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Journal of Pathology j Pathol 2004 204 460 469 Published online in Wiley InterScience wwwintersciencewieycom DOI IO 002lpath65 Review Article Wiskott Aldrich syndrome protein and the cytoskeletal dynamics of dendritic cells Yolanda Calle l lSiUeCi ani Chou Adrian Thrasher2 and Gareth Eones Randall Diwsion ofCell andVlolecular Biophysics King39s College London Guy39s Campus London SEl lUL UK 2Molecular lmmunology Unit lnstitute oanild Health University College London London WClN lEl l UK Correspondence to Professor Gareth Ejones Motility and Cytosllteleton Group Randall Diwsion ofCell andVlolecular Biophysics King39s College London New llunt39s llouse Guy39s Campus London SEl lUL UK Email garetnoneslltcl ac ullt Abstract The regulated migration and spatial localization of dendritic cells in response to environ mental signals are critical events during the initiation of physiological immune responses and maintenance of tolerance Cells de cient in the Wiskott Aldrich syndrome protein WASP have been used to demonstrate the importance of the dynamic remodelling of the actin based cytoskeleton during the selective adhesion and migration of these cells Unlike most cell types macrophages dendritic cells and osteoclasts utilize a specialized adhesive array termed the podosome in order to migrate Podosomes are composed of many of the same structural and regulatory proteins as seen in the more commonly found focal adhesion but are unique in their requirement for WASP Without WASP podosomes cannot form and the affected cells are obliged to use focal adhesions for their migratory activities Once activated by a series of upstream regulatory proteins WASP acts as a scaffold for the binding of the potent actin nucleating protein complex known as Arp23 This article reviews the available evidence that suggests that failures in the regulation of the actin cytoskeleton may contribute signi cantly to the immunopathology of the Wiskott Aldrich syndrome Copyright 2004 Pathological Society of Great Britain and Ireland Published by John Wiley amp Sons Ltd Additional material for this review article is available in Wiley InterScience at httpwww3 intersciencewiley comcgiebinj about1 130suppmatthtm Keywords WASP dendritic cells macrophages actin cytoskeleton cell migration Introduction Understanding cell motility its mechanisms and its consequences is central to understanding many of the body s defence and healing processes as well as disease processes such as cancer invasion and metastasis For these reasons the eld of cell motility and its control has attracted a conceited effort from many of the world s leading cell and molecular biologists over the past two to three decades Progress in unravelling the molecular basis of cell motility and the signalling pathways that control it has been rapid 12 Yet the complexity of the role of cell locomotion in development healing and cancer the complexity of the signalling pathways and the shear variability of the experimental phenomena have meant that many of the big questions are still unresolved Over recent years it has emerged that a major key to understanding how cells move and respond to their environment lies in the dynamics of disassembly relocation and reassembly of cytoskeletal structures within the cell New methods of microscopy for imag ing live cells coupled with rapid developments in u orescent proteins 3 are already revealing the sites of interactions between molecules in the signalling path ways that control cytoskeletal dynamics 4 Perhaps more importantly new uorescence imaging methods are also beginning to shed light on the supramolecular dynamics of the cytoskeleton 56 A general picture has emerged of the migrating cell as a highly polarized entity in which the complex regulatory pathways that control the cytoskeleton are spatially and temporally segregated according to function 7 Cell interactions with the extracellular matrix and surrounding cells also determine cell behaviour and fate Formation of cell cell and cell matrix adhesions allows cells to sense their environment and respond to signals that regulate most aspects of cell life includ ing survival proliferation and differentiation 8 The extracellular matrix and the surface of neighbouring cells provide molecular cues that both promote and regulate cell motility by providing foci for the assem bly of the adhesion points required for the generation of traction forces necessary for cell migration 9 In nearly every case examined cell locomotion involves a regulated cycle of behaviour 10 The initial response of a cell to a migration stimulus is to polarize and extend protrusions that come to de ne the direction of locomotion These protrusions take the form of broad lamellipodia and more often than not are seen in association with spikelike protrusions beyond the cell s leading edge called lopodia Both structures Copyrigit 2004 Pathological Society of Great Britain and Ireland Published byJohn Wiley amp Sons Ltd WASP and the cytoskeleton are formed as a result of actrn polymenzatron and are stabrlrzed by adtesron erther to the extracellue lar matrrx or to nerghbourrng cells by transmembrane receptors that are also lrnked to the actrn cytoskeleton 1011 Cell progressron also requrres the drsassembly of adhesrons at the rear of a moyrng cell allowrng tarl detachment and cytoplasmlc retractlon of the posterlor margrn Whrle many aspects of thrs cycle are shared the detarls can drffer srgnr cantly between cell types In thrs reyrew we wrll descrrbe the adhesrye and motrle behayrour of dendrrtrc cells DCs and how defectrye assembly of the cytoskeleton rn these cells leads to farlures of cell adhesron and mrgratron key parameters rn the development of an acqurred rmmune response DCs are cntrcal for the generatron and rntegratron of physrologrcal rmmune responses and for marntenance of perrpheral tolerance 12 The controlled operatron of these processes whrch requrres rnteractron wrth other rmmune effector cells rs entrrely dependent on mlgratlon from sentlnel sltes to secondary lymphatlc organs drrected by chemotactrc srgnals 13 and sucr cessful communrcatron between DCs and lymphocytes wrthrn these trssues 14 The outcome of such events rs largely detennrned by the actryatron state of the DC and the specr c envlronment wrthrn whrch rnteractron occurs Berng at the rrght place at the rrght trme rs therefore of crrtrcal rmportance The cytoskeleton of DCs Motrlrty rs closely lrnked to the dynamrc organlzar tron of actrn mrcro laments and tubullnebased mlcror tubules Most cells have welledeveloped mrcrotubule systems that help stabrlrze the arrays of mlcro lae ments that tend to predomrnate at the cell perrphery 10 The actrn cytoskeleton rs also the anchorrng component of the adhesron plaques of connectrye tlsr sue cells to extracellular matrrx proterns whether rt be at the nascent adhesrons formed at the very edge 46 of an adyancrng cell called focal complexes or the more mature focal adhesrons that develop from them 15 In most cell types the focal adhesrons are assor crated wrth large bundles of mrcro laments termed stress bres that can erther pass rnto the marn cell body or run along the basal surface of the cell to lrnk adhesron srtes lo 1n stark contrast to the sltr uatron found rn the majorlty of resrdent connectrye trssue cells mrgratrng DCs along wrth macrophages and mlgratoryrphase osteoclasts possess nelther stress bres nor large focal adhesrons Frgure 1 Instead the cytoplasm contarns a delrcate tracery of mlcror lament bundles that concentrate at a serres of dlse Crete fool at the substratum lnterface These structures termed podosomes 17 are wrdely drstrrbuted on the lower surface and rn the case of actryely mrgratory cells can be Seen to concentrate at the Zone between lamellrpodra and lamellae of polarrzed cells Frgures 1 and 2 Images of xed cells farl to show the hrghly dynanrc nature of podosomes rn mrgratrng DCs and macrophages rn fact they appear and then fragment and drsperse agarn oyer rnteryals of a few mrnutes as the cell margrn extends durrng the locomotory cycle 18 and see supplementary Vldeo Durrng thrs pro cess actln ls constantly belng exchanged lnto and out of the lamentous core of the podosome 19 where rt ls presumed to lnteract wrth a varlety of structural and srgnallrng moretres such as talrn paxrllrn yrnculrn and Src famrly proterns 7 proterns also known to regulate focal adhesron structure and srgnallrng 20723 Attachment to the substratum and subsequent Slgr nallrng events are rnrtrated by subswammrspecl c members of a large famrly of rntegrrns that are brought together to form a dense attachment plaque at the base of each focal adhesron or focal contact 915 Inter grrns are srmrlarly requrred at podosomes to medrate the attachment of cells to therr substratum but rn thrs case therr arrangement rs drfferent At least rn DCs mlgratory osteoclasts and macrophages the rntegrrns appear to be arranged as crrcumferentral rrngs around Figure I Cytoslceletal organlzatlon ot normal dermal broblasts and dendntlc cells A Dermal broblasts lncubated wrth phalloldln whlch blrlds Fraaln to demonstrate large bundles ot actlrl laments red makng up the stress bres anchored at then dlstal tlps lrto tocal adhesrons contalnlngnnculln green B lmmature dendntlc cells assemble actlrl laments red at the ad arlarlg cell marglns and can be seen tn the retractlon bres towards the rear ot the cell but no stress bres are tormed lnstead most Fractlrl ls located at dlscrete punctate structures located on the substratum lmertace These are podosomes The suppomng array ot mlcrotubules lnthese cells ls shown ln green Path011004204 4507th 461 Y Calle et aI Figure 2 Dtstrtbuttoh ot podosomes at the leadmg edge of mtgratmg cells Podosomes oah be touhd under most otthe cell but clusters concentrate behmd the lamelltpodta ot mtgratmghumah macrophages AeC ahd mouse osteodasts D4 Cor ocahzamon of saw laments A ahd eGFPeWAEP B m the podosomes of human macrophages Panel C ts a merged tmage otA ahd B The aotm core ts rtoh m WASP Osteoolasts were stamed wtth phallotdm to show actln laments D and ahttehhoulm ahttsera were used to vlsuahze vthouhh E Merged tmages ot hactm ahd vlnwhn are shown m pahel F note the arrangement otymoulm around the aotm core of the podosomes enlarged mset the acttn lament core of the podosome thure 3 20 and we nd that human DCs selecttyely recrutt members of the 32 group of tntegrtn heterodtmers to fonntng podosomes 24 Another major dtfference between conyenttonal focal adhestons and podosomes ts the arrangement of WlskottrAldrlch syndrome pros tetn WASP Whtle acttye WASP or tts ubtquttous homologue NVWASP ts found concentrated at or near the spreadtng lamelltpodtal edge tn many cell types 252o as far as we are aware there have been no reports to date that show WASP or NVWASP to be located tn focal adhestons In contrast leukocyte podosomes do recrutt WASP 27729 see thure 2 whtle NVWASP ts present tn the podosomeellke adhee SW5 structures of srcetransformed connecthe tlssue cells 30732 A restrtcted number of other cell types have recently been reported to possess podosomeellke structures 33735 though tt seems that there ts no absolute requtrement for NVWASP to be recrutted to these podosomes for thetr fonnatton 35 The Wiskott Aldrich protein WASP The WlskottrAldrlch syndrome WAS gene encodes a 5023man actd tntracellular protetn WASP exprer ssed excluslvely ln haematopoletlc cells 36 WASP Patho1004204 4507459 ts one member of a dtsttnct famtly of protetns that parttctpate tn the transductton of stgnals from the cell surface to the acttn cytoskeleton 37 Other members of thts famtly tnclude the ubtquttously expressed NVWASP mentloned earller and SCAR suppressor of Grproteln coupled cycllceAMP recep tor rst found tn 3 genetlc screen lrt chryoslglmm dISCOIdEMm A mammallan homologue of SCAR was subsequently descrtbed by two groups one retatned the name SCAR the other named the proteln WAVE VtASPefamlly Verprollnrhomologous protetn and both acronyms are now wtdely used Mammals also express two further lsoforms of SCARWAVE brmgr mg the total number of WASP famtly members to ve WASP NVWASP and SCARMAVElrS 38 The WASP famtly protetns are organtzed tnto evoe luttonary conseryed modular domatns thure 4 The Cetermlnus conslsts of a module responslble for blndr mg to and also acttyattng the ArpZ3 complex a potent nucleator of acttn polymerlzatlon 3940 Thts module ts made up of a WASP homology 2 WH2 sequence followed by ashort central C sequence and a tenntnal actdtc A sequence WH2 domatns btnd monomertc acttn 41 whtle the c and A sequences btnd to and acttyate the Arp23 complex NVWASP has a Very stmtlar Crtel39Tnmus domatn structure to WASP except for the presence of an addtttonal WH2 WASP and the cytoskeleton 46 Figure 3 Dlstrlbutlon of In lntegrlns around the aonn core of podosomes of rnouse lmmature dendnno ceHs Aeq and macrophages D4 Cells were stalned uslng anusera to detect In lntegrln subunlts green panels A and D and pHaHoldln to show Feaaln red panels B and E Merged Wages are shown In panels c andF As wnh vlnwhn the recrulted lntegrlns form an annular nng around the 3am core enlarged lnsets Mammalian WASP family WASP I l Starl I NVWASP wch WM VmHnernrh 8mm Scat ll Fromm IV W a loo amino acids Figure 4 Domaln organlzauon of the mammahan WASP famlly modl ed horn an onglnal by Dr Laura Macheskywlth permlsslon ln the lnacuve state WASP and NVWAEP are thought to exlst In an autolnhlbltory conformauon wnh the rnoleoule folded In such a Way as to enable a seaole lnteracuon between the CNS and c domalns SCAR protelns do not have an autolnhlblted conformauon WHl WASP hornology l domaln B baslc domaln CRlB Cdon and Rac lmeractwe domaln W WASP homologyz domaln c central domalnA acldlc domaln SHD Scar homology domaln domaln Flgure 4 Thls Cetel39mmal module confuse mgly termed lhe WA WCA or VCA module In dlffen mg publlcatlons IS recognlzed as the mlnlmal reglon requlred to bmd and actlvate the Arp23 complex 4042 The Net mlm of WASP famlly protelns are much more dlvergent Each contams a central prollnserlch reglon that acts as a blndlng slte for yanous SH3 src homology 3 domalnecontammg protelns but a domaln at the Netel mlnus of each proteln de nes Whether u 15 classed as a WASP or a SCAR Both WASP and NVWASP contam the WASP homology 1 WHl domaln a close relatlve of the EnaVasp homology 1 EVHl domaln of the EnaVASP proteln famlly The WHl domaln bmds to the WASP lntsracte mg proteln WI that regulates WASP or NVWASP actlvlty 4344 WASP and NVWASP also possess a blndlng domaln CRIB also called GED for the GTP bound actlvated form of the small GTPase de2 as well as a baslc sequence that bmds to phosphatldylle nosltol 45eblsphosphats PIPz 4546 In contrast the SCARWAVE protelns have a unlque Netermlnal domam commonly referred to as the SCARWAVE homology domaln SHDWHD for Whlch no men acuons have yet been dlscovsrsd SCARs also lack any CRIB domalns so cannot dlrectly bmd meme bers of the small Rho GTPase famlly of acun rage ulators Patho1004204 4507459 464 Discussion of the molecular details of WASP inter actions with Arp23 together with their roles in ini tiating actin polymerisation is beyond the scope of this review and only a brief overview is given here For a more detailed account the reader is referred to cited publications and reviews 47 50 When not activated WASP and NWASP exist in an autoinhib ited state in which the CRIB domain interacts with the C domain In this state WASP and NWASP are not able to activate the Arp23 complex because the WCA domain cannot properly interact with the A1p23 complex As previously mentioned Cdc42 is an acti vator of WASP 45 binding in its GTPloaded state to the CRIB domain 51 This binding results in a destabilization of the autoinhibited conformation of WASP leading to its activation 52 Cdc42 is a key regulator of the actin cytoskeleton implicated in the production of lopodia at the cell margin 53 and in generating cell polarity 54 56 While Cdc42 has numerous other targets within leukocytes WASP and NWASP are unique in being able to directly stimulate actin assembly through its binding to Arp23 Cdc42 is itself activated downstream of surface receptors such as integrins 5758 cytokine receptors 54 G protein coupled receptors 59 or growth factor receptors 60 so is an ideal candidate for linking surface receptor activation to actin remodelling Nevertheless a wide variety of SH3 domaincontaining proteins have also been identi ed as WASP interacting proteins and more recent work has rmly established a role for phospho rylation of WASP in regulating its activity Phospho rylation of WASP has been previously demonstrated following Bcell receptor crosslinking and stimulation of the collagen receptor on platelets 6162 The pro tein tyrosine phosphatase P39IPPEST has been shown to dephosphorylate Y291 of WASP the speci city of this interaction possibly being mediated by the adapter protein PSTPIP which binds directly to WASP 63 The location of Y291 adjacent to the CRIB within the hydrophobic core of the autoinhibited structure has led to speculation that phosphorylation has an important role in the regulation of WASP activity More recently the Src family tyrosine kinase Hck has been shown to directly induce phosphorylation of WASP Y291 independently of Cdc42 resulting in a change in physical properties and consequently a shift in electrophoretic mobility 64 Furthermore a synthetic phosphomimicking mutant Y291E which introduces a negative charge into the core at the interface between two hydrophobic sheets enhances actin polymerization both in vitro and in viva suggest ing that posttranslational modi cation in the form of Y291 phosphorylation has a direct regulatory function 64 Similar ndings implicate phosphorylation of the analogous tyrosine of NWASP Y256 as a key reg ulator of neurite outgrowth 65 An L270P mutation in the CRIB domain of WASP was recently shown to result in enhanced Cdc42independent activity of the protein in vitro presumably through disruption of jPathoI 2004 204 460 469 Y Calle et a the autoinhibited structure which manifested clini cally as an Xlinked form of severe congenital neu tropoenia SCN 66 The mechanisms underlying the haematological abnormalities are however unknown at present 47 Whatever the mode of release of WASP from its autoinhibited state once this has occurred full binding of the WCA domain of WASP to Arp23 is achieved 67 and actin lament assembly rapidly follows 148 Patients lacking WASP suffer from Wiskott Aldrich syndrome WAS a rare inherited Xlinked recessive disease approximate incidence 1 in 250000 indi viduals in the European population characterized by immune dysregulation and Inicrothrombocytopenia In the absence of haematopoietic stem cell transplanta tion many patients with classical WAS die in child hood and early adulthood from haemorrhage infec tion or malignancy The mechanisms responsible for the pathophysiology of WAS are directly linked to de cient actin organization in haematopoietic cells However the biological defects that have the most profound effect on the functioning of the immune sys tem as a whole have not yet been clearly determined Based on observed defects of chemotaxis and migra tion which are described later in this review an attrac tive hypothesis is that many features of WAS result from aberrant cell traf cking which may only need to be subtle to produce profound disturbances of immune cell development functioning and homeostasis 68 Equally there is good evidence for defective TCR sig nalling 69 and for attenuation of phagocytosis 70 suggesting that the complex immune dysregulation of WAS re ects multicompartmental abnormality The underlying mechanism for Inicrothrombocytopenia is less clear but is partially correctable by splenec tomy indicating that clearance of abnormal platelets by this organ is important Understanding both how WASP family proteins regulate the cytoskeleton and of how these processes in uence the functioning of the haematopoietic system may have important impli cations for treatment of this disorder and perhaps for the development of novel treatments of other immuno logical diseases 477172 Podosomes are regulated by WASP In the case of DCs macrophages and migratory phase osteoclasts it is evident from the preceding discussion that podosomes must be a particularly active site of WASP familymediated actin lament assembly but since NWASP is also expressed in such cells our unpublished data and NWASP can be recruited to podosomes of nonhaematopoietic cells 3035 determining the precise role of either of these proteins in mammals can be dif cult Our approach to this problem has largely relied on the existence of the naturally occurring human gene knockout for WASP which gives rise to the WAS disorder with supporting work coming from work with a WASP null mouse WASP and the cytoskeleton model where WASP knockout results m yarrous abnonnalrtres assocrated wrth the rmmune system rncludrng a reductron m the numbers of lymphocytes platelets and thymocytes reduced phagocytrc actryrty m neutrophrls and defectrye cylosksletal functron 6973 We and others have found that WASP plays an essentral role m the fonnatron of podosomes m monocyterderlved DCs 2428 and therr llnsagsr related companrons the macrophages 2974 and osteoclasts 75 In human DCs and macrophages the absence of WASP results m the complete abrogatron of podosomes whrle m the murrne system DCs and macrophages from WASP null mrce are largely deyord of podosomes Frgure 5 However we can also detect a very low percentage of murrne cells drsplayrng drsorganrzed clusters of Fractln dots remrnrscent of poorly ordered podosomes our unpublrshed results The number of 39podosomes39 per cluster rs consldsrr ably reduced and each rs de crent m Fracun content when compared wrth normal murrne podosomes In addrtron yrnculrn rs not recrurted around the actrn core of these abnormal structures Hence rt seems that m the mouse system although some other pror terns from the WASP famrly yery lrkely NVWASP our unpublrshed data can at least partrally compenr sate for the actlnrpolymerlzlng actryrty of WASP they cannot compensate for the adaptor actryrty of WASP For example the recrurtrnent of yrnculrn to form anan lar assocratrons wrth the actrn core rs rmparred m WASPenull cells It rs thought yery lrkely that WASP specr cally controls spatrally and temporarrly the fore matron of podosomes by an unknown mechanrsm an rssue berng further rnyestrgated m our laboratory at present These obseryatrons were also con rmed m osteoclast podosomes where lt was seen that migrar tory WASPenull murrne osteoclasts were completely deyord of podosomes 75 Instead actrn and Vlnr culln often coelocallzed m scattered patches wrth 46S morphology remrnrscent of the focal adhesrons seen m eprthelral or broblastrc cells WASPenull osteoclasts also farled to assemble normal actrn sealrng rrngs and m therr place they drsplayed morphologrcally abnorr mal actn rrngs depleted of actrn and yrnculrn wrth very few drscrete podosomerllke structures wrthrn the rrng structures apparent dunng the bone resorptron phase of the osteoclast cell cycle 75 Although physrologrcal steadystate levels of bone resorptron are marntarned a major rmparnnent rs observed when WASPenull anlr mals are exposed to a resorptrye challenge Our results proyrde clear eyrdence that WASP rs a crrtrcal compo nent of podosomes m osteoclasts and agarn rndrcate a nonrredundant role for WASP m the dynamrc orgaan zatron of these structures 75 Paxrllrn talrn and members of the 32 rntegrrn subgroup are also assocrated m rrngs around the actrn core of podosomes m DCs and macrophages Frgure 3 Lack of podosomes m human WAS DCs results m 32 rntegrrn drspersal throughout the cell surface and a decrease m cell adherence to ICAMel a lrgand for 32 rntegrrns 24 Thrs rndrcates an essentral role for WASP m the regulatron of rntegrrn clusterrng m DCs that rs requrred for proper DC adherence to ICAMel Our prelrmrnary ndrrgs suggest that WASP rs playrng a role m proyrdrng the requrred platform for the organrzatron of rntegrrns m the cell membrane that mrght be a prereqursrte for the further actryatron of such rntegrrns m response to therrsubstrata orpossrble lnslderout srgnallrng events 76 For example when Wlldrtyps murrne osteoclasts were plated on bone slrces a greater percentage of cells formed typrcal osteoclastrc sealrng zones when compared wrth cells plated onto glass surfaces rndrcatrng that fonnatron and maturatron of actrn rrngs are favoured by a physrologrcal bone substratum Thrs response to the substratum was not observed m WASPenull osteoclasts 75 Figure 5 WASPerluH cells do not torm podosomes Actln organlzatlon ln mouse WASPenuH dendntlc cells A and macrophages slylsuallzed uslrlg phalloldln stalnlng Mouse WAEPrde clerlL macrophages and spleenrderlved dendntlc cells tall to assemble podosomes The Vast malorlty ot the cells n culture also tall to polanze correctly wtth a broad leadlng edge ct Flgures l4 and show hyperelongated morphology Also note the abnormal lateral prolectlorls a charaaerlstlcofWAEPrrluH cells ln both mouse and humans Path011004204 4507459 466 WASP and cell migration We have found that podosome assembly in normal human DCs is regulated during cell maturation After activation with LPS immature DCs become actively attached to substrata of extracellular proteins such as bronectin or ICAMl where they rapidly gener ate podosomes then polarize and initiate migration Somewhat unexpectedly after about 4 h in culture the cells begin to lose their podosomes become more rounded cease migration and eventually by about 8 h detach from the substratum in small clusters This time course of cell behaviour was correlated with the process of maturation as measured by standard mark ers such as HLADR and CD86 expression 24 Thus podosome assembly is restricted to the early stages of maturation and is lost as cells complete maturation instead the actin cytoskeleton is diverted to support the many ruf es and extensions to be seen on the sur face of the rounding cells These data support a role for podosomes not only in cell attachment to substrata but also in active migration which is restricted to an early phase of DC maturation We do not know yet why mat uration leads to podosome loss unpublished data from our laboratory have con rmed that mature DCs contain abundant WASP and that active Cdc42 and Rac can be used to generate lopodia and ruf es in mature cells 28 nevertheless we have been unable to regenerate podosomes in matured DCs Concomitant with the loss of podosomes 32 integrins instead become dispersed to the surface ruf es 2428 following which there is a signi cant decrease in the strength of DC adhesion to ICAMl 24 Although the pattern of rearrange ments of the DC cytoskeleton in spleenderived mouse DCs in response to LPSinduced maturation was some what different in the detail we also observed a similar temporal regulation of the assembly and disassembly of podosomes following DC maturation Calle et al manuscript in preparation These results show that podosome assembly is highly regulated during DC maturation and suggest a role for podosomes in the movement of activated immature DCs from peripheral tissues to the lymphatic vessels and peripheral lymph nodes as cells transmigrate across endothelial barriers rich in ICAMl 1377 WASP is also essential for maintaining normal cell morphology during directional migration in DCs and macrophages A high percentage of both human and murine normal DCs and macrophages in culture have a polarized morphology with a distinctive broad lamel lipodium at the leading edge and a narrow contractile tail Figures 1 and 2 In contrast a high percentage of WAS DCs and macrophages display a characteris tic spindle like or hyperelongated morphology lack ing a clear leading lamellipodium Instead the front and rear of the cells are usually terminated by two or three bluntpointed protrusions When viewed in a timelapse movie WAS cells also display another abnormality of protrusive behaviour irregular and transient projections of the lateral margins 28 Much jPathoI 2004 204 460 469 Y Calle et al the same irregularity of cell shape is observed in the murine WASnull system unpublished observations see Figure 5 suggesting that the absence of WASP creates a somewhat disordered actin cytoarchitecture that fails to sustain the development of a stable cell front The irregular and hyperelongated morphology of WASnull cells could be the direct result of a lack of podosomes since these structures do preferentially assemble near the broad leading edge However we have found that not all WASPexpressing myeloid cells necessarily possess podosomes For example many normal murine bone marrowderived macrophages fail to elaborate podosomes and instead have dynamic adhesion structures similar to focal complexes Never theless these cells are not hyperelongated 78 so the mere absence of podosomes fails to explain why WAS DCs and macrophages cannot sustain the polarized development of leading lamellipodia The explanation is more likely to be found in the proposed role for WASP and its homologue NWASP in the dynamic assembly of actin at the cell margin 264879 It is likely that the ability to make WASP and NWASP dependent podosomes in a few cell types is a special ized functional development of the universal role of these proteins in cell protrusion The lack of normal leading lamellipodia in WASP null cells does have one major consequence that may not be related to podosomes an impaired chemotactic response It has long been recognized that appropri ate cell polarization is fundamental to the initiation of the chemotactic migration of a wide variety of cell types from motile Dictyostelium 80 through to mammalian tissue cells 81 and leukocytes 82 If the mechanisms generating cell polarity are dis turbed then it is found that while cell migration may still be observed the directed motile response to chemoattractants is grossly impaired 5483 The small GTPase Cdc42 is regarded as a critical media tor in generating cell polarity 56 so it is tempting to relate loss of appropriate Cdc42 signalling through WASP to potential defects in leukocyte chemotaxis As described earlier Cdc42 was rst described as a regulator of lopodia formation 84 though it is by no means the only protein capable of generating these structures WASP or at least its close homologue NWASP is also known to locate to lopodia when in its activated Arp23binding state 79 and these structures are commonly believed to have some role in sensing the immediate environment of cells 85 We have shown that WASPnull cells fail to generate normal lopodia suggesting a link between failures of a Cdc42 signalling mechanism to lopodia forma tion through an absence of WASP 86 We 7487 and others 88 have also shown that WASP regulates DC and macrophage cell polarization towards leuko cyte attractants In a more detailed study using time lapse recordings of normal and WAS macrophages observed directly while migrating under a gradient of the macrophage chemoattractant CSFl MCSF we found that WAS cells were unable to respond WASP and the cytoskeleton to the chemotactic stimulus More recently we were able to restore the normal chemotactic response of WAS cells to CSFl after expressing fulllength human WASP in WAS macrophages 74 These data provide strong evidence for the role of WASP in regulating the chemotactic responses of leukocytes at least in response to activation of cytokine receptors The sit uation is less clearcut for surface receptors that sig nal through heterotrimeric G protein complexes We were unable to measure any defect in the chemotac tic response of WAS neutrophils to potent agonists such as IL 8 or fMLP 87 and to date we have also failed to measure consistent chemotactic defects in immature WAS DCs when tested against the RAN TES chemokine unpublished data It may be that sig nalling via GPCR to chemotaxis pathways does not absolutely require the involvement of WASP but can and does utilize NWASP or even the SCAR proteins that are also associated with protrusive behaviour of cells 89 but clari cation of these speculations awaits further study Outlook The work just described is carried out using cell cul ture systems but veri cation of a role for WASP in the translocation of DCs from peripheral sites to lymph nodes following activation with antigen comes from very recent work on mice We have shown that WASPnull DCs exhibit defects of cell migration at multiple levels in viva including emigration from skin traf cking to draining lymph nodes and also in spa tial localization within the lymph node de Noronha etal submitted There are clearly many ways in which these defects could contribute to immune dys regulation Inef cient delivery of antigen to lymphoid tissue would compromise the development of phys iological immune responses and activation without proper migration to a regulated microenvironment could result in the initiation or propagation of ectopic in ammatory processes The maintenance of toler ance and the homeostatic regulation of naive and memory Tcell populations which require to vary ing extents interaction between MHC and TCR to preserve numbers and function might also be com promised Together abnormalities of DC migration and of migration of T and B lymphocytes are also likely to be responsible for the observed disorganiza tion of microarchitecture in secondary lymphoid tis sue of WASPnull animals such as for example the poorly organized follicles and reduced marginal zone B cells that we have catalogued Supplementary material Two supplementary videos Movie1 and Movie 2 may be found at the website httpwww3interscience wileycomcgibinjabout1130suppmatthtm 467 Movie Normal human macrophage Interference re ection microscopy IRM reveals the adhesion sites of living cells to the substratum as black areas while other parts of the cell appear as various shades of grey This sequence shows the rapid appearance and disappearance of podosomes the black dots at the leading edge upper left of an elongated cell migrating to the left of the frame The leading edge and lateral marng of the cell can be seen to icker white these are ruf es on the plasma membrane Movie is 10 minutes 60 frames captured at 10 second intervals Movie 2 WAS macrophage There are no podosomes in this elogated cell The black areas are sites of focal complex attachment to the substratum Note the lack of adhesion turnover at the tips of the cell and the robust adhesion areas on the lateral margins There is very little active membrane ruf ing in this case Movie is 10 minutes 60 frames captured at 10 second intervals Acknowledgements This work was supported by the Medical Research Council and The Wellcome Trust References 1 Pollard TD Blanchoin L Mullins RD Molecular mechanisms controlling actin lament dynamics in nonmuscle cells Annu Rev Biophys Biomol Stmct 2000 29 5457576 2 Burridge K Wennerberg K Rho and Rac take center stage Cell 2004 116 1677179 3 LippincottSchwartz J Snapp E Kenworthy A Studying protein dynamics in living cells Nature Rev Mol Cell Biol 2001 2 4447456 4 Anilkumar N Parsons M Monk R Ng T Adams JC Interaction of fascin and protein kinase Calpha a novel intersection in cell adhesion and motility EMBO J 2003 22 539075402 5 Salmon WC Adams MC WatermaniStorer CM Dualiwavelength uorescent speckle microscopy reveals coupling of rnicrotubule and actin movemens in migrating cells J Cell Biol 2002 158 31737 6 Watanabe N Mitchison TJ Singleimolecule speckle analysis of actin lament turnover in lamellipodia Science 2002 295 108371086 7 Small IV Rottner K Kaverina I Functional design in the actin cytoskeleton Curr Opin Cell Biol 1999 11 54760 8 Giancotti FG Ruoslahti E Integrin signaling Science 1999 285 102871032 9 DeMali KA Wennerberg K Burridge K Integrin signaling to the actin cytoskeleton Curr Opin Cell Biol 2003 15 5727582 10 Ridley AJ Schwartz MA Burridge K etal Cell migration integrating signals from front to back Science 2003 302 170471709 11 Verkhovsky AB Chaga OY Schaub S Svitkina TM Meister JJ Borisy GG Orientational order of the lamellipodial actin network as demonstrated in living motile cells Mol Biol Cell 2003 14 466774675 12 Banchereau J Steinman RM Dendritic cells and the control of immunity Nature 1998 392 2457252 quthoI 2004 204 460 469 468 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Kobayashi H Miura S Nagata H et al In situ demonstration of dendritic cell migration from rat intestine to mesenteric lymph nodes relationships to maturation and role of chemokines JLeukoc Biol 2004 75 4347442 Guermonprez P Valladeau J Zitvogel L Thery C Arnigorena S Antigen presentation and T cell stimulation by dendritic cells Annu Rev Immunol 2002 20 6217667 Small JV Stradal T Vignal E Rottner K The lamellipodium where motility begins Trends Cell Biol 2002 12 1127120 Cramer LP Organization and polarity of actin lament networks in cells implications for the mechanism of myosinibased cell motility Biochem Soc Symp 1999 65 1737205 Adams JC Regulation of protrusive and contractile cellimatrix contacs J Cell Sci 2002 115 2577265 Evans JG Correia l Krasavina 0 Wagon N Matsudaira P Macrophage podosomes assemble at the leading lamella by growth and fragmentation J Cell Biol 2003 161 697 Destaing O Saltel F Geminard JC Jurdic P Bard F Podosomes display actin turnover and dynamic selfrorganization in osteoclass expressing actinrgreen uorescent protein Mol Biol Cell 2003 14 407 7416 Pfaff M Jurdic P Podosomes in osteoclastilike cells structural analysis and cooperative roles of paxillin prolinerrich tyrosine kinase 2 Pyk2 and integrin alphaVbeta3 J Cell Sci 2001 114 277572786 Ochoa GC Slepnev VI Neff L et al A functional link between dynamin and the actin cytoskeleton at podosomes J Cell Biol 2000 150 F3777F390 Babb SG Masudaira P Sato M Correia l Lim SS Fimbrin in podosomes of monocyteiderived osteoclass Cell Motil Cytoskeleton 1997 37 3087325 Carragher NO Frame MC Focal adhesion and actin dynamics a place where kinases and proteases meet to promote invasion Trends Cell Biol 2004 14 2417249 Burns S Hardy SJ Buddle J Yong KL Jones GE Thrasher AJ Maturation of DC is associated with changes in motile characteristics and adherence Cell Motil Cytoskeleton 2004 57 1 18 7132 Labno CM Lewis CM You D et al Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP Curr Biol 2003 13 161971624 Lorenz M Yamaguchi H Wang Y Singer RH Condeelis J Imaging sites of NiWASP activity in lamellipodia and invadopodia of carcinoma cells Curr Biol 2004 14 6977703 Binks M Jones GE Brickell PM Kinnon C Katz DR Thrasher AJ Intrinsic dendritic cell abnormalities in Vfrskotk Aldrich syndrome EurJImmunol 1998 28 325973267 Burns S Thrasher AJ Blundell MP Machesky L Jones GE Con guration of human dendritic cell cytoskeleton by Rho GTPases the WAS protein and differentiation Blood 2001 98 1 14271 149 Linder S Nelson D Weiss M Aepfelbacher M WiskottiAldrich syndrome protein regulates podosomes in primary human macrophages Proc Natl Acad Sci U SA 1999 96 964879653 Mizutani K Miki H He H Maruta H Takenawa T Essential role of neural WiskottiAldrich syndrome protein in podosome formation and degradation of extracellular matrix in srci transformed broblass Cancer Res 2002 62 6697674 Abram CL Seals DF Pass 1 et al The adaptor protein Fish associates with members of the ADAMs family and localizes to podosomes of Smrtransfor med cells J Biol Chem 2003 278 16844716851 Linder S Aepfelbacher M Podosomes adhesion hotispos of invasive cells Trends Cell Biol 2003 13 3767385 Kaverina l Stradal TEB Gimona M Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp237 dependent dernovo actin polymerization at discrete microdomains J Cell Sci 2003 116 491574924 Moreau V Tatin F Varon C Genot E Actin can reorganize into podosomes in aortic endothelial cells a process controlled by Cdc42 and RhoA Mol Cell Biol 2003 23 680976822 jPathoI 2004 204 460 469 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Y Calle et a Spinardi L Rietdorf J Nitsch L et al A dynamic podosomeilike structure of epithelial cells Exp Cell Res 2004 295 3607374 Derry J M Ochs HD Francke U Isolation of anovel gene mutated in WiskottiAldrich syndrome Cell 1994 78 6357644 Snapper SB Rosen FS The WiskottiAldrich syndrome protein NASP roles in signaling and cytoskeletal organization Annu Rev Immunol 1999 17 9057929 Takenawa T Miki H WASP and WAVE family proteins key molecules for rapid rearrangement of cor1ical actin laments and cell movement 7 Cell Sci 2001 114 180171809 Machesky LM Mullins RD Higgs HN et al Scar a WASpi related protein activates nucleation of actin laments by the Arp23 complex Proc Natl Acad Sci U SA 1999 96 373973744 Machesky LM Gould KL The Arp23 complex amultifunctional actin organizer Curr Opin Cell Biol 1999 11 1177121 Paunola E Mattila PK Lappalainen P WH2 domain a small versatile adapter for actin monomers FEBS Lett 2002 513 92797 Machesky LM Insall RH Scar1 and the related WiskottiAldrich syndrome protein WASP regulate the actin cytoskeleton through the Arp23 complex Curr Biol 1998 8 134771356 Mar neermles N Rohatgi R Anton lM et al WlP regulates N7 WASPimediated actin polymerization and lopodium formation Nature Cell Biol 2001 3 4847491 Anton lM de la Fuente MA Sims TN et al VJIP de ciency reveals a differential role for VJIP and the actin cytoskeleton in T and B cell activation Immunity 2002 16 1937204 Higgs HN Pollard TD Activation by Cdc42 and PIP2 of WiskottiAldrich syndrome protein VNASp stimulates actin nucleation by Arp23 complex J Cell Biol 2000 150 131171320 Rohatgi R Ho HY Kirschner MW Mechanism of NiWASP activation by CDC42 and phosphatidylinositol 457bisphosphate J Cell Biol 2000 150 129971310 Thrasher AJ WASP in immuneisystem organization and function Nature Rev Immunol 2002 2 6357646 Carlier MF Le Clainche C Wiesner S Pantaloni D Actinrbased motility from molecules to movement BioEsSays 2003 25 3367345 Weaver AM Young ME Lee W Cooper JA Integration of signals to the Arp23 complex Curr Opin Cell Biol 2003 15 23730 Millard TH Sharp SJ Machesky LM Signalling to actin assembly via the WASP NiskottiAldrich syndrome protein7family proteins and the Arp23 complex Biochem J 2004 380 1717 Aspenstrom P Lindberg U Hall A Two GTPases cdc42 and rac bind directly to a protein implicated in the immunode ciency disorder WiskottiAldrich syndrome Curr Biol 1996 6 70775 Rohatgi R Ma L Miki H et al The interaction between NiWASP and the Arp23 complex links Cdc42dependent signals to actin assembly Cell 1999 97 2217231 Allen WE Jones GE Pollard JW Ridley AJ Rho Rac and Cdc42 regulate actin organization and cell adhesion in macrophages J Cell Sci 1997 110 7077720 Allen WE Zicha D Ridley AJ Jones GE A role for Cdc42 in macrophage chemotaxis J Cell Biol 1998 141 114771157 Nobes CD Hall A Rho GTPases control polarity protrusion and adhesion during cell movement 7 Cell Biol 1999 144 123571244 EtiennerManneVille S Cdc42 i the centre of polarity J Cell Sci 2004 117 129171300 Price LS Leng J Schwartz MA Bokoch GM Activation of Rac and Cdc42 by integrins mediates cell spreading Mol Biol Cell 1998 9 186371871 EtiennerManneVille S Hall A Rho GTPases in cell biology Nature 2002 420 6297635 Benard V Bohl BP Bokoch GM Characterization of rac and cdc42 activation in chemoattractantistimulated human neutrophils using a novel assay for active GTPases J Biol Chem 1999 274 13 198713 204 Kurokawa K Itoh RE Yoshizaki WASP and the cytoskeleton 61 62 63 64 65 66 67 68 69 70 71 72 73 74 Baba Y Nonoyama S Masushita M et al Involvement of WiskottiAldrich syndrome protein in Bcell cytoplasmic tyrosine kinase pathway Blood 1999 93 200372012 Gross BS Wilde JI Quek L Chapel H Nelson DL Watson SP Regulation and function of WASp in platelets by the collagen receptor glycoprotein VI Blood 1999 94 416674176 Cote JF Chung PL Theberge JF et al PSTPIP is a substrate of PTPiPEST and serves as a scaffold guiding PTPiPEST toward a speci c dephosphorylation of WASP J Biol Chem 2002 277 297372986 Cory GOC Garg R Cramer R Ridley AJ Phosphorylation of tyrosine 291 enhances the ability of WASp to stimulate actin polymerization and lopodium formation J Biol Chem 2002 277 45115745121 Suesugu S Hattori M Miki H et al Sustained activation of N7 WASP through phosphorylation is essential for neurite extension Dev Cell 2002 3 6457658 Devriendt K Kim AS Mathijs G et al Constitutively activating mutation in WASP causes Xrljnked severe congenital neutropenia Nature Genet 2001 27 3137317 Kim AS Kakalis LT AbdulrManan N Liu GA Rosen MK Autoinhibition and activation mechanisms of the WiskottiAldrich syndrome protein Nature 2000 404 1517158 Lacout C Haddad E Sabri S et al A defect in hematopoietic stem cell migration explains the nonrandom Xichromosome inactivation in carriers of WiskottiAldrich syndrome Blood 2003 102 128271289 Zhang J Shehabeldin A da Cruz LA et al Antigen receptor induced activation and cytoskeletal rearrangement are impaired in WiskottiAldrich syndrome proteinrde cient lymphocytes J Exp Med 1999 190 132971342 Lorenzi R Brickell PM Katz DR Kinnon C Thrasher AJ WiskottiAldrich syndrome protein is necessary for ef cient IgGi mediated phagocytosis Blood 2000 95 294372946 Klein C Nguyen D Liu CH et al Gene therapy for Wiskotti Aldrich syndrome rescue of Tcell signaling and amelioration of colitis upon transplantation of retrovirally transduced hematopoietic stem cells in mice Blood 2003 101 215972166 Pivniouk VI Snapper SB Kettner A et al Impaired signaling via the highraf nity IgE receptor in WiskottiAldrich syndrome proteinrde cient mast cells Int Immunol 2003 15 143171440 Snapper SB Rosen FS Mizoguchi E et al WiskottiAldrich syndrome proteinrde cient mice reveal a role for WASP in T but not B cell activation Immunity 1998 9 81791 Jones GE Zicha D Dunn GA Blundell M Thrasher A Restorai tion of podosomes and chemotaxis in WiskottiAldrich syn 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 469 drome macrophages following induced expression of WASp Int J Biochem Cell Biol 2002 34 8067815 Calle Y Jones GE Jagger C et al WASp de ciency in mice resuls in failure to form osteoclast sealing zones and defecs in bone resorption Blood 2004 103 35273561 Cram EJ Schwarzbauer JE The talin wa the dog new insighs into integrin activation Trends Cell Biol 2004 14 55757 Mar anontecha A Sebastiani S Hopken UE et al Regulation of dendritic cell migration to the draining lymph node impact on T lymphocyte traf c and priming J Exp Med 2003 198 6157621 Jones GE Prigmore E Calvez R et al Requirement for PI 37 kinase gamma in macrophage migration to MCP71 and CSF71 Exp Cell Res 2003 290 1207131 Ward ME Wu JY Rao Y Visualization of spatially and temporally regulated NiWASP activity during cytoskeletal reorganization in living cells Proc Natl Acad Sci U S A 2004 101 9707974 Iijima M Huang YE Devreotes P Temporal and spatial regula7 tion of chemotaxis Dev Cell 2002 3 4697478 Raftopoulou M Hall A Cell migration Rho GTPases lead the way Dev Biol 2004 265 23732 Rickert P Weiner OD Wang F Bourne HR Servant G Leukoi cytes navigate by compass roles of PI3Kgamma and is lipid producs Trends Cell Biol 2000 10 4667473 Vanhaesebroeck B Jones GE Allen WE et al Distinct PI3Ks mediate mitogenic signalling and cell migration in macrophages Nature Cell Biol 1999 1 69771 Nobes CD Hall A Rho Rac and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress bers lamellipodia and lopodia Cell 1995 81 53762 Franz CM Jones GE Ridley AJ Cell migration in development and disease Dev Cell 2002 2 1537158 Thrasher AJ Burns S Lorenzi R Jones GE The WiskottiAldrich syndrome disordered actin dynamics in haematopoietic cele Immunol Rev 2000 178 1187128 Zicha D Allen WE Brickell PM et al Chemotaxis of macrophai ges is abolished in the VfiskottiAldrich syndrome Br J Haematol 1998 101 6597665 Linder S Higgs H Hufner K Schwarz K Pannicke U Aepfeli bacher M The polarization defect of VfiskottiAldiich syndrome macrophages is linked to dislocalization of the Arp23 complex J Immunol 2000 165 2217225 Yan C Mar neeruiles N Eden S et al WAVE2 de ciency reveals distinct roles in embryogenesis and Racimediated actin based motility EMBO J 2003 22 360273612 quthoI 2004 204 460 469
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