Dynamic Coupling of ALCAM to the Actin Cortex Strengthens Cell

cecs abu nvriyMdclCne,GetGotpen2,62 GA 6525 28, Grooteplein Netherlands. Geert The Center, Nijmegen, Medical University Radboud Sciences, os eRiet cell te Joost strengthens cortex actin CD6 the to to adhesion ALCAM of coupling Dynamic ARTICLE RESEARCH ß eevd2 uut21;Acpe aur 2014 January 4 Accepted 2013; August 20 attributed. Received properly is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This [email protected]) ([email protected]; correspondence for *Authors Zu ETH Engineering, mechanotransduction 1 called different process initiate applied 2012). to a Gardel, externally cell and (Schwarz the transduce signals; into integrins, cytoskeleton biochemical the and through cadherins forces CAMs, Several as 2012). Gardel, such and (Schwarz 2003). with cytoskeleton interactions Springer, actin through and adhesionthe affinity and/or (Carman avidity of CAM membrane regulate plasma Cells (avidity) level, the distribution expression in Cell the receptors and on motility. depends (affinity) and cell conformation cohesion, regulated and tightly facilitating is signaling by adhesion organization transmembrane tissue play development, in and attachment roles cell mediate important (CAMs) molecules adhesion Cell INTRODUCTION synapse, tethers Immunological Membrane adhesion, Cell CD6, force ALCAM, Atomic microscopy, spectroscopy, force actin Single-cell synapse. the WORDS: immunological KEY and the at CD6 adhesion between DC–T-cell cell link strengthen stabilize mechanical to cortex a adhesion, cortex how form DC–T-cell for and the framework contacts to stiffens a propose proteins contribute We adaptor ALCAMs adhesion. ALCAM through cell linking strengthens cortex and Furthermore, actin interactions. the to the cytoskeletal depend and tethers actin membrane sites plasma adhesion on anchor to to ALCAM ALCAM of of cortex. propensity recruitment actin by the the to influenced ALCAM contrast, not of By domains is intracellular bonds the of ALCAM–CD6 linking of the certain affinity processes the with analysis that SCFS constructs reveal improved and ALCAM mutations tail expressing cytoplasmic cells fluorescence The of adhesion. force reflection cell combination ALCAM–CD6-mediated single-cell total-internal examine combine to with microscopy we Here, (SCFS) and stress. spectroscopy resist mechanical bonds contacts. CD6 ALCAM–CD6 to DC–T-cell cell how and sustained adapt about (DC) known to leukocyte is contribute cell little cell activated However, dendritic T a a the on on molecules synapse, (ALCAM) molecule immunological adhesion the At ABSTRACT eateto uo muooy abu nttt o oeua Life Molecular for Institute Radboud Immunology, Tumor of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,19–66doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1,2, ¨ ih atntas 6 08Bsl Switzerland. Basel, 4058 26, Mattenstrasse rich, ,JneHelenius Jonne *, 2 eateto issesSineand Science Biosystems of Department 2 ioStrohmeyer Nico , 2 lsadaCambi Alessandra , n xeinesersrse of stresses shear experience and h ebaepoia ieadaKE etd oi at motif peptide KTEA a at and (PCRD) site domain proximal positive-charge-rich membrane of tail a the cytoplasmic short contains The al., Although 2010). with al., et ALCAM proteins, et 2004). adaptor (Fehon (Zimmerman via cortex al., interact, actin site the can et tail actin-binding cytoplasmic direct its Zimmerman 2004), a 2000; lacks adhesion al., al., ALCAM cell et strengthens et Riet cytoskeleton D, te using (Nelissen cytochalasin by actin 2004; of cytoskeleton, actin concentrations al., the low the et of activation Zimmerman example, For and 2000; 2007). al., ALCAM et (Nelissen between interactions remain synapse investigated. immunological be the to at DCs adhesion which ALCAM by regulate mechanisms that molecular the hypothesis antigen-recognition However, resisting the onwards. the from step by adhesion to facilitating contacts and led stress DC–T-cell shear have stabilize 2008), bonds al., ALCAM–CD6 force et (SCFS) atomic (Helenius spectroscopy 2004; using assays force and by single-cell insights adhesion al., (AFM)-based bonds, These cell microscopy 2007). ALCAM–CD6 et strong al., of et facilitate Riet quantification (Hassan to (te stress and mechanical stimulation 2006) whereas under al., DC–T-cell cells, needed et are Zimmerman T optimal and contacts and cell–cell for initial, DCs prolong the bonds Dustin, of to ALCAM–CD6 2006; contribute adhesion al., mainly et antigen-independent bonds (Zimmerman ICAM-1–LFA-1 CD6 2007). and an ALCAM function-associated of between consisting by lymphocyte dimer encoded integrin chain an and (LFA-1, adhesion for intercellular antigen-1 (ICAM-1) the are essential between contacts form molecule-1 therefore, DC–T-cell that bonds that by is, show stabilized studies contacts. shear microscopy DC–T-cell confocal withstand maintaining and to establishing ability The ta. 04 utn 07.A h muooia yas,DCs synapse, immunological (Gimferrer the At synapse 2007). highly Dustin, the immunological 2004; to the al., recruited et of are CD6 interface DC–T- and Hassan establishing organized ALCAM 1995; Upon al., both 2006). al., et contact, et (Bowen cell 2010). Zimmerman activation 2004; al., cell al., T cells et et T during by Weidle role expressed a (CD6) see 6 play cells differentiation review of dendritic cluster by a with expressed (DCs) ALCAM for (van of tumors 2001, interactions of Heterotypic types al., many the et in 1995). of metastasis Kempen and al., protein associated migration et cell transmembrane are with (Bowen trans-interactions I ALCAM–ALCAM CAMs type Homotypic of a superfamily is immunoglobulin CD166) as known rsnigstsa peso pt 0.5 to up of that, speeds naı at indicate sites presenting tissue, antigen-specific studies an lymphoid microscopy start within thereby Intravital and response. cells immune T to antigens present elahso n iceia sasso dynamic show assays biochemical and adhesion Cell h ciae ekct elahso oeue(LA,also (ALCAM, molecule adhesion cell leukocyte activated The 1 alG Figdor G. Carl , ITGAL and v el cnDsfrantigen for DCs scan cells T ¨ve < ITGB2 .5N/m 0.05 1 m n ailJ Mu J. Daniel and epciey swl as well as respectively) , / Mme ta. 2004) al., et (Mempel m/s 2 Wofe l,2007). al., et (Woolf nvitro In a ller ¨ and 1595 and 2, b *

Journal of Cell Science up()eet] h eodtp fubnigeet cuswe ebaettesaeple u ftecl ihcaatrsi ogpaeu fcon of plateaux long characteristic [den with increases cell force the subst the of the as out from unbind pulled cell and are the cortex tethers of cell membrane separation the when to the occurs (T)-events]. anchored During events tether remain cell curve. unbinding un as The receptors force–distance cur (iii) of force. [denoted surface approach force–distance detachment sequentially type force cell the the This break second First, as of substrate (D). The occur. to line) and recorded referred events (J)-events]. dashed cell is is unbinding jump cantilever the (green curve molecular the between region force–distance of on formed contact exerted a types bonds the force two and increases, adhesive fitting D) cell maximum by in The the th determined (D) curve on or (iv). is strain retract separated b (blue) the red completely of arrows, ICAM-1-Fc As are arrows, force black (green), in signature. substrate red preset by GaHuFc openings and adhesion a by (denoted four (black), cell reaching (marked of approach a BSA until substrate one to the substrate i.e. the over the corresponds During coating, from onto cell S1B). control pressed retracted the Fig. a is is positioning material with cell by cell the supplementary contact probed D), see into is in quality brought cantilever curve (coating is ConA-coated approach (red) cell a CD6-Fc the on Subsequently, ligand-coating immobilized (i). cell ALCAM-specific glass K562 on a mask of adhesion PDMS The (C) significant). not EERHARTICLE RESEARCH 1596 combining ALCAM By express mutations. that strengthening tail cells cytoplasmic in specific K562 cytoskeleton with on constructs SCFS actin used the we of adhesion, this role the and adhesion A.C., and C.G.F. cell unclear. and ALCAM–CD6-mediated remains Kanger, ezrin adhesion strengthen ezrin proteins S. and adaptor with regulate J. the syntenin-1 Harkes, associate whether R. However, to unpublished). J.t.R., seems Tudor, al., ALCAM et ezrin- (C. Barreiro 1998; Specifically, al., the et (Yonemura S. 2002). CAMs whereas PCRD link J. the can PDZ-binding through unpublished), Harkes, actin proteins 2005; to adaptor R. of A.C., al., I family J.t.R., (ERM) et and Tudor, radixin-moesin Gimferrer adaptor class C. C.G.F. 1997; 2008; the Kanger, al., Coffer, the of and et domain Beekman (Grootjans recognizes PDZ KTEA motif The syntenin-1 1A). (Fig. protein C-terminus the f mutations (1 alanine coefficient and to Manders’ position threonine of proximal of means membrane positions by the the S2), at indicate Fig. PCRD material Asterisks the (supplementary site. shows microscopy binding confocal (bottom) PDZ-domain by ALCAM-WT the determined of is tail which cytoplasmic SCFS. C-terminus, the using the ALCAM- into by at zoom motif mutations A tail KTEA-binding (top). cytoplasmic the CD6 with of constructs domain ALCAM membrane-proximal of Analysis 1. Fig. oivsiaehwACMC6bnsetbihcell establish bonds ALCAM–CD6 how investigate To D h.ACMGIhsn yolsi albtaGIaco.()Clclzto ftedfeetACMcntut ihsnei- n zi,as ezrin, and syntenin-1 with constructs ALCAM different the of Colocalization (B) GPI-anchor. a but tail cytoplasmic no has ALCAM-GPI Thr. ebaettesfre.Atgte,tedt ugs a suggest data the Altogether, of number the formed. lowers and tethers adaptor substrate by the membrane with cortex contact tightens substrate, cellular actin the cortex. the to the actin recruitment ALCAM the to influences stiffens proteins ALCAM but affinity linking unlinked bond Furthermore, affect of not tethers. does ALCAM–CD6 membrane amount stressing bonds mechanically of that considerable formation show experiments the the The a to through linked adhered however, were ALCAMs ALCAMs cortex; most cell that actin ALCAM–CD6-mediated observed of mechanisms We regulation analyze molecular adhesion. the the to to contribute of procedures that ALCAM microscopy determination fluorescence allowed The new reflection (TIRFM) total-internal substrates. with and active data combination SCFS functionally interactions in mechanical with the constructs cell measuring the while of imaged cantilever AFM an be to attached could cells microscopy, optical and AFM < ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal N(i.Atrapee otc iernigfo . o10s the s, 120 to 0.5 from ranging time contact preset a After (ii). nN 2 A LA id oC6truhisotrI oant the to domain Ig outer its through CD6 to binds ALCAM (A) 5 ulclclzto n 0 and colocalization full 5 oe *** none; P , .01 n.s., 0.0001; i h cell the til or rate, stiffness tdas oted lack ve thin a e stant

Journal of Cell Science yokltn(eRe ta. 07.Hwvr hte ci linking actin whether However, 2007). al., actin et the Riet by (te regulated cytoskeleton is adhesion cell ALCAM–CD6 Heterotypic different capabilities with actin-linking constructs ALCAM of Characterization RESULTS cell ALCAM–CD6-mediated contacts. how DC–T-cell stabilizes for adhesion framework mechanical ARTICLE RESEARCH iadfrwihK6 el ontepesrcpos–was – receptors express not a do – cells albumin ICAM-1-Fc K562 serum to which bovine and for to (GaHuFc) ligand anti-human-Fc adhesion goat unspecific (BSA), probing of By strength (supplementary cell, times. same the the contact substrates with substrates ligand-coated predefined specific was various and for unspecific cantilever, S1A,B) with AFM Fig. contact K562 an material single to glass into a attached in adhesion, brought cell masks ALCAM, quantify PDMS expressing To four-segment cell 1C). (Fig. using discriminate dishes by improved Petri to assay we Therefore, SCFS necessary adhesion. ALCAM-mediated the cell was to examined and interactions it ALCAM–CD6-mediated unspecific specific we Hereto, of contribution different the CD6. linking, between show to constructs actin adhesion ALCAM of the degrees that observed Having different constructs of ALCAM adhesion cell ALCAM–CD6-mediated the proteins. Probing for cytoskeletal account with ALCAM levels of expression colocalization Thus, different of S1D). that lack Fig. exclude did material can cells (supplementary K562 we of ALCAM untransfected levels express whereas similar Fluorescence- not surface, expressed their constructs 2008). at ALCAM ALCAM transfected the cells Coffer, of K562 each that and showed with binding analysis influence sorting (Beekman would cell binding, activated PDZ syntenin-1 for and known membrane of the not to motif close not that a located unlikely in did is is which PCRD it T556A, Furthermore, mutation the 1B). the in PCRD Fig. (Fig. the binding mutation ezrin of material T556A influence charge the supplementary positive unaltered, the not 1B; remained because did (Fig. expected, ALCAM-GPI As ezrin microscopy whereas S2A,B). actin, with confocal or colocalize syntenin-1 Indeed, to ALCAM- either of bind ezrin. with colocalization would S. less nor ALCAM-GPI revealed J. syntenin-1 whereas Harkes, syntenin-1, neither 1A) R. (Fig. ALCAM- of in J.t.R., predicted binding T581A position we Tudor, mutation Therefore, the unpublished). C. membrane-proximal that A.C., 1998; and the C.G.F. al., Kanger, at et to binds ezrin (Yonemura cytoplasmic PCRD member the family Harkes, of ERM the the unpublished) R. whereas A.C., J.t.R., ALCAM, and of Tudor, tail C.G.F. C. Kanger, al., 2008; S. et Coffer, (Grootjans J. to site and PDZ-binding thought the Beekman is at syntenin-1 1997; motif protein KTEA the adaptor to The bind 1A). (ALCAM- Fig. domain (see cytoplasmic GPI) and ALCAM (ALCAM- transmembrane the of tail lacks that form PDZ- cytoplasmic the (GPI)-anchored in the residue is glycosylphosphatidylinositol alanine at which of to motif latter residue the binding T581A, threonine the and T556A two to (ALCAM- mutations, with linker fused 5-amino-acid ALCAM (eGFP) a GFP), protein using ALCAM-WT fluorescent C-terminus (ALCAM-WT), intracellular green constructs ALCAM These enhanced 1A). (WT) with (Fig. wild-type constructs transfected were stably ALCAM cells different K562 into non-adherent with insight used further we give process, To this unclear. remains ALCAM modulates D h ol nlec the influence would Thr D h rALCAM-GPI or Thr D h) n a and Thr), h aoiyo h neatosbtenACMepesn cells ALCAM-expressing CD6. thus, between and and, interactions force the ALCAM–CD6-mediated detachment of majority the the specific of 66–77% that for accounts adhesion conclude we Therefore, yfso h F a oteC-terminus. the unaffected to are tag similar GFP ALCAM the of showed fusion interactions by cells the dynamics, K562-ALCAM-WT and adhesion K562- and Because ALCAM-GPI 1). the ALCAM-GFP (Table ALCAM-WT of that than respectively, adhesion faster, showed force 2E) in (Fig. ALCAM- detachment express increase functions normalized that exponential time-dependent cells the single of Fitting with forces 2E). adhesion (Fig. detachment unspecific ALCAM the ALCAM- this of subtracted from we component component adhesion, specific cell the mediated describe unspecific be of to To adhesion forces their background. considered detachment we CD6, to lower specifically much showed 1.5 cells K562 5.4 with control cells K562-ALCAM-GPI and xrsigACMcntut htahr oC6 agn from cells ranging K562 CD6, of to forces adhere 2.24 detachment that the constructs ALCAM contrast, expressing By cells. adhesion background K562 the of (0.75 constitutes low force detachment were unspecific CD6 K562 the control to of and bound each substrates cells expressing control to 2B cells bound Fig. of constructs forces the 1D). ALCAM detachment cell, (Fig. the the determined that of was shows strength force adhesion detachment the maximum characterize To material S1E). supplementary 2A; Fig. K562 (Fig. cells of ALCAM–CD6-mediated ICAM-1- K562 untransfected those or and GaHuFc from Fc) (BSA, depth, substrates specific unspecific and to adhering shape cells in of Averaged both 2010). differ, interactions al., in curves et recorded (Friedrichs insight adhesive gives force–distance process were which de-adhesion of The substrate the shape into the and examined. S1C). 1D), (Fig. cell curves was force–distance the Fig. between substrates adhesion interactions material cell CD6-Fc-coated ALCAM–CD6-mediated supplementary to specific the 1C; Thereafter, (Fig. determined usrt otc einb IF.Atrbigi otc with contact in being cell– After the TIRFM. imaging by region while the this contact SCFS from into substrate AFM-based cell insight using K562-ALCAM-GFP gain substrate single To CD6 their a rearrange. and detached CAMs cortex we the actin process substrate, the a from to cell linkages a of detachment Upon CD6 membrane from of tethers unbinding one-by-one shows AFM-TIRFM a 4.7 reached 5.8 adhesion K562-ALCAM- with with CD6, cell adhered to the adhesion cells specific contact WT of of case the the s of In 120 plateau. plot after A that 2C). showed (Fig. (i.e. study time force ( detachment To contact mean the with types, 2007). cell increased all al., adhesion) times For contact s. et after 0.5–120 membrane constructs K562 spanning Riet ALCAM of plasma different forces te detachment expressing the cells the is measured 2004; we cytoskeleton at dynamics, al., actin ALCAM movement the et ALCAM, receptor the (Zimmerman of to, in move case or the at, implicated be In must site. CAMs adhesion contact cell to contribute To avidity ALCAM–CD6-mediated of the dynamics reveal forces detachment Contact-time-dependent 6 6 . N eas nrnfce 52clscno adhere cannot cells K562 untransfected Because nN. 0.1 .4n o3.28 to nN 0.84 6 ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal 6 ... eahetfre vrcnattm Fg 2D) (Fig. time contact over forces detachment s.e.m.) D . N K562-ALCAM- nN, 0.4 h oteC6sbtaews28ad18times 1.8 and 2.8 was substrate CD6 the to Thr 6 .1n,wr infcnl higher. significantly were nN, 1.21 6 . N 52ACMGPcells K562-ALCAM-GFP nN, 0.3 D 6 h el ih6.4 with cells Thr .4n;mean nN; 0.34 6 . N ncomparison, In nN. 0.3 6 ...This s.d.). 6 . nN 0.4 1597

Journal of Cell Science otesbtae hra h elbd eaae esof tens extensions fluorescent separated body, revealed cell body movies the of TIRFM cell unbinding The the the after substrate. that the whereas from and substrate, micrometers attached curve partially the remained force–time cell 3B; to the The (Fig. that a showed 1). ms/image by analyses 200 TIRFM Movie and at 3A) material images (Fig. TIRFM curve supplementary of force–time series a in continuous recorded was process EERHARTICLE RESEARCH 1598 the 100 retracting of upon distance detached a was over cell s the 20 s, for 5 cantilever for substrate the m .Ti detachment This m. ni h odrpue,tette ihrw otecl oyand body extended cell the to are withdraws the tether tethers the to ruptures, membrane bond the force the to the until 3). outward tether Consequently, membrane 2, an the substrate. applies Movie anchor on which cantilever material bonds, structures ALCAM–CD6 the filamentous supplementary as of 3C; observed Krieg (Fig. Retraction be 2008; substrate can al., and the substrate et 2008) (Helenius the al., tethers et to These membrane 1–3). attached are Movies material extensions remained (supplementary seconds membrane several for plasma the of ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal N (left, ALCAM-WT expressing of cells curves force–distance Averaged time. (A) contact on cellular force the detachment of Dependence 2. Fig. (right, r-P rcnrlK6 el ( cells K562 control or -GPI or - -GFP, of ALCAM-WT, forces expressing detachment cells the Box- of (B) plots adhesion. whisker wider e.g. and – – stronger deeper show CD6-Fc from ALCAM-WT cells of detachment the detecting curves force–distance shape, specific in whereas similar very look and ICAM-1-Fc) GaHuFc (BSA, curves Control force–distance averaged. and their force at maximum aligned Force– were substrates. curves the distance of each on the cells of characteristics detachment the different highlight curves The substrates. (blue) ICAM-1-Fc-coated GaHuFc or (black), (green) BSA (red), CD6-Fc to niae h 57%qatls h center the quartiles, 25–75% the indicates *** condition. each for analyzed curves force–distance the of give number brackets angle in Numbers substrates. BSA, ICAM-1-Fc-coated CD6-Fc, or with GaHuFc time contact s 10 xrce rmta hw nC. in was shown E that and from D extracted in Data 1). CD6 Table to (see cells ALCAM-expressing of binding rates extracts curves) (dashed the Fitting data CD6. to adhering and ALCAM constructs different expressing cells of K562 forces detachment the Normalized to (E) fits data. exponential single Dashed show plateau. curves a approach contact and with time increase CD6 from cells K562 ( increases. Mean cells (D) K562 control or -GPI - -GFP, cells ALCAM-WT, single expressing of force detachment substrates, the CD6-Fc-coated to increasing time With contact (C) mean. square the the represents and median the represents line FD § 52, N FD N § 6 C 34, 5 ... eahetfre of forces detachment s.e.m.) 3 rcnrlK6 cells K562 control or 13) N C 5 0 deigfr1 s 10 for adhering 10) P , .01 h box The 0.0001. N C D § h or Thr 0 after 10) D Thr

Journal of Cell Science ist h aagvni i.2 n i.4 eutdi xoeta ieconstants, time exponential in resulted 4C Fig. and 2E Fig. in given data the to Fits oto 52cls15.8 cells K562 Control 52ACMGI14.7 K562-ALCAM-GPI al .Rtso nraei eahetfreadtteigrtsfrtedfeetACMcntut xrse nK6 cells K562 in expressed constructs ALCAM different the for rates line tethering cell and ALCAM-expressing force detachment in increase of Rates 1. Table ARTICLE RESEARCH CS(i.1)rpeetteubnigo Asta nhrthe anchor that CAMs by of recorded unbinding T-events the represent whether 1D) debated bleached signal- (Fig. been low SCFS including has a It and lacking reasons, ratio. sensitivity be to-noise optical several insufficient might for molecules, curves GFP images force–distance TIRFM of S3). in from Fig. material detachment events supplementary the some 3A,B; by Unbinding the Fig. (indicated 3B), in images to (Fig. arrows TIRFM substrate numbered recorded the attributed curve simultaneously from the in be force–time tethers membrane can the (T)- ALCAM-GFP-containing tether events In a indicating unbinding 1D). drops (Fig. cantilever the event to applied force the a K562-ALCAM- 52ACMGP41.2 K562-ALCAM-GFP 52ACMW 41.5 K562-ALCAM-WT aefrcnrlK6 el rmFg D eut r mean are Results 2D. Fig. from cells K562 control for Rate D h 23.6 Thr t dtcmn force) (detachment K (s) 6 6 6 6 6 . .8 21.9 0.981 2.5 . .7 12.7 0.970 2.9 . .9 18.7 0.992 2.5 . .7 39.3 0.979 8.7 0809845.6 0.968 10.8 6 s.d. a t obndSF n IF aaso htti s ned the indeed, is, The this 2008). that show al., data et case. TIRFM (Helenius and substrate SCFS the combined to tethers membrane h ci yokltnitrcin ntte yais When dynamics. tether of on influence interactions the in of in cytoskeleton interested tens mechanism actin were of adhesion we the particular, this distances In study over detail. to CD6 more us to prompted adhere micrometers to tethers exploit K562-ALCAM-GFP membrane expressing cells that observation The tethering modulate membrane linking ALCAM-mediated cytoskeletal and time Adhesion K o h ifrn LA osrcswt nacrc ie yadjusted by given accuracy an with constructs ALCAM different the for , ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal R 2 otc rjs a otcnat epciey with respectively, loses contact, substrate. body lost the cellular has the just that or moment contact the and at (top) (bottom) 2 3 Movie Movie False-color material (C) supplementary arrows. from numbered images the by force–time indicated and as images curve be TIRFM can in tethers observed of simultaneously the release from The cell substrate. ALCAM-GFP CD6-Fc the images retracting TIRFM while of recorded marked Selection events (B) unbinding arrows). detail of (numbered in subset shown a is with rectangle) (bottom), where (gray curve occur force–time events the unbinding of section TIRFM The of stream images. continuous expressing a cell recording K562 while a ALCAM-GFP withdrawal of and substrate to CD6-Fc-coated approach the from the upon recorded curves (red) cell to tethers events. individual adhesion of TIRFM unbinding time-lapse the with correlates SCFS Combining 3. Fig. (tethering) t K (s) 6 6 6 6 6 A oc–ie(lc)adheight–time and (black) Force–time (A) . 0.989 2.8 . 0.968 2.6 . 0.906 6.9 . 0.985 6.7 080.976 10.8 R 2 1599 R 2 .

Journal of Cell Science eahetfre(i.1) o 52clsepesn the expressing increased cells T-events of K562 number For the constructs, 1D). ALCAM (Fig. T- different of force number occurred detachment by the main that counted substrate the we separating events Accordingly, the after tethers. recorded from membrane force that they the body assumed of body we cell all, Therefore, cell cortex. not the actin if the from most, to far bound be displaced cannot are molecules ALCAM ARTICLE RESEARCH 1600 that contact cells of K562 s for 10 present after were Strikingly, T-events 4A). more (Fig. considerably time contact with . 10 m fe ecigtemaximum the reaching after m . 10 m sgnrtdby generated is m LA bonds. ALCAM cells), < ALCAM-GFP that or implies the WT tethering background background ICAM-1-Fc), of a level suggesting This and lower, 2.8 around much GaHuFc of was level (BSA, T-events of substrates number control all (5.6 - ALCAM-WT or expressing cells with xrse LA-P (14.1 ALCAM-GPI expressed 0 ACMGIcls fteTeet r eitdby mediated are T-events the of cells) (ALCAM-GPI 80% D h (7.4 Thr ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal 6 .) o nrnfce 52cls swl sfor as well as cells, K562 untransfected For 0.7). 6 . ( 2.4 6 .. -vnsprrtato Fg 4B). (Fig. retraction per T-events s.d.) o 52clsepesn ALCAM. observed expressing is cells shift K562 a for panel) right s, 50 of 120 data and (merged times contact cells. long K562 For control for and ALCAM-GPI or ALCAM- ALCAM-GFP, expressing ALCAM-WT, cells K562 similar for s show distributions 5 panel) and left 2 time, 0.5, contact of data contact (merged short times at T-events tether from of forces Histograms A (E) in merged. displayed are set All data (right). s the 120 from or T-events (left) 2 of for times occur contact T-events which position at the (distance) showing curves 1). Table Decay in (D) given are (dashed values fit lines, exponential single a is with and fitted time contact per on T-events depends of retraction s. number 10 mean of The time (C) contact a after ICAM-1- substrates or Fc GaHuFc cell BSA, a CD6-Fc, of from retraction per detected events LA cnrl rmC6F substrates ( CD6-Fc from (control) ALCAM ALCAM- GFP, ALCAM- cell ALCAM-WT, K562 either a expressing of retraction per T-events observed of plots Box-whisker adhesion. (A) cell in tethers of membrane formation the modulates of ALCAM assembly Intracellular 4. Fig. en B ennme ( number Mean the (B) represents mean. square the the and represents median line center the 25–75% quartiles, the indicates box The curves analyzed). force–distance of number the N < C 6 § 1 (ALCAM- 61% .;mean 1.1; 0 ubr ie nbakt show brackets in given numbers 10, 6 D h,ACMGIo no or ALCAM-GPI Thr, .) GP(5.2 -GFP 0.5), 6 ... compared s.e.m.) < D 2 (ALCAM- 52% h el)and cells) Thr 6 ... fT- of s.e.m.) 6 D Thr 0.4)

Journal of Cell Science 00 a ta. 03.Teeoe eeiiecl adhesion cell repetitive Therefore, 2013). events. uncorrelated al., as treated al., were et et measurements Friedrichs 2007; Dao of al., memory et 2010; no (Zarnitsyna shows events adhesion between adhesion cell used previous the are cycles, times on adhesion delay effect sequential when no that had demonstrates times This Fig. contact S4A). material than (supplementary longer forces detachment or with ALCAM-specific to substrate equal a from times ALCAM-GPI delay expressing cell repeated Moreover, a 4E). of (Fig. detachment pN 50 and 25 between ranging forces ue efe ftemmrn Cose l,20)o single a or 2005) al., et membrane substrate. the (Cross from forces 2000) requires the al., membrane et (Oesterhelt from the domain the the transmembrane of in cell resides leaflet from that the molecule outer GPI-anchored of extracted a extracting detachment However, are during membrane ALCAMs transmembrane-anchored measurements. adhesion cell contributed our distribution to ALCAM in differences that possibility excluded the we Therefore, S2C,D). Fig. material (supplementary CD9 EERHARTICLE RESEARCH rpriso h lsammrn n ci otx n the plasma and the the cortex, on actin of depends and out membrane force pulled plasma tether are the The they of 2001). as properties (Sheetz, force maintain constant membrane they because near clamps force a native are bond tethers ALCAM–CD6 Membrane the of kinetics Unbinding of microdomains in distributions confocal the of ( resolution the microscope at imaged when et However, (Gilsanz the 2013). ALCAM al., of through influence domain Ig membrane extracellular also plasma the with the might interaction at CD9 ALCAM of member micro-organization family 2009). tetraspanin al., et Zanten The (van domains lipid GPI-enriched the plasma in avidity because reside in expected they changing difference be the might a by ALCAMs particular, GPI-anchored on CD6 of In distribution to formation. ALCAM tether adhesion membrane of cell and alter distribution and the membrane which influence linking, actin might of degrees modulating different the show by However, constructs system adhesion. ALCAM background the cellular of and levels complexity expression the reduced non-adherent cells in constructs K562 ALCAM different of expression Stable influence adhesion contacts cell repetitive nor distribution ALCAM Neither oc n h ubro ehr e elwsfound was increase which cell CD6, to per time. the bound contact to cell–substrate molecules correlate tethers with force ALCAM detachment of and of that tethers number conclude of number we number the Therefore, both S4B). the part Fig. material minor (supplementary detachment and cell a between correlation weak only force a ALCAM adhesion, contributes cell the total 1). tethering to of (Table force membrane each detachment of expressing rates Although the number cells to the similar for were which constructs at increased rates tethers exponential the single increasing of Surprisingly, with at 4C). fitted tethers (Fig. was of substrate functions tethering the number how be with the determine time can To time, contact that 1B). contact 2000) (Fig. with cortex al., develops An actin et ALCAM the (Nelissen constructs. to GPI-anchored domain linked the ALCAM cytoplasmic that other a is lacks behavior the this Cells expressing for T- 4C). explanation many cells (Fig. as substrate as twice CD6 almost events the had with ALCAM GPI-anchored cell expressing the of time contact tmgtb pcltdta P-nhrdo single or GPI-anchored that speculated be might It et epotdtema ubro -vnsaantthe against T-events of number mean the plotted we Next, < . 0.25 0 N hc r ihrta h vrg tether average the than higher are which pN, 100 m ) l LA osrcssoe similar showed constructs ALCAM all m), < 0.2 m m 2 n ooaie with colocalized and yolsi alde o le h fiiy(nidn ae fthe of rate) the (unbinding through CD6. affinity data for cortex the domain our actin alter ALCAM the 4E), extracellular not to Fig. does ALCAM and tail of 2 cytoplasmic linking (Table the forces that unbinding shows similar unbinding contact-time- identical at near that the thus, rates on and, based speculate properties Nevertheless, forces. cortex tether time alter we the actin cortical constructs, contact in expressing changes ALCAM dependent cells increasing for other with However, the S4C). force prominent Fig. second material a became (supplementary by For indicated which is increase. tethers peak, this simultaneous membrane two the explain of of cells, unbinding not unbinding K562-ALCAM-GPI simultaneous does stronger-tethering The the tethers trends). membrane for supplementary control multiple (see S4C contact- forces the Fig. marked tether of a material the was of exception there increase cells, the time-dependent ALCAM-GPI and With cells cells. K562 K562 untransfected D 45.8 to 53.4 increased and force tether transmembrane anchor mean the that of bonds domain. independent ALCAM–CD6 ALCAM is of intracellular tethers rate have unbinding membrane cells means this K562 the further, step from ALCAM that one Taken pulled the properties. of tethers mechanical similar independent membrane that, the suggest construct, forces tether similar entte oc o nrnfce el a 29.1 was cells untransfected for force tether mean D ihteC6sbtae h ea uvswr itdwith resulting fitted The were 4D). curves (Fig. ( decay functions constants the decay decay times contact substrate, exponential s) (120 CD6 simple determine long and To the s) tethers. (2 with the short after anchor rate that unbinding the the bond(s) depict of T-events curves decay rate of of These unbinding kinetics number 4D). unbinding (Fig. mean distances the the pulling against analyze plotted we To 2008). bonds, the ALCAM–CD6 al., of et the properties of (Krieg unbinding strength the bond the reflects on 2008). and depends al., bond tether et receptor–ligand the Krieg the of 1996; anchoring lifetime al., bond the et the However, (Hochmuth on substrate not the but to pulled, tether are they which at speed ods,fo hc h eircldsrbsteubnigrate unbinding the describes reciprocal ( the which from bond(s), (5 ftette nrae EasadClewo,2007). lifetime Calderwood, the and tether, (Evans the membrane s increases to a 120 tether of the anchored at load of the are whereas share If tethers tethers. bonds bond, membrane multiple most single ALCAM–CD6 anchor s, bonds one ALCAM–CD6 2 multiple only of because, by be time large might substrate This as contact 2). twice a (Table almost s being after 120 time after contact determined those of as s 2 after determined osrcshv h aeubnigrt fmmrn tethers, membrane of rate unbinding same k the have constructs qiiru ( equilibrium o 52clsepesn LA-To 34.7 of ALCAM-WT showed expressing forces s) tether (0.5–5 cells mean with times (maximum K562 panel) contact left tether for 4E, short of (Fig. peaks Histograms after overlapping examined. recorded were rate 1978). (Bell, forces unbinding forces rate) the loading tether force unbinding, (i.e. Therefore, force induced applied force the on of depends model Bell the o el xrsigec fteACMcntut ihthe with constructs ALCAM the of each expressing cells for k off h f37.4 of Thr h,40.6 Thr, off fe ogcnattms(010s i.4,rgtpnl,the panel), right 4E, Fig. s; (50–120 times contact long After h idn htteclsepesn aho h ALCAM the of each expressing cells the that finding The m osntdcaeta ntesddsoito ae at rates dissociation unstressed that dictate not does , al ) tiigy efudams qa nidn rates unbinding equal almost found we Strikingly, 2). Table , /)t eeltema ieieo h ALCAM–CD6 the of lifetime mean the reveal to m/s) 6 21p o LA-F,46.4 ALCAM-GFP, for pN 12.1 ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal 6 6 ..,ACMGPo 41.8 of ALCAM-GFP s.d.), k 32p o LA-P n 31.3 and ALCAM-GPI for pN 13.2 0 6 off . N LA-P f36.4 of ALCAM-GPI pN, 9.8 r lotesm eas,a tpltdby stipulated as because, same the also are ) d K eedvddb h ercinspeed retraction the by divided were ) 6 68p o ALCAM-WT, for pN 16.8 6 52p o ALCAM- for pN 15.2 6 07p,ALCAM- pN, 10.7 6 6 26p.The pN. 12.6 6 . N The pN. 9.9 . Nfor pN 8.6 6 . pN 9.1 1601 k off

Journal of Cell Science otettlahso ftecl (mean cell the of adhesion total the to 14.1% i otedt ie nFg Dalwdt prahteepnnilconstants, exponential the approach to allowed 4D Fig. in given data the to fit A ytema ehrfre h rdc a oprdwt the with compared accounted was tethers membrane 16.3% product that for The showing force force. detachment tether multiplied total and mean fits the exponential the by from extrapolated were force) oto 52cls21.3 cells K562 Control al .Dcyrt n iscaincntnsdtrie rmtte itiuinplots distribution tether from determined line constants cell dissociation ALCAM-expressing and rate Decay 2. Table ARTICLE RESEARCH 1602 binding. CD6 upon stiffness cell modulates and adhesion cell ALCAM- ALCAM-GFP, strengthens ALCAM-WT, expressing cortex actin cells the K562 to binding ALCAM 5. Fig. of number The 0 substrate. distance to and at tethers tethers cell force tethers membrane membrane between detachment of of formed mean contribution number adhesion the the the the estimated and we 4E), 4D) 2C), (Fig. (Fig. (Fig. force retraction per tether events mean the Using to tethers adhesion membrane cell ALCAM-mediated of contribution The 52ACMGI10.2 K562-ALCAM-GPI K562-ALCAM- eemn h nidn rates, unbinding the determine 57%qatls h etrln ersnstemda n h qaerpeet h mean. the represents square the *** cells. and K562 median control the represents and ALCAM-GPI line expressing center cells the with quartiles, contrast 25–75% in CD6 with contact after significantly 52ACMGP10.1 K562-ALCAM-GFP ih uvsbfr is otc ihC6F n ubr61 hratr 52cls( cells force– K562 approach thereafter. the 6–12 of number region and contact CD6-Fc the fitting with linearly contact by first extracted deformation) before to curves resistance (i.e. ALCAM-eight stiffness expressing cell cells the K562 of for plots Box-whisker same forc retraction (C) averaged the expres the nearly cells of stays for – elasticity it region whereas The contact e.g. CD6, (B) – peak. ( slope detachment elasticity linear The maximum S5B. the the Fig. fitting material supplementary by on in presented measured aligned was were times contact curves different force–distance averaging, at constructs For ALCAM different time. contact s 50 of brackets) 52ACMW 10.3 K562-ALCAM-WT 6 . o ALCAM- for 1.1 6 . o LA-T 14.8% ALCAM-WT, for 2.6 D h 10.8 Thr D k h n 27.9% and Thr off fteC6seii od omdb ahACMcntut(e et.Rslsaemean are Results text). (see construct ALCAM each by formed bonds CD6-specific the of , m ie tmxmmdetachment maximum at (i.e. m d K ( 6 6 6 6 6 m )a s 2 at m) . 0.234 2.7 . 0.489 0.1 . 0.462 0.1 . 0.496 0.2 . 0.487 0.2 6 6 6 . o ALCAM-GFP, for 1.6 ..,idpnetof independent s.d.), . o ALCAM-GPI for 0.5 D h rACMGI( ALCAM-GPI or Thr 6 ... fK6 el xrsigACMWadACMGPicessuo ogrcnattm with time contact longer upon increases ALCAM-GFP ALCAM-WTand expressing cells K562 of s.e.m.) D h rACMGI(ihnarneo – nN/ 1–2 of range a (within ALCAM-GPI or Thr k off N (s C d § 2 6 6 6 6 6 K otc ie hs ehr otiuecnieal oALCAM– to adhesion. considerably cell contribute CD6-mediated tethers Thus, time. contact fteaeae oc–itnecre(i.5) eod the Second, 5A). (Fig. curve of force–distance for comparison averaged 5A; cells the (Fig. First, of at K562-ALCAM-WT averaged S5A). aligned with distances and were Fig. cells times force K562-ALCAM-GPI material contact force–distance detachment and supplementary features, maximum constructs common force– all their the reveal K562 of of the To curves construct shape ALCAM expressed. the which cells on that depends showed curves distance experiments cortex SCFS actin the Our modulates and attaches ALCAM 1 .3 19.1 0.030 .0 18.3 0.004 .0 19.1 0.006 .1 15.8 0.011 .1 17.2 0.010 fdfeetACMcntut.Teeepnnilcntnswr sdto used were constants exponential These constructs. ALCAM different of , 0 eegnrtdb averaging by generated were 10) )at2s N C § 0 xrsigACMW,ACMGPo ALCAM- or ALCAM-GFP ALCAM-WT, expressing 10) ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal . 7.5 m d hwdhwtteigifune h shape the influenced tethering how showed m K P , ( 6 6 6 6 6 m .01 .. o infcn.Tebxidctsthe indicates box The significant. not n.s., 0.0001; )a 2 s 120 at m) . 0.261 0.2 . 0.273 0.1 . 0.262 0.1 . 0.317 0.1 . 0.290 0.1 N FD m ,idctdb h rysae area). shaded gray the by indicated m, § A vrgdfredsac uvsof curves force–distance Averaged (A) 2(xc ubr r ie nangle in given are numbers (exact 52 6 k s.e.m. off (s 2 6 6 6 6 6 1 itnecre,telast the curves, distance 0.003 0.001 0.002 0.002 0.002 t10s 120 at ) –itnecurves e–distance D h stiffen Thr igthe sing

Journal of Cell Science (mean sepce,ti irpino h otxdcessthe decreases cortex the 35.5 of from T-events disruption of forces this cells rupture K562 expected, of of number As S5C,D,E). the elasticity Fig. material increased (supplementary the 1.9-fold and tethers membrane 1.6-fold reduced addition ALCAM-WT the (LatA) express by that A linkages material cortex latrunculin supplementary actin 5B; of of (Fig. Disruption cortex S5A,B). and actin Fig. time-dependent the cells to K562-ALCAM-WT ALCAM This linking dynamic of the for 5B). to correlates (Fig. only which cells, K562-ALCAM-GFP observed time but was s contact increase 5 below with times contact differences at increased small The were outwards. elasticities pulled cell was between syntenin-1, membrane and via ALCAM- plasma cortex cortex the the for only to stiff attach not whereas the could which pulled outwards, ALCAM-GPI, A bonds actin 1B). membrane (Fig. ALCAM the syntenin-1 plasma the to protein stressing adaptor linked the force was by elasticity ALCAM-GFP) probably cortex, (and tail in cytoplasmic the ALCAM-WT those that An and difference is of forms -GFP, cell. ALCAM and twofold mutated the ALCAM-WT the expressing expressing almost of cells deformability the the between higher for a explanation indicate values lower 1.8 1.6 ALCAM-GFP, for 52ACMGIcls itn hs iersoe eeldcell 3.0 revealed K562-ALCAM- of slopes maximum elasticities linear of these the Fitting those cells. before K562-ALCAM-GPI than steeper force or were detachment cells cells K562-ALCAM-WT for K562-ALCAM-GFP recorded curves force–distance ARTICLE RESEARCH itnecreaaye,ealdu oepoemolecular explore to us enabled force– analyses, new of The the tail cortex. with curve actin together cytoplasmic TIRFM, the distance and to the SCFS ALCAM in of of combination binding mutations the how affect ALCAM explored we Here, cortex. actin DISCUSSION stiffening the a with to strengthens ALCAM proteins, ALCAM affixing adaptor of by together, by adhesion association mediated Taken probably the to cortex. cortex, that actin contribute actin suggest signaling, results the through these of or stiffening directly, ligand-induced Fig. might material supplementary which 1B; S2), (Fig. ezrin ALCAM- is candidate because probable other protein syntenin-1 adaptor ALCAM- an when using than and cytoskeleton stiffness actin ALCAM-GFP the stiffen cell Fig. might ALCAM-WT, material of (supplementary Thus, CD6 levels S5G). to bound higher constructs adhesion with ALCAM cell of cells slightly levels K562 higher correlated Moreover, control stiffness. and change ALCAM-WT, not ALCAM-GPI did expressing expressing CD6 2012). cells cells the contrast, SCFS - with Gardel, K562 an or cycles of that -GFP and contact beginning revealed the sequential (Schwarz at substrate after stiffness and cytoskeleton cell experiment the actin being of Cell the Comparison influenced is 1C). the strongly (Fig. of is cell deformation, substrate resist region by the glass to ability stiffer while contact its or much stiffness, i.e. the Cell the – cell. against from the pressed curve quantified of force–distance stiffness be approach the whether can for questioned observed stiffness on we is depends expressed, dependency cells is such K562 construct of ALCAM elasticity which the that cytoskeleton determined actin Having the stiffen bonds ALCAM–CD6 6 . nN/ 0.1 6 .. Set,20;Sne l,2005). al., et Sun 2001; (Sheetz, s.d.) D h nrae tfns infcnl Fg C.By 5C). (Fig. significantly stiffness increased Thr m o LA-P (mean ALCAM-GPI for m 6 . nN/ 0.2 6 . nN/ 0.1 m o LA-T 3.0 ALCAM-WT, for m m o ALCAM- for m D h antln oi.A it. to link cannot Thr 6 . Nt 21.7 to pN 9.8 6 ....Nt that Note s.e.m.). D 6 h el or cells Thr D . nN/ 0.1 D h,and Thr, 6 h and Thr . pN 6.9 D Thr m m ci otx(i.4.Teubnigrt fALCAM–CD6- of unbinding/dissociation rate an unbinding revealed of the rate The tethers to membrane ALCAM 4). (Fig. linking anchored upon cortex unaffected of was actin affinity Riet the CD6 te that for confirmed 2004; of experiments ALCAM al., SCFS affinity Our et the 2007). Zimmerman not al., 2000; et but al., avidity cortex et the (Nelissen affects actin ALCAM that actin the and to ALCAM ALCAM Our adhesion. linking cell adhesion. strengthens that and is establishes cell finding ALCAM–CD6-mediated primary of mechanisms elt h LA–D nidn aeo .006 s 0.40–0.63 of rate unbinding ALCAM–CD6 the to well teghnn deinwt oe rbblt ofr tethers. in form results to cortex probability actin lower the a of to with roles ALCAM adhesion complementary linking strengthening The in ruptured. ezrin are and membrane syntenin-1 the for target on possible ALCAM a with is and cortex connects the Ezrin to cortex. site PKC proximal the membrane to strengthens a ALCAM at Syntenin-1 affixing ALCAM cortex. tightly actin by ezrin the bonds proteins to ALCAM–CD6 adaptor ALCAM the linking a with syntenin-1 CD6, formed, and to rapidly bound is When complex (B) supramolecular The cis-interactions domains. recruitment. using ALCAM ALCAM ALCAM extracellular allow of with to microdomains cortex to scaffolds have actin CD9 they the tetraspanin cortex, and or actin ezrin is the ALCAM Because to ALCAM either ALCAM CD6. – release of to cell tail T recruited cytoplasmic the a and link e.g. cortex syntenin-1 – actin cell the CD6-expressing from a released with contact makes – tension. ALCAM–CD6-mediated under strengthening adhesion and Establishing 6. Fig. id oC6 LA elnst h ci otx hc further which faster cortex, 6). actin (Fig. times the adhesion cell to 2–3 ALCAM strengthens re-links once ALCAM that sites CD6, conclude to we contact binds observations these CD6 From 2). (Fig. to with recruitment constructs is ALCAM displayed ALCAM- be ALCAM cytoskeleton. – linkage first cortex actin must actin that deficient but the minutes demonstrated within from site CD6 released contact CD6 express to the to that adhere recruited cells and K562 of ALCAM Contact- measurements avidity. the ALCAM that force modulates highlight time-dependent resonance actin further to results plasmon ALCAM SCFS of Our surface linking 2004). al., by et determined (Hassan been had ale tde aedmntae nascainbetween association an demonstrated have studies Earlier a mdae usd-nsgaigudrtninwe ekrernlinks ezrin weaker when tension under signaling outside-in -mediated < ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal . s 0.5 2 1 o h LA–D od hsvlecompares value This bond. ALCAM–CD6 the for A hna LA-xrsigcl ..aDC a e.g. – cell ALCAM-expressing an When (A) D h n LA-P – ALCAM-GPI and Thr 2 nvitro in 1 which 1603

Journal of Cell Science deinsts(i.5,) owihtepeec fezrin of ALCAM- presence by illustrated the the – strengthens which part thereby minor to a and 5A,B), only cortex (Fig. contributes lowers actin cortex, sites the actin for the adhesion of to crucial ALCAM elasticity not links the and is ALCAM latter of motif the therefore, 1B); PKC (Fig. syntenin-1 to PKC that shown have 2001; studies al., et Previous (Ng 2006). partners interaction Larsson, to binding conformation their their changing altering and both by transduction activities, PKC ERM e.g. influence signal – can isoforms – (PKC) cortex, al., C et kinase Titushkin cell protein 2010; Interestingly, al., 2013). the et (Fehon stiffness of cellular and can pathways structure proteins ERM 2009). the al., et modulate (Hao cortex cellular actin the the connects to that membrane binding ezrin initial supports sites PCRD otc ra rgeiglclatnplmrzto n PIP and virus co- polymerization the and actin towards local receptor respectively) triggering viral CXCR4, area, the and contact of (CD4 polarization receptor cellular (Gordo induced entry and binding EERHARTICLE RESEARCH LA ihC6i o nw.Tu,teemgteitan of exist 1604 might interactions there Thus, heterotypic PKC 2004). known. between in not al., interplay is involved unrecognized et cytoskeleton-dependent CD6 with is (Zimmerman in ALCAM ALCAM Rac-1 interactions role of homotypic Whether no these modulation cytoskeleton- have during avidity the (Zimmerman GTPases However, A by 2004). Rho-like latrunculin activated or al., D be et cytochalasin can agent Rac-1 disrupting Rac-1 adhesion, GTPase cell Rho-like at the as stimulates activating such (Sala-Valde and by cells, synapse, T immunological polymerization in the actin and structures edge actin leading polarized the for essential is 1 (Gordo production idn oi.Bsdo h bevto htALCAM-WT-, that observation PDZ- the C-terminal C-terminal on - altered and a the -GFP- Based to of motif. well presence equally binding the binding syntenin-1 tag, by S. Moreover, eGFP unaffected J. unpublished). Harkes, appeared A.C., previous R. binding and with 1B; J.t.R., C.G.F. (Fig. agrees Tudor, Kanger, studies which (C. colocalization measurements S2), in FRET-FLIM Fig. confirmed material was supplementary syntenin- ezrin with constructs and ALCAM The these syntenin-1. 1 of and association ezrin the as of unpublished). such loss proteins, A.C., eliminates the adaptor ALCAM-GPI and of of of binding Tudor, C.G.F. tail the cytoplasmic C. Kanger, motif the 2005; of S. truncation PDZ-binding al., J. The et Harkes, the (Gimferrer R. to ALCAM J.t.R., of syntenin-1 tail of cytoplasmic binding the Zmemne l,20) oee,tectpamcti of tail cytoplasmic the However, 2004). ALCAM of al., modulation et avidity (Zimmerman cytoskeleton-dependent in role ytnn1cnpa ptoeprlrgltr oei actin CD4 in in role regulatory documented spatiotemporal as a remodeling, play can Syntenin-1 insights elucidated. with be PKC new complex to remains supramolecular by Our regulated a 2004). is forms ALCAM, which al., ezrin, et that suggest (Zimmerman upon phosphorylated PKC activation be of to PKC ability target the direct and motifs a phosphorylation not is ALCAM nterctpamcti ttemmrn rxmlst Fg 1A). (Fig. (PIP site these 4,5-bisphosphate proximal phosphatidylinositol membrane of of enrichment the The at binding PCRD tail the cytoplasmic exposed their ligand that in constructs ALCAM that cytoskeleton. expressed actin cells the These concluded of stiffness the affected we constructs ALCAM 5C), (Fig. CD6 h ALCAM- The h ALCAM- The a mdae inln.Snei-,wihbnst h PDZ the to binds which Syntenin-1, signaling. -mediated ´ D ta. 02.Drn ooyi ALCAM-mediated homotypic During 2012). al., et s h-xrsigK6 el tfee fe idn to binding after stiffened cells K562 Thr-expressing D D ´ h osrc sdhsamtto htprevents that mutation a has used construct Thr h osrc losbnigt zi u not but ezrin to binding allows construct Thr -lnoe l,21) utemr,syntenin- Furthermore, 2012). al., et n-Alonso ´ -lnoe l,21) Syntenin-1 2012). al., et n-Alonso a Fg B yamcaimthat mechanism a by 6B) (Fig. + a a-,snei- and syntenin-1 Rac-1, , a el uigHIV-1 during cells T eas tlcsPKC lacks it because a ly a plays D Thr. 2 )at a 2 h e CSaayi rcse ple nti td revealed study this 2009). al., in et applied Hao processes 2008; analysis al., ERM SCFS et as new Krieg 2005; The such al., proteins, et involves (Sun adaptor membrane molecules to and/or plasma membrane the proteins plasma to the transmembrane linkage linking (Hong This or membrane releasing cytoskeleton. tether by the the and and of 2012) Thereby, bonds composition al., tethered lipid et constant. regulate the to altering able site (Krieg by are stall forces to Cells attachment cell 2008). the al., the allow et and at energy absorb tethers applied membrane force the otoldtruhernascae inln.Teefindings speculatively, These is, signaling. which ezrin-associated site, contact through cellular are to controlled the tethers binding ALCAM stiffens membrane stress, mechanical that CD6 to probability exposed When the formed. actin lowers the by to ezrin strength ALCAMs linking total cortex, by the mediated However, to probably linkages. contribution is ezrin affixing limited that weaker process a CD6, by cell a with in syntenin-1, matures strengthens with cortex by cell Adhesion and contact the avidity. the to site of ALCAM increasing to contact plasma time by the recruited the increasing adhesion at and in an accumulates cortex With ALCAM molecules the site. from ALCAM contact released CD6, are with comes membrane ALCAM expressing contact cell a When into 6). (Fig. CD6 to adhesion a unknown. is has contacts DC–T-cell 5C). tethering at (Fig. role membrane membrane functional plasma ezrin, ALCAM–CD6-mediated the and of Whether ALCAM–CD6-binding properties changing by the of induced and stiffening cortex, to attributable actin possibly the is times which contact 4E), the (Fig. increased extending upon to However, CD6 with 2008). al., 2001; (Sheetz, et compositions Krieg similar have tethers membrane these membrane of forces forming similar that < cells required of K562 constructs via from ALCAM probability tethers 2009) different membrane al., expressed Pulling the et 6). 4, lower Hao (Figs 2008; tethers to by al., et and facilitated (Krieg ALCAM membrane is ALCAM- the the to cortex by actin demonstrated actin As the 6). this D (Fig. that to ezrin hypothesize and We ALCAM syntenin-1 4). of (Fig. ALCAMs formed ALCAM, anchorage GPI-anchored When tethers as many such 3). as cortex, were (Fig. actin twice the that force– to bonds tethers link the not membrane ALCAM–CD6 could in of T-events by unbinding time the anchored first to the curve ALCAM–CD6- for distance colocalized with during TIRFM we combining that SCFS, By formed tethers. and formed bonds are adhesion ALCAM–CD6 cell tethers mediated membrane that usrtsoe ogdsacs(pt eea 100 several to to cells (up attach distances tethers Membrane long 2012). over al., substrates et Sundd 2000; (Grabovsky al., tethers et anchor bonds P-selectin–PSGL-1 or VCAM-1 etohl orl ln h nohlu,weeintegrin- where endothelium, the along roll to neutrophils Therefore, of independently S2). 6). membrane (Fig. interactions Fig. plasma cortical the actin material at (supplementary ALCAM organizes cells CD9 K562 in CD9 ta. 03.I el,alo u LA osrcssoe similar showed of constructs ALCAM sizes by (Gilsanz our of ALCAM microdomain all of ALCAM cells, ALCAM domain In 2013). Ig al., extracellular et containing the with interacting microdomains the tetraspanin-enriched at adhesion ALCAM–CD6-mediated synapse. regulating immunological in ezrin h osrc,tepeec fernmgtb ufcett link to sufficient be might ezrin of presence the construct, Thr 7p tsotcnattmswt D ( CD6 with times contact short at pN 37 ncnlso,ordt upr oe o ALCAM-mediated for model a support data our conclusion, In ebaetteigi motn o ekctsand leukocytes for important is tethering Membrane ersai D a eetyfudt c sasafl in scaffold a as act to found recently was CD9 Tetraspanin ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal . 0s h oc eurdt ulmmrn tethers membrane pull to required force the s, 50 < 0.2 < u f6(i.e. 6 of out 1 m m # 2 ) hsipisthat implies This s). 5 n ooaie with colocalized and m )adkeep and m) < a 16%) 4 b 1 –

Journal of Cell Science eesee t4000clsm 62 eoemeasurements. before h 16–24 cells/ml 400,000 at seeded were Bea h ehrad) 52cl ie htsal expressed stably that lines cell ALCAM- Invitrogen K562 from ALCAM-GFP, Netherlands). purchased ALCAM-WT, were The antibiotics and (Breda, serum the media, Culture of cultures and lines stabilization Cell METHODS and AND MATERIALS formation cells. T the with formed synapse to on immunological ALCAM contributes how of mechanism DCs the of understanding our increase ARTICLE RESEARCH na netdmcocp Osre.1 es) h eprtr was temperature The mounted Zeiss). Instruments) (Observer.Z1, 37 JPK at microscope maintained Module, inverted CellHesion an microscope on force with atomic an II using performed (Nanowizard was cells living using SCFS spectroscopy force Single-cell 150- a using Instruments) Petri Precision (35-mm World dishes glass-bottomed dish, on made were coatings different Four Substrates CO 0.5% 5% with under atmosphere 1640 supplemented in humidified RPMI modified FBS] a growing Iscove’s [75% 5% 25% in cells mixture and containing antibiotic-antimycotic (FBS) medium K562 medium serum al., a bovine Briefly, Dulbecco’s et fetal in 2004). 10% (Nelissen cultured containing al., previously were et described suspension as Zimmerman maintained 2000; and generated ihGHF,tefnlse a kpe n o S-otn nya2% a only used. BSA-coating was for solution and TSM skipped containing was BSA step final the GaHuFc, with 5 or Systems) 5 with incubated was olwdb tao n,atrafnlrnei il- ae,lf odry. water to Milli-Q left water, with Milli-Q rinsed in 4 rinse at thoroughly incubation final overnight then a an after and Following as sulphuric and, M h ethanol prepared 18 1 by pure followed were for in immersion cantilevers (Sigma) by acid ConA-coated cleaned were 2010). cantilevers (Sigma) follows: al., (ConA) A et concanavalin with (Friedrichs coated Bruker) (NP-O, cantilevers H80.Atrwsigwt S ufr h noee ls surface glass uncovered the buffer, TSM with washing After 8.0). pH a lce ihTMbfe otiig2 S yicbtn tfor it incubating by BSA 2% 37 containing at min buffer 30 TSM with blocked was 37 ntecs fC6F-adIA--ccaig t ite l,2007). al., et 4 Riet at (te overnight ICAM-1-Fc-coatings incubated were and dishes CD6-Fc- glass of Briefly, method three-step case a using the prepared were in coatings different four The mm. 2 ntuet)wt e-on t38 at set-point a with Instruments) 2 MTi-C,10m al MCaCl mM 1 NaCl, buffer TSM mM 150 in Tris-HCl, ImmunoResearch) mM Jackson (20 (GaHuFc; anti-human-Fc usrt,apyn Nfrpeeie ieitras(.–2 s). (0.5–120 intervals the time with predefined contact 5 different at into for retracted to was cell cell nN cell the the 2 Subsequently, pushing cantilever-bound (partly) applying after the cells substrate, measured data of When was and min. Adhesion cantilevers substrates 5–10 experiments, discarded. for during cantilevers were cantilever the to the from allowed was detached to cell the strongly Thereafter, microscopy. adhere DIC using by ( The monitored HEPES). softly FBS mM pushed without (+10 < was 7.4 medium pH cantilever culture at ConA-coated in antibiotic-antimycotic 0.5% chamber with perfusion but the in 2011). suspension al., The et attachment day. 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HPUC-2- TIRF, had (Laser (coupler, condenser OZ TIRFM fiber slider 3S-488-3.5/125-SAS-4, mode a to single connected QPMJ-A3A, a into fiber, Coherent) (Sapphire488-4.5AS-11; mW, laser solid-state 50 a LP, by emitted 488 beam a coupling through a achieved and Instruments) JPK 200, (CellHesion 100 SCFS AFM-based Zeiss) an (Observer.Z1, with microscope fitted inverted an using performed was TIRFM TIRFM-SCFS Combined n h CL irsoi mgn etefrteueo hi facilities. their of use the for comments, Centre constructive Imaging and Microscopic encouraging NCMLS for the members and laboratory our thank We Acknowledgements One- 8. OriginPro using [ analyzed curves associate statistically exponential phase Detachment were algorithms. T-events (WaveMetrics) and force–distance 6 forces Pro from Igor determined force–distance in-house were using analysis, stiffness curves cell Force-step force– and software. of averaging, JPK correction curve with baseline curves after determined distance were forces Detachment processing Data scanning laser confocal a 63 a with using performed LS5) Leica (CLSM, was microscope always Analysis were controls Isotype-specific included. Pharmingen). BD 555370, number umrzsteenmesfreeyexperiment. every for numbers these summarizes anWinyUsaitcltssb sn rpPdPim5. by Prism determined GraphPad were using by Significances tests cell. statistical the the U in resulting Mann–Whitney of 1D) deformation Fig. nN, to 0.2–1.5 resistance range force–distance (force region approach contact linear (F- Therefore, the force-indentation into substrate. converted between glass were compressed curves is and cell cantilever the which the in curves force–distance and approach maximum giving function ( Gaussian extraction a sigma Mean by 8. fitted OriginPro were using histograms fitted force were rate) retraction by divided lgi Ao nIae ht:/s.nonhgvi/ n elcsthe reflects for CD9 and or actin ezrin, (http://rsb.info.nih.gov/ij/) cell. syntenin-1, each with ImageJ colocalizing ALCAM actin of in amount (Invitrogen); the using JACoP antibodies calculated were coefficients anti-mouse plug-in Manders phalloidin. goat by stained was and rabbit conjugated and 2% for isotype-specific Alexa-Fluor-647- staining using buffer secondary performed in was min, CLSM ezrin and 5 fixed syntenin-1 in for quickly PBS step in were blocking Triton 0.5% cells with a permeabilization after PBS, and, ice-cold (PFA) paraformaldehyde in washing extensive by %BA 0m lcn)t iiieuseii idn.Pthn was Patching 12 binding. at unspecific minimize incubation to by glycine) induced mM 10 BSA, 3% uvs( curves eehretdadsandwt 10 a with stained and Cells harvested through. bleed were avoid to sequentially collected were Signals objective. otm-eedn eahetfreadtteigcre.Exponential curves. tethering [ and curves force decay detachment time-dependent to aidadpromdi w usqetsre ndfeetaesof areas different ( on cells of series number subsequent The two substrate. in the performed and varied pcfcsmls–a 4 at – samples 5 specific with – (AZN-L50) ALCAM against 6 A.5alnFURojcie(es) IF luiainwas illumination TIRFM (Zeiss). objective aPlan-FLUOR NA1.45 s N ora fCl cec 21)17 5510 doi:10.1242/jcs.141077 1595–1606 127, (2014) Science Cell of Journal FD qassd) h elcre tfns a eemndusing determined was stiffness cortex cell The s.d.). equals nlzdaedtie.Splmnaymtra al S1 Table material Supplementary detailed. are analyzed ) y 5 y 0 +A ˚ ,atrpoogdicbto nCS ufr(PBS, buffer CLSM in incubation prolonged after C, N exp( ˚ o .Atrrmvn non antibodies unbound removing After h. 1 for C 2 t / t K y 5 ]t uuae ieie (distance lifetimes cumulated to )] N y m 0 /lo nioyaantC9for CD9 against antibody of g/ml C m +A esrdadforce–distance and measured ) /lcnetaino antibody of concentration g/ml 6 N (1 A.0olHXP APO PL HCX oil NA1.40 2 d exp( uvsadfte over fitted and curves ) 2 t / t K ) eefitted were ))] k catalog ; 1605

Journal of Cell Science isn,A,Sa A., Gilsanz, a,J . i,Y,Kulk . eel .E,Rlaa,B .adSa,S. Shaw, and L. B. Rellahan, E., K. Debell, M., Kruhlak, Y., G., Liu, Degeest, J., J. A., Hao, Smets, G., Reekmans, P., Zimmermann, J., J. Grootjans, ifre,I,Iba I., Gimferrer, Farno M., Mittelbrunn, M., Calvo, I., Gimferrer, J. D. Muller, and J. Helenius, A. J., Bretscher, and Friedrichs, I. A. McClatchey, G., R. Fehon, A. D. Calderwood, and A. E. Evans, L. M. Dustin, M. C. Franz, and C. Gonnermann, P. L., Dao, J. Rieu, and B. Roux, F., Ronzon, B., Cross, C., W. A. T. Springer, Wang, and V. R., C. Carman, A. Malacko, B., Modrell, X., Li, D., D. Patel, A., M. Bowen, I. G. Bell, rbvk,V,Fiesn . hn . lis .A,Pld . iao,G., Cinamon, A., Peled, A., D. Bleijs, C., Chen, S., Feigelson, V., Grabovsky, Gordo ...... adDM icse h eut n omne on commented and results the manuscript; discussed the manuscript. D.M. edited and the J.H. C.G.F. D.M. A.C., data. and N.S., interpret J.H. J.H., to J.t.R, J.t.R., helped tools. and paper; software experiments the analytical during wrote developed assisted and data N.S. analyzed and experiments, J.H. performed and designed J.t.R. contributions Author interests. competing no declare authors The interests Competing ARTICLE RESEARCH 1606 J. P. Coffer, and M. J. Beekman, Vicente-Manzanares, C., M. Montoya, M., J. Serrador, M., Yanez-Mo, O., Barreiro, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.141077/-/DC1 online available material Supplementary material Supplementary for PMC in Deposited research. National release. this Swiss immediate supported a also and 91110007] Foundation NWO-ZonMW Science number Medium [grant NWO An Investment 269019]. Sized the number by Grant [Advanced supported Council is a C.G.F. Research and RGP0027/2012]. European NWO-83609002]; number number [grant grant [grant Frontiers subsidy is Human Meervoud A.C. NWO 293-2011]. an Biology ASTF by Molecular number supported European [grant a fellowship Research and short-term Scientific 680-47-421]; Organization for number Organisation grant Netherlands [Veni the (NWO) by supported is J.t.R. Funding imran . ai,G,Vvs . Serra-Page J., Vives, G., David, P., Zimmermann, and M. E. Lafuente, G., C. Figdor, W., G. Swart, R., Caban Reyes, Y., Pineda, 20) hshlps -eitdhdoyi fPP eessEMproteins ERM releases PIP2 membrane. of lymphocyte hydrolysis from C-mediated Phospholipase (2009). domains. cytoplasmic Du ersai CD9. tetraspanin spectroscopy. force single-cell by Protoc. components Nat. matrix extracellular to adhesion proteins. ERM of role the cortex: adhesion. kinapses. and synapses spectroscopy. force single-cell comparative Recognit. directly by adhesion matrix supported and spectroscopy. force phosphatase AFM alkaline by GPI-anchored membranes between force anchorage underemphasized? conformation al. and adhesion affinity et leukocyte-cell U. activated ligand. Francke, CD6 of M., a characterization (ALCAM), J. molecule and Pesando, mapping, M., Cloning, Neubauer, H., Marquardt, 618-627. deinmlcl ne lwconditions. flow under cell 1 vascular molecule endothelial adhesion on rolling and tethering leukocyte stimulates Kooyk chemokines van al.; et R. R. Y Lobb, T., Lapidot, F., Arenzana-Seisdedos, F., Baleux, entry. 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