Review Article

Water Flux in Cell Motility: Expanding theMechanisms of Membrane Protrusion

Vesa M. Loitto,* Thommie Karlsson, and Karl-Eric Magnusson

Division of Medical Microbiology, Department of Clinical andExperimental Medicine, Linkoping University, Sweden

Transmembrane water fluxes through aquaporins (AQPs) are suggested to playpivotal roles in cell polarization and directional cell motility. Local dilution bywater influences the dynamics of the subcortical actin polymerization and directsthe formation of nascent membrane protrusions. In this paper, recent evidence isdiscussed in support of such a central role of AQP in membrane protrusion forma-tion and cell migration as a basis for our understanding of the underlying molecu-lar mechanisms of directional motility. Specifically, AQP9 in a physiological con-text controls transmembrane water fluxes driving membrane protrusion formation,as an initial cellular response to a chemoattractant or other migratory signals. Theimportance of AQP-facilitated water fluxes in directional cell motility is under-scored by the observation that blocking or modifying specific sites in AQP9 alsointerferes with the molecular machinery that govern actin-mediated cellular shapechanges. Cell Motil. Cytoskeleton 66: 237–247, 2009. ' 2009 Wiley-Liss, Inc.

Key words: aquaporins; cell motility; polarity; morphology; filopodia

INTRODUCTION

Water is the key solvent for the processes of life.The lipid bilayer membrane that encloses all living cellsand separates them from other cells and the extracellularenvironment is, however, generally impermeable towater and polar solutes. In response to extra- or intracel-lular signals, cells therefore employ an array of efficienttransport mechanisms to allow rapid passage of waterand ions. The exact nature of the water transport mecha-nism remained unknown until Peter Agre and co-workersisolated a channel-forming, integral membrane proteinof 28 kDa [CHIP28; Preston et al., 1992], now calledaquaporin-1 [AQP1; Agre et al., 1994]. The discovery ofthe AQP resulted in the Nobel Prize in chemistry for Pe-ter Agre in 2003. Because the entry and release of waterfrom cells and tissue are fundamental processes of life, itis not surprising that AQP have been found throughoutnature. In humans alone, there are at least 13 differentAQP-like proteins [King et al., 2004; Verkman, 2005]. Itis now recognized that several of these AQPs play

pivotal roles in altering cell morphology [de Baey andLanzavecchia, 2000; Loitto et al., 2007], and current evi-dence also suggests that fluxes of water are directlyinvolved in directional cell motility [Loitto et al., 2002;Saadoun et al., 2005a,b; Cao et al., 2006]. The purposeof this paper is to summarize observations on AQPs in

*Correspondence to: Vesa M. Loitto, Ph.D., Division of Medical

Microbiology, Department of Clinical and Experimental Medicine,

Linkoping University, Linkoping SE-581 85, Sweden.

E-mail: vesa.loitto@liu.se

Contract grant sponsors: Wenner-Gren Foundation, The Swedish

Research Council (Medicine), The Swedish Society of Medicine, The

Magnus Bergvall Foundation, The Clas Groschinsky Memorial Foun-

dation, The Faculty of Health Sciences, Linkoping University.

Received 13 October 2008; Accepted 20 February 2009

Published online 4 April 2009 in Wiley InterScience (www.

interscience.wiley.com).

DOI: 10.1002/cm.20357

' 2009 Wiley-Liss, Inc.

Cell Motility and the Cytoskeleton 66: 237–247 (2009)

general and AQP9 in particular supporting a direct rolefor water in cell motility and the formation of cell protru-sions.

CELL MEMBRANE PROTRUSIONAND CELL MOTILITY

Cell migration is a multistep process involvingnumerous extra- and intracellular soluble signaling com-ponents. Initially, cells extend protrusions in the direc-tion of migration in response to soluble or bound signals.These protrusions become transiently fixed when stableadhesions form at their leading edge, and then the cellbody follows the protrusion using the stable adhesions astraction points. When these are released at the rear of thecell, the tail retracts [Lauffenburger and Horwitz, 1996].Movement over an extended distance occurs throughmany repetitions of this cycle in a both random anddirectionally persistent manner. Single motile cells, suchas fibroblasts and neutrophils, rapidly change their mor-phology by extending and withdrawing membrane pro-trusions, which vary in size and shape: some are broadand flat lamellipodial extrusions while others are long,thin filopodial formations.

One current idea of how cells protrude relies on thedendritic-nucleation model of actin polymerization[Mullins et al., 1998], where actin filaments (F-actin)form a branched network physically pushing the mem-brane forward. It has been suggested that treadmilling ofthe branched actin filament array consist of repeatedcycles of dendritic nucleation, elongation, capping, anddepolymerization of filaments [Borisy and Svitkina,2000; Pollard et al., 2000; Svitkina et al., 2003]. Thegeometrical organization of actin filaments differs inlamellipodia and filopodia; in lamellipodia actin fila-ments close to the membrane are organized into anextensive network of Y-branches [Svitkina et al., 1997;Svitkina and Borisy, 1999], while in filopodia the cross-linking activity of accessory proteins, like fascin, bun-dles the actin filaments into a tightly packed, parallelarrays that project forward to the membrane [DeRosierand Edds, 1980]. Membrane protrusion relies on the ac-tivity of the Rho family GTPases, mainly Rac1, Rac2,and Cdc42, which activate Arp2/3-mediated branchingand rapid actin polymerization [Welch and Mullins,2002]. Filopodial protrusion have been implicated inseveral fundamental physiological processes, of whichcell migration is perhaps the best characterized [Mattilaand Lappalainen, 2008]. For filopodial initiation a con-vergent elongation model has been proposed [Svitkinaet al., 2003]. Here some filaments within the lamellipo-dial dendritic network acquire a privileged status bybinding anticapping molecules, and where the barbed

ends of privileged filaments gradually cluster and formthe tip complex of the future filopodia.

Cdc42 is among several other proteins playing acritical role in the formation of filopodial protrusions[Nobes and Hall, 1995]. Thus, microinjection of acti-vated Cdc42 induces filopodial formation in fibroblasts[Kozma et al., 1995; Nobes and Hall, 1995]. It binds andactivates, via phosphatidylinositol-4,5-bisphosphate, theWASP and N-WASP proteins [Machesky and Insall,1998; Welch and Mullins, 2002], as well as IRSp53,which recruits the Ena/VASP family protein Mena[Krugmann et al., 2001; Lanier et al., 1999] to the filopo-dial tip and thereby protects elongating filaments fromcapping [Bear et al., 2002]. The lipid phosphatase-related protein 1 [LPR1; Sigal et al., 2007], Rho in filo-podia [Rif; Ellis and Mellor, 2000], myosin-X [Berg andCheney, 2002] and the formins [Peng et al., 2003] areother proteins implicated in filopodial formation. Themechanisms for filopodial formation have recently beenreviewed by Mattila and Lappalainen [2008].

It has been speculated that polymerization of actinfilaments is combined with thermal flexing of the fast-growing filament tips, allowing filaments to grow whenthey bend away from the membrane [Mogilner andOster, 2003]. When they spring back to the original posi-tion, the lipid bilayer is supposedly pushed forward.Thus, the filament end must apparently be free for poly-merization to occur, and its growth may not per se pro-duce force. The dendritic-nucleation-based membraneprotrusion is further highly dependent on rapid transportof available building blocks such as G-actin, which isusually in complex with monomer-binding proteins suchas thymosin and profilin. Actin molecules released fromthe base of the cortical actin meshwork and diffuse backto the polymerizing tips to continuously replenish thispool. Zicha et al. [2003] recently questioned specificallyhow actin polymerization at the leading edge of lamellaeand the growing tip of filopodia is refueled. By meas-uring diffusion rates they argued that diffusion of vitalcomponents is by itself too slow to provide basis formembrane protrusion, which rather rely on an activeforce such as hydrostatic pressure originating fromcontractions in the rear of the cell.

OSMOSIS AND CELL MOTILITY

An early and durable hypothesis relating to amoe-boid cell motility suggests that cytoplasmic streamingdrives membrane protrusions [Bray, 2001]. Indeed, inmost motile cells, such as neutrophils and fibroblasts, adramatic and vigorous internal streaming can beobserved. The amoeboid cell contracts the outer, corticallayer of cytoplasm selectively in certain regions andthereby squeezes a stream of more fluid cytoplasm into

238 Loitto et al.

the advancing membrane protrusion. Accordingly, rearretraction precedes front protrusion in polarized fibro-blasts [Dunn and Zicha, 1995]. Furthermore, in fish epi-thelial keratocytes a myosin II- and Rho kinase-depend-ent flow of F-actin aligns with the prospective axis ofmovement [Yam et al., 2007]. Further support comesfrom observations of regional hydrostatic pressurechanges, which do not equilibrate throughout the cyto-plasm on scales of 10 lm and 10 s [Charras et al., 2005].The idea of pressure-driven membrane protrusions hasbeen tested in many experiments and the overall conclu-sion is that these pressures probably are necessary, butnot sufficient to achieve expansion of lamellipodia [cf.Bray, 2001].

Another proposition is that a localized and direc-tional liquefaction or solation of the cortical actin gelallows for osmotic expansion and provides forcerequired for protrusion [Oster and Perelson, 1987]. Inthis regard, the actions of actin severing proteins need tobe taken into consideration. Among these are gelsolinand cofilin, which sever and uncap actin filaments.Hereby free barbed ends are generated for subsequentactin polymerization [Condeelis, 2001; Stossel, 1993].Gelsolin and cofilin are thus critical regulators of actindynamics and protrusive activity in cells. The idea is thatthere is a ‘turgor’ pressure resisted by tension in cyto-plasmic filaments. At the leading edge, cycles of briefand local swelling in a transiently disrupted actin fila-ment gel powers an outward expansion of the membraneto a new equilibrium size [Condeelis, 1992; Stosselet al., 1999]. It is indeed known that local changes inactin filament dynamics due to for instance Ena/VASPactivity directly cause changes in cell morphology[Lacayo et al., 2007].

Several lines of evidence suggest a mechanisticrole for osmosis and water fluxes in the migration ofcells, such as neutrophils. Putting cells in solutions ofincreasing osmolarity [Bryant et al., 1972; Dipasquale,1975; Rabinovich et al., 1980; Trinkaus, 1985] impaircell motility. It is further known that biological processesare affected by deuterium, which alters the osmotic prop-erties of living cells [Rastogi et al., 1971]. Accordingly ithas been shown that heavy water induce cell shapechanges and reduce motility of human neutrophils [Zim-mermann et al., 1988]. Osmolality also affects tumor cellpseudopod protrusions [You et al., 1996], and theoreticalanalyses show that an osmotic gradient elicits waterfluxes that promote movement of cells [Jaeger et al.,1999]. Moreover, fluxes of water can be examined usingself-quenching of fluorophores [Hamann et al., 2002;Solenov et al., 2004], a method that also has been usedto visualize water fluxes during neutrophil spreading andmotility (Fig. 1A–1C) [Loitto et al., 2002]. In the ‘hyper-loaded’ cells, fluorescence increased at morphologically

active sites such as at the pseudopodia in motile cellsand the cell edge of spreading cells (Fig. 1C). Theincreased fluorescence at morphologically active siteswas most likely the result of a dilution of the fluorescentcompounds generated by a local AQP-facilitated waterinflux [Loitto et al., 2002]. We have confirmed that thefluorescence originating from filopodial regions is notdue to membrane accumulation. This was done in twoways. First, cells were studied using differential interfer-ence contrast, confocal- and total internal reflection fluo-rescence microscopy. Second, we also labeled cells usingthe membrane dyes DiIC18-lipid and WGA-lectin, andin none of these experiments could we detect peripheralmembrane accumulation.

AQPAND CELL MOTILITY

Polarized Distribution of AQP

Human neutrophils express the aquaglyceroporinAQP9 [Ishibashi et al., 1998], which in immunofluores-cence labeling experiments is seen in morphologicallyactive regions such as the leading edge of motile cells(Fig. 1D) [Loitto et al., 2002]. Furthermore, whenexpressing green fluorescent protein (GFP)-taggedAQP9 in human promyelocytic HL60 cells differentiatedto become granulocytes, there was an essentially similarlocalization of AQP9 to the cell periphery with a prefer-ence to the leading edge in living cells (Fig. 1E). Indeed,several AQPs have now been shown to display a polar-ized distribution [Saadoun et al., 2005a,b; Cao et al.,2006].

AQP-Null Mice and AQP Expression

Phenotype analysis of AQP-null mice and expres-sion assays have recently been used as tools to show theinvolvement of various AQPs in cell motility. In thesestudies, tumor growth and cell migration was greatlyimpaired in AQP1-deficient cells [Endo et al., 1999;Saadoun et al., 2005a]. In kidney epithelial cells [Hara-Chikuma and Verkman, 2006], B16F10 melanoma and4T1 breast cancer cells [Hu and Verkman, 2006] cellmotility was AQP1-facilitated. Also AQP4-overexpress-ing cells showed increased cell motility [Saadoun et al.,2005b; Auguste et al., 2007]. In migrating CHO cells,AQP1 became polarized to the front end of cells [Saa-doun et al., 2005a], and a similar leading edge localiza-tion of AQP4 was observed in motile astrocytes [Saa-doun et al., 2005b]. In dermal fibroblasts [Cao et al.,2006], corneal epithelia [Levin and Verkman, 2006], andkeratinocytes [Hara-Chikuma and Verkman, 2008], cellmigration was AQP3-dependent. Moreover, expressionof AQP5 in NIH3T3 cells [Chae et al., 2008] and AQP1in malignant astrocytes increased motility [McCoy and

Water Flux in Cell Motility 239

Figure 1.

240 Loitto et al.

Sontheimer, 2007], and AQP1 knockout chondrocytesshowed reduced motility and adhesion [Liang et al.,2008]. These findings give strong support to the involve-ment of water transport in directional cell motility.

A model for how AQPs are involved in facilitatingthe motile behavior of cells has been forwarded byPapadopoulos et al. [2008]. They suggest that AQPsfacilitate cell movement by accelerating rapid changes incell volume and shape, which are required when cellsmove through a narrow extracellular matrix. In principle,they describe the link between water flux through AQPsand facilitated cell movement to be dependent on local-ized changes in cellular osmolarity, together with anincreased influx of water at the leading edge, creating anincreased localized hydrostatic pressure. Similar to theAQPs, several ion transporters such as the Na1/H1

exchanger NHE1 and the anion exchanger AE2 also areknown to polarize to the leading edge of a moving cell[Grinstein et al., 1993; Denker et al., 2000; Klein et al.,2000; Lagana et al., 2000]. The presence of concentratedion transporters at these sites enables a local osmotic gra-dient to be created and regulated. This osmotic gradientmight be the actual driving force for localized water fluxthrough the AQPs. Charras et al. [2005] have previouslyshown that a local increase in hydrostatic pressureachieved by acto-myosin driven contraction is enough totear a fragment of the membrane free from the corticalactin cytoskeleton and induce formation of blebs. Thesame mechanism could be applied to describe AQP-facilitated lamellipodial formation and chemotaxis. Thewater flux over the membrane at the sites of lamellopo-dia will generate a localized increase in hydrostatic pres-sure, tearing the membrane free from the actin cytoskele-ton and thereby creating space for further elongation ofthe actin filaments through more rapid G-actin diffusionflow in the liquefied areas. The localized hydrostaticpressure is most likely localized by the compact corticalactin meshwork, at sites of water influx, restricting the

volume increase to the leading edge [Ito et al., 1987;Schwab et al., 2007].

Water Fluxes in Human Neutrophils

Mercury compounds are generally regarded astoxic and of little specificity in their effects on cells. Stillwe would like to forward evidence for a critical role inAQP function. Using ionic mercury (Hg21), a known in-hibitor of water permeability [Macey and Farmer, 1970]and AQPs [Preston et al., 1993], it has been shown thatdirectional cell motility is dependent on water fluxthrough AQPs [Loitto et al., 2002]. Following additionof Hg21, there was a rapid, dose-dependent cessation inall morphological shape changes and chemoattractant-stimulated cell motility [Loitto et al., 2002; Loitto andMagnusson, 2004]. It has furthermore been shown thatHg21 forms a mercaptide bond with Cys213 in AQP9[Ishibashi et al., 1998]. It has not been exactly deter-mined how this modification affects the pore structure,but Hg21-exposure leads to an initial influx of extracel-lular Ca21 followed by a complete efflux of the Ca21-sensitive fluorophore in human neutrophils loaded withthe cytosolic fluorophore Fura-2 [Loitto et al., 2002;Loitto and Magnusson, 2004]. Because AQP6 also dis-plays increased permeability after Hg21-exposure [Yasuiet al., 1999], it was proposed that AQP9 also opens upon exposure to Hg21, which is likely due to conforma-tion changes leading to severely altered transport speci-ficity. It is not likely that the low doses (<10 lM) ofHg21 used caused any membrane damage, because novisual effects on neutrophils exposed to Hg21 weredetected [Loitto et al., 2002; Loitto and Magnusson,2004].

It has previously been shown that polyethylene gly-cols (PEGs) increase membrane stability and decreasemembrane permeability in mast cell degranulation [Mag-nusson et al., 1982; Magnusson and Stendahl, 1984],which in retrospect could have been an effect of PEGs

Fig. 1. Visualization of water fluxes and AQP9 in cells. (A) Fluores-cence quenching can be used as a method to visualize water fluxes.

(B) BCECF self-quenches on prolonged loading. Here cells loaded for

a short time and then lysed show a drop in fluorescence intensity upon

addition of detergent, while the fluorescence intensity from cells

loaded to self-quenching concentrations increases on lysis. BCECF

was visualized using the pH-insensitive excitation wavelength (from

Loitto et al. [2002] with permission from J Leukoc Biol). (C) Loadingthe fluorophore BCECF into cells for prolonged time results in partial,

cytosolic self-quenching of the fluorophores. Under the microscope,

‘hyperloaded’ cells show increased fluorescence in morphologically

active cell regions (arrowheads in B), i.e., in pseudopodia and at the

cell periphery of radially spreading cells. Mechanistically the fluoro-

phores at the self-quenching concentration respond to minute, local

dilutions by transmembrane water influx resulting in increased fluores-

cence intensity at these sites. For more details see Loitto et al. [2002].

(D) Immunofluorescent labeling of AQP9 in primary, human neutro-

phils displays a localized distribution to the leading edge of morpho-

logically polarized cells. (E) In differentiated human promyelocytic

leukemia cells (HL60) transfected with GFP-tagged AQP9 a similar

localization to the cell edges is observed. In HL60, overexpression of

AQP9 also results in enhanced protrusive activity, with the formation

of numerous filopodia not normally observed in morphologically

polarized HL60. (F) GFP-AQP9 expression in several cell types, such

as C3H10T1/2 fibroblasts normally void of AQP9, yields a filopodial

phenotype with long, thin protrusions forming dorsally and all around

the cell periphery. Similar projections were not found in fibroblasts la-

beled with a membrane dye or expressing a membrane localizing

GFP. (G) Site-directed substitution of the potential PKC binding/phos-

phorylation site Ser11 to Ala reduces the relative number of filopodia.

For more details see Loitto et al. [2007]. Micron mark for leukocytes

5 lm, and fibroblasts 10 lm.

Water Flux in Cell Motility 241

on aquaglyceroporins. Based on permeability data frommixtures of PEG400 and PEG1000 it was furthermoreconcluded, that only PEGs with a molecular weight lessthan 370 can enter the cytoplasm of neutrophils[Dahlgren et al., 1985]. Because AQP9 is an aquaglycer-oporin permeable to both glycerol and water, it was sug-gested that PEGs -due to molecular resemblance to glyc-erol- use the AQP channels to enter neutrophils [Loittoand Magnusson, 2004]. Indeed, PEGs with molecularweights ranging from 200 to 600 were shown to inhibitneutrophil cell migration in a dose- and size-dependentfashion, whereas equivalent concentrations of the largerPEG1000 had negligible effects [Loitto and Magnusson,2004]. In accordance with a blocking mechanism ofaction, preincubation of human neutrophils with lowconcentrations of small-sized PEGs protected the cellsagainst Hg21-induced leakage [Loitto and Magnusson,2004]. Possibly, glycerol resemblance nature of ethyleneglycol allows transport through AQP9 while the poly-meric structure occasionally leads to incomplete passageand trapping during pore transport and thereby inhibitsHg21 from gaining access to the pore-localized Cys213.It is also conceivable that for a long PEG polymer it isharder for a terminal end to reach and enter the narrowpore opening in AQP9.

AQPAND MEMBRANE PROTRUSION

GFP-tagged AQP9 was recently expressed in vari-ous cell types normally void of this specific AQP [Loittoet al., 2007] to further assess whether AQP9 and waterfluxes are involved in the migratory behavior and mor-phological alterations of single motile cells. The cellsincluded were mouse C3H10T1/2 fibroblasts, rat bladderNBT-II carcinoma and mouse B16F10 melanoma cells.In all cells the expression of GFP-AQP9 induced the for-mation of numerous actin containing filopodial protru-sions both along the cell edges and on the dorsal surfaceof the cells (Fig. 1F) [Loitto et al., 2007]. While filopodiabeing extended at the cell edge occasionally remainedsubstratum-attached, dorsal filopodia could be observedthrough several focal planes. Furthermore, not onlyGFP-AQP9, but also integrins appears to accumulate insuch protrusions [Galbraith et al., 2007]. Also HL60cells overexpressing AQP9 formed filopodia (Fig. 1E),which is intriguing because granulocytes normally makefew if any of these. It was recently shown that Crypto-sporidium parvum infection recruits AQP1 to the attach-ment site in cholangiocytes, epithelial cells that line theintrahepatic bile ducts [Chen et al., 2005], whichenhanced host-cell membrane protrusion. Further sup-port of AQP involvement in such events comes frommigrating CHO cells, where AQP1 was associated with

increased turnover of cell membrane protrusions[Saadoun et al., 2005a].

Phosphorylation of AQPs

It has been proposed that phosphorylations of spe-cific residues result in an allosteric change of the AQPprotein, which either triggers a translocation of intracell-ularly located channels to the plasma membrane orchange the opening state of channels already inserted inthe membrane [Gunnarson et al., 2004]. AQP2 containsa protein kinase A (PKA) phosphorylation consensus siteat the carboxyl terminal domain, and the water channelactivity was shown to be stimulated by cAMP-dependentphosphorylation [Fushimi et al., 1993; Kuwahara et al.,1995]. Addition of forskolin (adenylyl cyclase activator),8-bromo-cAMP (nondegradable cAMP analog) or injec-tion of the catalytic subunit of PKA also increased theAQP1 water channel activity [Yool et al., 1996; Patilet al., 1997]. A PKC-regulated pathway has previouslybeen described for the eye, where phorbol esters reducedthe intraocular pressure [Mittag et al., 1987]. In AQP4-expressing oocytes, phorbol diesters produced around90% reduction in swelling [Han et al., 1998], and site-directed substitution of Ser156 in AQP5 reduced cellmotility of transfected NIH3T3 cells [Chae et al., 2008].In plants the mechanism of phosphorylation-dependentAQP gating has been explored in greater detail [Torn-roth-Horsefield et al., 2006]. Fluctuations in watersupply are handled by regulating the AQP channel, viz,achieving closure by dephosphorylation. In humanAQP9, Ser11 is a potential, intracellular PKC binding/phosphorylation site [PhosphoBase v. 2.0; Ishibashiet al., 1998]. At the molecular level, site-directed substi-tutions of Ser11 to Ala (S11A; Fig. 1G) and Ser222 toGly (S222G) reduced the AQP9-induced morphologicalchanges, i.e., cells expressing the S11A or the S222Gmutants formed fewer filopodial protrusions than thewild-type AQP9. In contrast to the AQP9 mutants andMock controls, cells transfected with wild-type AQP9became more sensitive to decreasing osmolarity. Thissuggests that both mutants had an impaired intrinsicwater conductance of the channel [Loitto et al., 2007].Ser222 is, however, localized extracellularly, close tothe pore-forming region of AQP9 and it is likely that thesubstitutions in this site reduce channel functioning byaltering conformation rather than phosphorylationcompetence.

AQP9, Cdc42, and PKCf

It was recently shown that cells expressing wild-type AQP9, have increased levels of activated, GTP-loaded Cdc42 compared to S11A mutants and Mock-transfected cells [Loitto et al., 2007]. The formation ofAQP9-induced filopodia was reduced by cotransfection

242 Loitto et al.

with an inhibitory GTP-Cdc42-binding N-WASP CRIBsynthetic analog [Loitto et al., 2007]. Further support fora Cdc42-AQP interaction comes from observations of adirect localization of Cdc42 to AQP1-enriched domains,shown in C parvum-induced membrane protrusions[Chen et al., 2004]. So, how is Cdc42 associated with thePKC activation/binding discussed above? Recently aninteracting signaling complex including Cdc42 and PKCwas discovered [Joberty et al., 2000], and where GTP-Cdc42 forms a ternary complex by associating with atyp-ical PKC, such as i/k or f, and two mammalian homo-logs of the C. elegans partitioning-defective proteinsPAR-3 and PAR-6. The complex has been found to beessential for asymmetric cell division, polarized growth,homeostasis of tight junctions and adhesion contacts andpossibly also for cell motility and lamellipodial forma-tion [Joberty et al., 2000; Lin et al., 2000; Qiu et al.,2000; Noda et al., 2001; Etienne-Manneville and Hall,2003; Nishimura et al., 2005]. Accordingly, when cellsexpressing AQP9 were treated with a myristoylatedPKCf inhibitor, a smaller number of cells with a filopo-dial phenotype were observed [Loitto et al., 2007].Moreover, in phosphorylation assays isolated wild-typeAQP9 but not S11A mutants became phosphorylated byactive, recombinant PKCf. These findings suggests thatAQP9-induced filopodial protrusion involve both PKCfand Cdc42 [Loitto et al., 2007], and given the describedinteraction between PKCf, Cdc42 and the Par proteins itis likely that AQP9 is either a target of the complex orcapable of regulating the activity or association of thecomplex. Still, other signaling pathways connectingAQPs to cell morphology alteration may be of impor-tance. In NIH3T3 cells, AQP5 expression was found tobe associated with Ras/ERK activation [Woo et al.,2008], and AQP5 bound to the SH3 domains of severalkey proteins associated with signal transduction includ-ing c-Src, Lyn, and Grap2, in a PKA-dependent manner[Chae et al., 2008].

MODEL OFAQP-INDUCEDMEMBRANE PROTRUSION

Physical Effects

The patterns of AQP-regulation of motility andmorphology support a proposal where expression ofwater channels leads to an increase in the basal waterinflux that initiates rapid actin filament elongation (Fig.2). Although the flow of water from the extracellularspace to the cytoplasm probably is minute in physiologi-cal buffers, a localized microdilution would be capableof generating a favorable milieu for actin filament elon-gation. First, it generates a steeper gradient and speedsup forward flux of components involved in actin poly-

merization, primarily G-actin. Recent findings haveshown that local hydrostatic pressure changes in thecytosol do not instantaneously equilibrate throughout thecell [Charras et al., 2005]. Indeed, cells may use polar-ized blebbing resulting from the internal hydrostaticpressure to generate force that facilitates a directionalmovement [Fackler and Grosse, 2008]. Second, a rela-tive increase in available building blocks forms an activepool that can immediately add onto preexisting barbedends. It is possible that the local increase in hydrostaticpressure could also break the filament-membrane an-chorage and expose more fast-growing filament ends.Third, if the water influx continues, actin filament elon-gation may outpace subsequent filament branching andactivity of capping proteins. In support of this notion, ithas been shown that siRNA inhibition of capping pro-teins resulted in increased filopodium formation [Mejil-lano et al., 2004]. Finally, not only G-actin but also othercomponents of filopodial protrusion, such as the anticap-ping proteins Ena/VASP [Bear et al., 2002], myosin-X[Berg and Cheney, 2002], actin filament-stabilizing pro-tein filamin A [Flanagan et al., 2001], and fascin [DeR-osier and Edds, 1980] will be recruited more rapidly bydiffusion to the site of localized dilution. Directional mo-tility is thus initiated at the chemoattractant receptor-level, leading to a subcortical accumulation of proteinsinvolved in building up the actin-based motility machin-ery. In combination with the initial activation-induceddepolymerization of the cortical F-actin meshwork andthe presence of concentrated ion transporters, the pro-teins of the motility machinery together with the distinctlocalization of AQPs form the basis of building up theosmotic gradient at the leading edge.

In summary, these findings favor a signal transduc-tion pathway depicted in Fig. 2. It works in concert withthe water transporting function of AQP9, and wherePKCf binds to Ser11 in AQP9, thereby transientlyincreasing local water influx. This stimulates local actindiffusion and polymerization and determines the locationof a nascent filopodium. Cdc42 interacts with PKCf andthese two molecules form a tetrameric complex togetherwith Par3 and Par6. Actin polymerization then followsaccording to the established signaling cascade for filopo-dial growth; with GTP-Cdc42 freeing WASP from auto-inhibition and activating IRSp53/Mena leading to Arp2/3 activation and actin filament elongation. Figure 2 pro-vides a testable working hypothesis along which futureexperiments can be based.

CONCLUDING REMARKS

In the short span of 15 years, our understanding ofwater channel functions has increased dramatically, andthe roles of AQPs in health and disease have become

Water Flux in Cell Motility 243

Fig. 2. Suggested model of AQP9-induced filopodial formation. (1)

Activation of cell morphological alterations are initiated at plasma

membrane microdomains where for instance G protein coupled recep-

tors (GPCRs) and water channels are enriched. Upon cellular activa-

tion, cytosolic signaling molecules such as the 2nd messenger cAMP

and protein kinase PKCf accumulate and become activated, respec-

tively. Both PKCf [Loitto et al., 2007] and cAMP [Pietrement et al.,

2008] have been shown to interact with AQP9. (2) PKCf phosphoryl-ates Ser11, in the water channel [Loitto et al., 2007]. (3) Activation of

AQP9 leads to a local water influx. (4) The dilution generates a

steeper gradient, and speeds up forward flux of for instance, mono-

meric actin (light gray circles). (5) A contraction of the cell cortex

induces a hydrostatic force within the cell and increases the forward

flux of monomeric actin further [Zicha et al., 2003]. Due to the

increased availability of building blocks, hydrostatic stretch activation

of intrinsic actin polymerization and possible softening of the cortical

network, actin filaments start to elongate. Concurrently, PKCf forms

a ternary complex together with Cdc42, Par3, and Par6 [Etienne-

Manneville and Hall, 2003]. (6) GTP-charged Cdc42 augments actin

polymerization by freeing WASP from autoinhibition and activating

IRSp53/Mena, leading to Arp2/3 activation and actin polymerization.

IRSp53, Mena, Scar, WASP, and other components of filopodial

growth form a filopodial tip cluster. The submembraneous space cre-

ated by water influx allows polymerization to outpace both capping

and branching, yielding long filopodial actin filaments. (7) The form-

ing filopodia is stabilized by increased exocytosis, feeding membrane

to the growing protrusion, and expression of cell adhesion molecules

such as integrins, providing anchorage to the substratum [Karlsson

et al., 2008].

244 Loitto et al.

increasingly well documented. Channel-mediated trans-membrane water movements are local, signal-regulatedand osmotically driven. The strong forces involved in os-mosis underscore the importance of tightly regulatedwater fluxes, which otherwise could be deleterious to thecells. A precise control of water homeostasis is also criti-cal in cell motility where even minor changes may resultin potentially deleterious compressive or expansiveforces. Thus, alterations in AQP expression or function-ing may be a potential cause of cell motility-relateddiseases, a topic that deserves further exploration.

ACKNOWLEDGMENTS

The authors also wish to express sincere gratitude toDr. Ken Jacobson for helpful comments on the manuscript.

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