implicated in promotingcell migration, as have Arf GAPs, suchas ASAP3 [14] and GIT1/GIT2 [15].

In this issue of Current Biology,D’Souza-Schorey and colleagues[16] describe a mechanism wherebyactivated Arf6 contributes to matrixdegradation during tumor cell invasion.Surprisingly, they provide evidence forArf6-mediated regulation of an entirelyunexpected class of membranetrafficking events: the release ofplasma membrane-derivedmicroparticles (Figure 1). Thesetumor cell-derived microparticlesappear to derive from regions of theplasma membrane that are distinctfrom traditional invadosomes, but,like invadosomes, these microparticlesare associated with matrix-digestingproteases.

Microparticles are one of a class ofmembranous structures released fromcells. Another such structure, the

Current Biology Vol 19 No 22R1038

exosome, is a lipid-delimited vesiclereleased by the fusion of an intracellularmultivesicular body with the plasmamembrane, spewing the containedvesicles into the extracellular milieu[17]. Microparticles, in contrast, arederived from outward budding andfission from the plasma membranedirectly, the inverse of the well-knowninward vesiculation of clathrin-coatedpits [17]. These microparticles containintegral membrane proteins derivedfrom their cells of origin and may alsocarry cargo of select intracellularproteins. Once thought to be theproduct only of dying cells,microparticles are now appreciatedalso to be products of livingcells — both normal and, mostoften, transformed [17]. Releasedmicroparticles can find their wayeven to the bloodstream, where theycan be used as markers of the cellsthat released them. However, themechanisms regulating microvesicleproduction by cells have remainedobscure.

In this new study, the role ofArf6-regulated pathways inmicrovesicle shedding fromthe LOX melanoma tumor cell modelis established [16]. Comparinghighly invasive LOXARF6-GTP cellswith less invasive LOXARF6-GDP

cells revealed that LOXARF6-GDP cellsaccumulated numerous vesicularstructures on the cell surface, whereasLOXARF6-GTP cells and wild-type LOXcells had far fewer of these structures.Instead, LOX and LOXARF6-GTP

cells accumulated microparticlesin the culture media, and thesemicroparticles were much reduced inthe culture media from LOXARF6-GDP

cells. Analysis of the microparticlespurified from the culture media showedthem to have a membrane bilayerwith phosphatidylserine on theextracellular face, and to beheterogeneous in size, consistentwith a plasma-membrane originrather than a multivesicular-body(exosome) origin. Tellingly, thesemicroparticles contained Arf6 proteinand cell-surface markers (b1 integrin),as well as matrix-digesting proteaseactivity. This suggests that tumorcells release matrix-digestingmicroparticles to create localizedclearing of the nearby matrixin advance of invadopodiaprotrusion.

How does Arf6 regulate microparticlerelease? Unexpectedly, Arf6 does not

function as part of a proto-vesicle coatin microparticle formation, as might beexpected if Arf6 had an analogousfunction to that of Arf1 in intracellularvesicle formation. Instead, Arf6 actsas a signaling intermediate toactivate a localized signalingcascade culminating in myosin lightchain phosphorylation (Figure 1).Determination of precisely how myosinlight chain activity leads to outwardmembrane scission will require futurework. Other important outstandingquestions include whether Arf6 isabsolutely required for microparticleformation and release (does Arf6depletion affect this?), and whatsignals upstream of Arf6 lead toits activation on the microparticlepathway. Clearly, the identificationof additional signaling componentsand pathways that regulate thisprocess may identify workableapproaches to therapeuticallytarget early metastatic cells.

Arf6 sits in the center of a webof interactions and signals thataffect a tumor cell’s ability to migrateduring metastasis (Figure 1), havingfunctions such as: directing matrixdegradation through microparticlerelease [16] and through the formationof invadosomes [7]; controllingdynamics of cytoskeletal and focaladhesion connections to extracellularmatrix through activation of Rac1[2,3]; promoting general cell migration,such as through the ArfGAPs ASAP3[14] or GIT1/GIT2 [15]; and alteringmembrane lipid composition andproducing lipid-derived messengers[1]. One apparently critical aspectof Arf6 function in these variouspathways is that it acts locally, sothat Arf6 activated in one location bya particular GEF is competent toperform certain tasks, but likely notothers [18]. This specificity suggeststhat it may be possible for particularArf6-mediated pathways to betargeted downstream of Arf6. Withthe current work, one additionalArf6-mediated pathway now needsto be considered as a potential target:the regulated release from tumor cellsof plasma-membrane microparticlescontaining matrix-digesting proteases.

References1. D’Souza-Schorey, C., and Chavrier, P. (2006).

ARF proteins: roles in membrane trafficand beyond. Nat. Rev. Mol. Cell Biol. 7,347–358.

2. Santy, L.C., Ravichandran, K.S., andCasanova, J.E. (2005). The DOCK180/Elmocomplex couples ARNO-mediated Arf6

activation to the downstream activationof Rac1. Curr. Biol. 15, 1749–1754.

3. Koo, T.H., Eipper, B.A., and Donaldson, J.G.(2007). Arf6 recruits the Rac GEF Kalirin tothe plasma membrane facilitating Racactivation. BMC Cell. Biol. 8, 29.

4. Tushir, J.S., and D’Souza-Schorey, C. (2007).ARF6-dependent activation of ERK and Rac1modulates epithelial tubule development.EMBO J. 26, 1806–1819.

5. Suzuki, T., Kanai, Y., Hara, T., Sasaki, J.,Sasaki, T., Kohara, M., Maehama, T., Taya, C.,Shitara, H., Yonekawa, H., et al. (2006). Crucialrole of the small GTPase ARF6 in hepatic cordformation during liver development. Mol. Cell.Biol. 26, 6149–6156.

6. Hashimoto, S., Onodera, Y., Hashimoto, A.,Tanaka, M., Hamaguchi, M., Yamada, A., andSabe, H. (2004). Requirement for Arf6 in breastcancer invasive activities. Proc. Natl. Acad. Sci.USA 101, 6647–6652.

7. Sabe, H., Hashimoto, S., Morishige, M.,Ogawa, E., Hashimoto, A., Nam, J.M., Miura, K.,Yano, H., and Onodera, Y. (2009). TheEGFR-GEP100-Arf6-AMAP1 signalingpathway specific to breast cancer invasionand metastasis. Traffic 10, 982–993.

8. Onodera, Y., Hashimoto, S., Hashimoto, A.,Morishige, M., Mazaki, Y., Yamada, A.,Ogawa, E., Adachi, M., Sakurai, T., Manabe, T.,et al. (2005). Expression of AMAP1, anArfGAP, provides novel targets to inhibitbreast cancer invasive activities. EMBO J.24, 963–973.

9. Tague, S.E., Muralidharan, V., andD’Souza-Schorey, C. (2004). ADP-ribosylationfactor 6 regulates tumor cell invasionthrough the activation of the MEK/ERKsignaling pathway. Proc. Natl. Acad. Sci.USA 101, 9671–9676.

10. Muralidharan-Chari, V., Hoover, H., Clancy, J.,Schweitzer, J., Suckow, M.A., Schroeder, V.,Castellino, F.J., Schorey, J.S., andD’Souza-Schorey, C. (2009). ADP-ribosylationfactor 6 regulates tumorigenic and invasiveproperties in vivo. Cancer Res. 69, 2201–2209.

11. Hu, B., Shi, B., Jarzynka, M.J., Yiin, J.J.,D’Souza-Schorey, C., and Cheng, S.Y. (2009).ADP-ribosylation factor 6 regulates gliomacell invasion through the IQ-domainGTPase-activating protein 1-Rac1-mediatedpathway. Cancer Res. 69, 794–801.

12. Li, M., Wang, J., Ng, S.S., Chan, C.Y., He, M.L.,Yu, F., Lai, L., Shi, C., Chen, Y., Yew, D.T., et al.(2009). Adenosine diphosphate-ribosylationfactor 6 is required for epidermal growthfactor-induced glioblastoma cell proliferation.Cancer, epub ahead of print.

13. Casanova, J.E. (2007). Regulation of Arfactivation: the Sec7 family of guaninenucleotide exchange factors. Traffic 8,1476–1485.

14. Ha, V.L., Luo, R., Nie, Z., and Randazzo, P.A.(2008). Contribution of AZAP-type Arf GAPs tocancer cell migration and invasion. Adv. CancerRes. 101, 1–28.

15. Hoefen, R.J., and Berk, B.C. (2006). Themultifunctional GIT family of proteins.J. Cell Sci. 119, 1469–1475.

16. Muralidharan-Chari, V., Clancy, J., Plou, C.,Romao, M., Chavrier, P., Raposo, G., andD’Souza-Schorey, C. (2009). ARF6-regulatedshedding of tumor-cell derived plasmamembrane microvesicles. Curr. Biol. 19,1875–1885.

17. Cocucci, E., Racchetti, G., and Meldolesi, J.(2009). Shedding microvesicles: artefacts nomore. Trends Cell Biol. 19, 43–51.

18. Donaldson, J.G., and Honda, A. (2005).Localization and function of Arf familyGTPases. Biochem. Soc. Trans. 33, 639–642.

Department of Medicine, Duke UniversityMedical Center, Durham, NC 27710, USA.*E-mail: Richard.Premont@Duke.edu

DOI: 10.1016/j.cub.2009.10.032