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Structurally diverse ion transport proteins anchor thespectrin–actin cytoskeleton to the plasma membrane bybinding directly to linker proteins of the ankyrin and protein 4.1families. Cytoskeletal anchoring regulates cell shape andrestricts the activity of ion transport proteins to specialisedmembrane domains. New directions are being forged by recentfindings that localised anchoring by ion transport proteinsregulates the ordered assembly of actin filaments and theactin-dependent processes of cell adhesion and motility.

AddressesDepartment of Stomatology, University of California, San Francisco,CA 94143-0512, USA*e-mail: barber@itsa.ucsf.edu

Current Opinion in Cell Biology 2002, 14:214–220

0955-0674/02/$ — see front matter© 2002 Elsevier Science Ltd. All rights reserved.

Published online 30 January 2002

DOI 10.1016/S0955-0674(02)00304-6

AbbreviationsAE1 anion exchanger 1ERM ezrin, radixin, moesinFAK focal adhesion kinaseFERM 4.1 ERMNHE Na+/H+ exchangerPIP2 phosphatidylinositol 4,5-bisphosphateIP3 inositol (1,4,5)-trisphosphateN-WASP neuronal Wiskott–Aldrich syndrome protein

IntroductionThe association of actin filaments with the plasma membrane provides mechanical stability, maintains cellshape and adhesion, and regulates dynamic surface protrusions,such as lamellipodia and filopodia, which are fundamentaldeterminants of motility and migratory potential. A num-ber of integral membrane proteins anchor actin filamentsand the cortical cytoskeletal network to the plasma membrane. These membrane anchors generally belong totwo distinct groups: adhesion molecules and ion transport proteins. The importance of cytoskeletal anchoring byadhesion molecules in regulating cell attachments is wellestablished [1]. This review explores the role of ion transport proteins as cytoskeletal anchors. The term ‘iontransport protein’ is broadly used to include ion channels,pumps and exchangers.

The role of ion transport proteins as anchors for the membrane skeleton was first characterised in the erythrocyte,in which the Cl–/HCO3

– anion exchanger AE1 docks thespectrin–actin cortical network by binding directly to thelinker proteins ankyrin and protein 4.1. In erythrocytes,this tethered complex is essential for membrane stabilityand cell shape determination. In non-erythroid cells,cytoskeletal anchoring assembles ion transport proteins in

specialised membrane domains, which restricts theirlocalised transport activity. Recent findings have revealeda new view on the functional importance of ion transportproteins as cytoskeletal anchors. While anchoring maintainsthe localisation of ion transport proteins, localized anchor-ing and ion transport activity in turn regulate the assemblyand organisation of actin filaments, cell adhesion and cell motility.

Key playersBasic components of a membrane-anchored cytoskeletalcomplex include an integral membrane protein, a linkerthat acts as a spacer and a tethered cytoskeleton that pro-vides stability, flexibility and polarity (Figure 1). A numberof structurally distinct ion transport proteins, including ionexchangers, ion channels and P-type ATPases, anchor thespectrin–actin cytoskeleton to the plasma membrane bybinding directly to ankyrin or to members of the protein4.1 family (Figure 1a). A recent review by Bennett and Baines [2••] includes a comprehensive description ofthe key players in anchoring complexes formed by ion transport proteins. As noted by Bennett and Baines [2••],assembled complexes of spectrin, ankyrin and protein 4.1are found exclusively in metazoans, which suggests thatthe membrane skeleton may have evolved as a componentof mechanically integrated tissues.

Cytoskeletal anchoring complexes assembled by ion trans-port proteins are analogous to those tethered by adhesionmolecules, including integrin binding to the 4.1 family mem-ber talin at focal contacts and CD44 binding to ankyrin andthe 4.1 family members band 4.1 and ERM proteins ezrin,radixin and moesin (Figure 1b). Talin, band 4.1 and ERMproteins contain an amino-terminal domain that binds transmembrane proteins, a central α-helical arm and a carboxy-terminal domain that binds actin or actin-associatedproteins (reviewed in [3]). The amino-terminal membrane-binding site is contained within a defined FERM (4.1-ERM)domain [4]. FERM domains act as modules to link cytoplas-mic proteins to the plasma membrane and are found in suchfunctionally diverse proteins as the focal adhesion kinase(FAK), the tumour suppressor proteins merlin and DAL-1,myosin VIIA and cytoplasmic tyrosine phosphatases.

Docking of the cytoskeleton to the plasma membrane is regulated by the localised abundance of phosphatidylinositol4,5-bisphosphate (PIP2) [5••]. The direct binding of PIP2 tokey players in membrane-anchored cytoskeletal complexes,including the β subunit of spectrin [6], ERM proteins [7]and ion exchangers [8,9], probably promotes their assembly.Crystal structures of a dormant moesin FERM−tail complex[10••] and of the radixin FERM domain complexed withinositol (1,4,5)-trisphosphate (IP3) [11•] reveal that theFERM domain is masked by an intramolecular association

Ion transport proteins anchor and regulate the cytoskeletonSheryl P Denker and Diane L Barber*

Ion transport proteins anchor and regulate the cytoskeleton Denker and Barber 215

between the amino and carboxyl termini. PIP2 binding at apleckstrin homology (PH) domain within the FERMdomain would induce a conformational change that exposesamino-terminal membrane-binding sites. This has beenconfirmed by Barret et al. [7], who found that mutations inthe PIP2-binding site of ezrin result in a redistribution ofezrin from actin-rich membrane structures to the cytosol.Binding of PIP2 to ion transport proteins might also induceconformational changes that unmask sites for bindingcytoskeletal linkers and stimulate transport activity.Mutations in the PIP2-binding site on the Na+/H+ exchangerNHE1 prevent binding of ERM proteins [12••] and impairtransport activity [9].

Functional significance: a view from the iontransport proteinLocalisation in membrane microdomains Cytoskeletal docking restricts ion transport proteins to specialised domains of the plasma membrane. AnkyrinGlocalises voltage-dependent Na+ channels at axon initial segments, which is necessary to generate local current and to propagate a self-generating action potential [13].

Cytoskeletal docking also localises the Na+/K+ ATPase at thebasolateral membrane of epithelial cells in mammals [14] andDrosophila [15], cardiac Na+ channels at the sarcolemma [16]and NHE1 at the leading edge of lamellipodia in fibroblasts(Figure 2; [12••]). Moreover, cytoskeletal docking throughlinker proteins may retain ion transport proteins constitu-tively at the membrane. The ubiquitously expressed NHE1isoform, which binds directly to ERM linkers to anchor thecytoskeleton, is not regulated by trafficking from intracellularstores [17]. In contrast, the epithelium-specific NHE3 isoform, which associates indirectly with ERM proteins andthe cytoskeleton through coordinate binding of an NHE regulatory protein [18], is acutely regulated by recruitmentfrom recycling endosomes [17].

Cytoskeletal docking also clusters ion transport proteinsinto higher-order complexes. The amino-terminal mem-brane-binding domain of ankyrin contains ankyrin repeatsfolded into subdomains that allow multivalent binding todifferent classes of membrane proteins. This permitsassembly of homocomplexes and heterocomplexes, includingthe clustering of AE1 tetramers [19] and multimeric

Figure 1

F-actin

Spectrin

Vinculin

Talin

ankyrin

AnkyrinAnkyrin

Cytoskeletal anchoring byion transport proteins

Ion exchangers Anion exchangers (AE1, AE2, AE3) Na+-H+ exchanger (NHE1) Na+-Ca+ exchangerIon channels Voltage-dependent Na+ channels Amiloride-sensitive Na+ channelsP-type ATPases Na+-K+-ATPase H+-K+-ATPase

Cytoskeletal anchoring byadhesion molecules

Integrin CD44

4.1, ERM 4.1, ERM

(b)

Linkers Ankyrin Protein 4.1 ERM proteins

(a)

α β

Current Opinion in Cell Biology

Membrane–cytoskeleton anchoring complexes. A basic anchoring complexincludes a membrane anchor (ion transport protein or adhesion molecule),a linker (ankyrin or a member of the protein 4.1 family) and a cytoskeletalnetwork of actin and spectrin. (a) Generic complex anchored by iontransport proteins. The association of these proteins with linkers and thecytoskeleton is described by Bennett and Baines [2•• ], except for the

recently identified anchoring by the Na+/H+ exchanger NHE1 [12•• ] andthe H+/K+ ATPase [54]. (b) Structurally analogous anchoring complexesare mediated by adhesion molecules, such as integrin binding to the4.1-like linker talin at focal adhesion sites, and CD44 binding to bothankyrin and ERM proteins. One distinction is that protein 4.1 and ERMproteins bind directly to actin, but talin binds indirectly through vinculin.

216 Cell regulation

complexes of Na+ channels and neurofascin at axon initialsegments [20], and the Na+/K+ ATPase and E-cadherin atthe basolateral domain of epithelial cells [14,21].

Regulating transport activityCytoskeletal anchoring and organisation play key roles inregulating the activity of ion transport protein. Disruptingthe association with spectrin or actin impairs the activity ofNa+ channels in cardiomyocytes [16] and epithelial cells [22],of store-operated Ca2+ channels in endothelial cells [23], ofa renal Na+/K+ ATPase [24] and of NHE1 in fibroblasts [9].How cytoskeletal anchoring regulates transport activity isunclear, but it could reflect a dependence on localisation[12••], assembly into higher-order complexes [19] or a possible direct mechanical effect of cytoskeletal elementson transport proteins [24]. Distinguishing between thesepossibilities will require approaches for targeting ion transport proteins to specific membrane domains indepen-dently of their cytoskeletal association.

Organisation of the actin cytoskeleton also plays a critical rolein the ability of transport proteins to mediate an osmoregula-tory response (reviewed in [25]). As suggested by Cantiello[26], the actin cytoskeleton might act as a volume sensor, withcross-linked actin filaments forming a sensory elastic gel thattransmits a physical regulation of plasma membrane transportproteins. Alternatively, osmotic activation of transport proteins can be mediated by signalling through the low molecular weight GTPase RhoA and the RhoA-associatedkinase ROCK [27,28,29•]. How RhoA is activated by changesin cell volume and how it mediates osmotic activation oftransport proteins remain unknown. One possibility is thatphosphorylation of components of the anchoring complex byROCK promotes their assembly. The direct phosphorylationof NHE1 by ROCK stimulates transport activity and is critical for Rho-dependent cytoskeletal remodelling [30].Additionally, ROCK phosphorylates ERM proteins [31],which is likely to promote their recruitment to the plasmamembrane [2••,32], probably through a conformation-induced unmasking of membrane binding sites that is

coordinately regulated by PIP2 binding. Hence, ERM proteins, like N-WASP (neuronal Wiskott–Aldrich syndromeprotein) [33], could act as coincidence detectors downstreamof Rho-family GTPases. Additional mechanisms for activationof ion transport proteins by Rho include a ROCK-dependentmyosin-based contractility [34,35] and altered mechanicalcoupling by changes in actin filament assembly. An importantnext step is to determine whether osmotically inducedchanges in the actin cytoskeleton are secondary to activationof Rho GTPases.

Functional significance: a view from thecytoskeletonAssembly of adhesion complexesOur view of cytoskeletal anchoring by ion transport proteins has recently been expanded to include an effect ofthe localised anchoring complex on actin-dependent processes.A number of ion transport proteins are either structurally orfunctionally associated with cell adhesion. Beta subunits of voltage-dependent Na+ channels contain an extracellularimmunoglobulin domain that mediates homophilic cell adhesion and an intracellular domain that binds ankyrin [36•]. Multivalent binding by ankyrin in turn coordinatelyassembles Na+ channels and the adhesion molecules neuro-fascin and NrCAM at the neuronal synapse [20]. In epithelialcells, ankyrin binding also coordinately assembles theNa+/K+ATPase and E-cadherin at the basolateral membrane[14,21], and mutations that inhibit Na+/K+ATPase activityimpair cell adhesion [37]. In fibroblasts, although NHE1 is notstructurally associated with adhesion molecules, its cyto-skeletal anchoring plays a critical role in the assembly of focal complexes. In NHE-null fibroblasts, integrin-inducedassembly of focal adhesions is impaired but is rescued by stable expression of either wild-type NHE1 [12••,38] or amutated NHE1 that lacks transport activity, but not byNHE1 with mutations in the cytoplasmic domain thatprevent ERM binding and disrupt cytoskeletal anchoring[12••]. As described below, an anchor-dependent assemblyof actin filaments likely mediates the effects of NHE1on cell adhesion.

Figure 2

Localisation of NHE1 and ezrin in fibroblasts.The coordinate clustering of NHE1 and ezrinat the leading edge of lamellipodia infibroblasts is regulated by their direct binding,but not by NHE1 activity. Panels showlocalisation of NHE1 and ezrin in NHE-nullfibroblasts expressing wild-type NHE1(PSN1), NHE1 with a point mutation thatprevents ion translocation (PSN1–E266I) andNHE1 with alanine substitutions in thejuxtamembrane region of the cytoplasmicdomain that impairs ERM binding(PSN1–KR/A). Reproduced withmodifications from [12•• ].

PSN1 PSN1-E2661 PSN1-KR/A

NHE1

Ezrin

Current Opinion in Cell Biology

Ion transport proteins anchor and regulate the cytoskeleton Denker and Barber 217

Cytoskeletal organisation The established importance of AE1 in regulating erythrocyteshape and membrane integrity suggests that its cytoskeletalanchoring or transport activity regulates assembly of thesubmembrane cytoskeletal network. Deletion of the AE1gene in two animal models has given conflicting results onwhether AE1 regulates cytoskeletal organisation. In cattlewith a naturally occurring deletion of AE1, erythrocyteshave decreased levels of spectrin and ankyrin and a dis-torted membrane skeleton containing disordered filamentsof uneven length and width [39]. In contrast, erythrocytesof mice with a targeted deletion of the AE1 gene have normal levels of spectrin and ankyrin and a normal sub-membrane cytoskeletal organisation [40]. This may bedue to the concomitant upregulation of membrane proteins that compensate for the loss of anchoring by AE1.Compensatory upregulation of AE2 and NHE1, whichboth anchor the cytoskeleton, as well as downregulation of AE1, has been shown in tissues from mice null for theAE1-associated band 4.2 protein [41]. Additionally, thedecreased membrane stability in AE1-null cells couldreflect loss of a major structural component because AE1 constitutes more that 25% of the total erythrocyte membrane protein.

An alternative approach has been used in fibroblasts todetermine that anchoring by NHE1 regulates cytoskeletalorganisation. In fibroblasts engineered to express NHE1with mutations that prevent ERM binding, both NHE1and ezrin are redistributed from the leading edge of lamel-lipodia to a uniform localisation along the plasmamembrane (Figure 2). In these cells, the ordered assembly

of focal adhesions and actin stress fibres is impaired andactin filaments that normally extend to the leading edge oflamellipodia are absent [12••]. These findings are consistentwith a role for ERM proteins in focal complex formationand actin filament assembly [42]. In fibroblasts expressinga mutated NHE1 (E266→I, single letter amino acid code)that lacks transport activity but can bind ERM proteins,NHE1 and ezrin coordinately cluster at the leading edge of lamellipodia (Figure 2) and the assembly of focal adhesions and actin filaments is normal. Hence, NHE1 hasdistinct, but related, functions as an ion transport proteinregulating intracellular pH and cell volume and as a struc-tural anchor regulating cytoskeletal organisation (Figure 3).

MotilityThe dual functions of NHE1 raise the question of why atransport protein is used as a cytoskeletal anchor at theleading edge of lamellipodia if transport activity is notrequired for anchorage or for actin filament assembly. Oneanswer is that ion transport is necessary for cell motility.Pharmacological inhibitors of NHE1 activity inhibitchemotaxis of leukocytes [43,44] and the migration ofendothelial [45] and epithelial [46–48] cells. Mutations inNHE1 that block either its anchoring or its transport function impair fibroblast migration, but the migratoryphenotypes are distinctly different (S Denker, D Barber,unpublished data). Blocking NHE1 activity impairs de-adhesion and retraction of the trailing edge. In contrast,blocking NHE1 anchoring impairs polarity at the leadingedge and persistence of a primary lamellipod. In motilecells, actin filaments are protrusive structures about themembrane and are oriented parallel to the direction of

Figure 3

Dual functions of ion transport proteins.A speculative model depicting distinct, butrelated, functions of ion transport proteinsbased on recent findings of NHE1 actions[12•• ,30]. A structural role in cytoskeletalanchoring regulates the localisation andclustering of ion transport proteins, celladhesions and the organisation of actinfilaments. Localised clustering of ion transportproteins restricts their activity to specialisedmembrane domains, resulting in localised ionand solute fluxes and changes in cell volume.Integrins and G-protein-coupled receptors(GPCR) regulate the activity of ion transportproteins and the assembly of anchoringcomplexes. These actions can be mediated byRho GTPases and their downstream kinases,such as ROCK, which directly (solid line) orindirectly (dashed line) act on key players inthe anchoring complex. Both structure- andactivity-based actions of ion transport proteinsregulate cell motility through distinct, butundetermined, mechanisms.

Rho GTPases

ROCK

Integrin GPCR

Cell motility

Anchoring complex Ion transport protein Linker Cytoskeleton

Transport activity Ion and solute fluxes Cell volume

Cytoskeletal anchoring Localisation and clustering Cell adhesions Actin filament organisation

Current Opinion in Cell Biology

α β

218 Cell regulation

movement, which is important for generating tension andmembrane protrusion; however, in the lamellipodia offibroblasts with impaired NHE1–ERM binding these filaments are absent [12••]. In addition to NHE1, theCl–/HCO3

– anion exchanger AE2 [48] and P-type ATPaseshave been shown recently to regulate cell migration[21,44], although it is undetermined whether this is mediated by their anchoring of ankyrin and the cytoskeletonor by their transport activity.

How the activity of ion transport proteins regulates migrationis unclear. Actin polymerisation at the leading edge is currently thought to be a sufficient driving force for membrane protrusion [49,50], and localised ion fluxescould regulate the activities of proteins that modify actinpolymerisation, such as cofilin or gelsolin, which are Ca2+-and pH-sensitive. Alternatively, as presented by Schwab[51•], the spatially restricted activity of ion transport proteins might create localised changes in cell volume thatregulate cell migration. According to this model, migrationis regulated by cycles of localised swelling at the protrusivefront from the osmotically obligated water uptake associatedwith ion transport activities and by localised cell shrinkageat the trailing edge. Consistent with this model, atomicforce microscopy has revealed cyclic swelling and shrink-age at the trailing edge of migrating MDCK-F epithelialcells [52]. Whether localised volume changes at the frontcan promote membrane protrusion remains to be deter-mined, although a model presented by Oster and Perelson[53] suggests that protrusion might be driven by the gelosmotic potential of the lamellipodium actin meshwork.Measuring volume changes in protrusive structures will bea challenge because of their dynamic nature and thin struc-ture. This challenge might be met by using standing-wavefluorescence microscopy, an interference-based opticalsystem that was used recently to measure actin assembly atthe leading edge of lamellipodia [50]. An appealing theoryis that actin polymerisation provides the driving force formembrane protrusion, but that coordinated movement isregulated by localised volume changes induced bycytoskeleton-anchored ion transport proteins.

ConclusionsRecent findings compel us to re-evaluate the significanceof cytoskeletal anchoring by ion transport proteins.Although docking is clearly important for assembling transport proteins in specialised membrane domains andrestricting their localised activity, a reciprocal action wherebyion transport proteins regulate dynamic remodelling of thecytoskeleton is now evident. The current challenge is todetermine aspects of cytoskeletal remodelling regulated byeither the anchoring of actin filaments or the localised iontransport. Of particular interest is how ion transport pro-teins regulate cell migration. Although an accepted modelfor directed migration suggests that the force of actin poly-merisation at the leading edge drives membrane protrusion,it is unclear how this accounts for docking of actin filamentsto the leading-edge membrane. One possibility is a cyclic

docking and release that is regulated by the activity of ion transport proteins or by local thermal fluctuations.Regardless of the mechanism, the critical message is that iontransport proteins have a significant role in dynamic remod-elling of the actin-based cytoskeleton.

AcknowledgementsWe thank Evangeline Leash for editing the manuscript and members of theBarber laboratory for their comments and suggestions.

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