ehnsniiecnei 3hemichannels 43 connexin mechanosensitive integrin ih ar* aulA iule,SrsaBraadJa .Jiang X. Jean and Burra Sirisha Riquelme*, A. Manuel Batra*, Nidhi 14-3-3 ARTICLE RESEARCH ß eevd1 pi 03 cetd6Otbr2013 October 6 Accepted 2013; April 19 Received work { the to equally San contributed Center, authors Science USA. *These Health 78229-3900, Texas TX of Antonio, University Biochemistry, responsible of Department is and (Cx) osteocytes connexin bone is the in molecules physical abundant those is of sensing which One 43, them of changes. allows the signals molecules capable detect mechanical of to to presence are sensitivity the and to cells location a owing whose As primarily these bone 2010). in fact, al., alterations et of Jacobs producing 2004; in matter factor involved (Hughes-Fulford, types osteocytes signal-generating response cell main of major the the the network are is Previous osteocytes the that bone and over behavior. the flow cell within fluid embedded bone that regulate suggest is the studies that through bone forces generated are the mechanical signals process, biochemical environment. osteoblasts, surrounding important its this cells, Several from forces bone During mechanical of to osteoclasts. subjected types and three the osteocytes turnover with bone of coordination process in remodeling the orchestrates tissue Bone INTRODUCTION Hemichannel Connexion, 14-3-3, WORDS: KEY 14-3- of 3 role scaffolding unidentified, Together, previously Golgi. a the define in results occur these to likely is interaction the that integrinsuggesting and A Cx43 brefeldin of by association Golgi the the reduced of Disruption 14-3-3 Golgi. the of in loading absence accumulated Cx43 mechanical the by in transport attenuated forward was on effect stimulatory increase This an with only not 14-3-3 associated in was which surface of the movement to the Cx43 promoted loading Mechanical membrane. integrin plasma and Cx43 of transport The stress. mechanical upon 14-3-3 HC of Cx43 of absence opening the for required HC; integrin that Cx43 and show Cx43 we of both Here, opening unknown. is Cx43 the mechanism underlying induces the from however, and released stimulation mechanical modulators integrin the that signaling shown small recently have These by We (HC). remodeling. hemichannels mediated and are formation bone events for important loading mechanical is by activated osteocytes in signaling Intracellular ABSTRACT ebaefrtefraino ehnsniieH nosteocytes. in HC mechanosensitive of formation the for membrane uhrfrcrepnec ([email protected]) correspondence for Author h 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,1716doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. nassigtedlvr fC4 n integrin and Cx43 of delivery the assisting in a h ,adbokdH pnn.Frhroe tdcesdthe decreased it Furthermore, opening. HC blocked and 5, eesbtas t neato ihC4 n integrin and Cx43 with interaction its also but levels h aiiae lsammrn eieyadfnto of function and delivery membrane plasma facilitates h rvne h neato ewe x3and Cx43 between interaction the prevented a neatwt 14-3-3 with interact 5 a rmteGliaprtst the to apparatus Golgi the from 5 h n h aoiyo the of majority the and h a n hsitrcinis interaction this and , ih14-3-3 with 5 a oteplasma the to 5 a senses 5 h further , a 5. hwdpeiul htfudfo ha tes(FS promotes We (FFSS) stress 1993). shear Goodenough, flow in fluid and connexons that (Musil hexameric previously into showed network assembled proteins, trans-Golgi membrane be other less to the the molecules reported of of is majority passage the Cx43 maintain the Unlike kDa. allow 2003). mainly 1 HC Paul, than and the HC and junctions (Goodenough between gap surface. environment molecules Both external cell small its and of the cell exchange to on join and of connexins connexon communication six docking when a the formed from are form HC formed (connexons). are HC two channels two between in These communication cells. (HC) direct are junctions adjacent mediating hemichannels Gap channels 2008). and Civitelli, only 2012b; junctions the al., et gap (Batra of cells these formation the for nlsssoe htintegrin that directly showed can analysis binding it and I 14-3-3 C-terminus mode with cytoplasmic interact 14-3-3 the consensus at a motif sequence contains that connexin only 14-3-3 Cx43 proteins, two of partners binding integrin possible and the investigating By complex. 2012a). al., a et (Batra loading mechanical to response in conjunction HC integrin in Cx43 interaction, of this activation the and the cells with integrin increases osteocytic that in demonstrated FFSS Cx43 recently with (Siller-Jackson We that HC 2008). Cx43 al., of shown et expression Siperko, surface previously of and cell and have (Silver assembly activation on complex We directly to protein–integrin effect 2003). surface their leading exert cell or the conformation alter pathways can forces messenger protein they protein-kinase-C-mediated secondary mechanical matrix, integrin extracellular a that the Given deform the by to 2007). known ATP also al., are et to of osteocytes (Genetos in release pathway membrane plasma the the on trigger that HC as reported Cx43 such study osteocytic opens Another factors and FFSS 2005). anabolic al., of osteocytes et release primary (Cherian the prostaglandins in to leading HC cells, Cx43 MLO-Y4 of opening the 02,hdn rsrigsrcua ois(akn)for (masking) motifs structural upon protein sorting target or the on hiding (Yaffe, tasks (clamping) conformations 2002), three certain following stabilizing perform the interaction: can protein 2006; unique of al., This 1995). et al., any (Gardino target et proteins Xiao 1995; separate its al., on et with or Liu either sites protein 14-3-3 binding same two of the the to on Interaction dimer al., the 1997). et of Shikano binding al., requires 1996; protein al., II et et mode (Muslin Yaffe I, III 2005; mode mode new – relatively mammalian motifs conserved a evolutionarily different binding and conserved an seven (Liu three is possesses kDa with It which 30 isoforms 1995). proteins, of al., of mass et family Xiao molecular either 1995; a as al., exist with et proteins heterodimers 14-3-3 or domain. homo- cytoplasmic the in motif ntepam ebaet omtemcaosniieHC mechano-sensitive the form to membrane plasma the on 5 h ao usini o osC4 oasml ihintegrin with co-assemble Cx43 does how is question major The a { htcudsrea cfodn rti o these for protein scaffolding a as serve could that 5 h h mre saptnilcniae x3i the is Cx43 candidate. potential a as emerged Pr ta. 06.I diin u database our addition, In 2006). al., et (Park a a locnan 433binding 14-3-3 a contains also 5 ,i eurdfrteoeigof opening the for required is 5, a interacts 5 137

Journal of Cell Science otos D uiidGTC4C ln )o His–14-3-3 or 1) (lane GST–Cx43CT Purified (D) controls. x3adintegrin and Cx43 neato ewe integrin between interaction onasywr muolte ihete niC4 nioy(ae1 ranti-14-3-3 or 1) (lane antibody anti-Cx43 either with His–14-3-3 immunoblotted of were down assay down Pull beads. magnetic with conjugated anti-integrin 14-3-3 anti-14-3-3 with either with immunoprecipitated with immunoblotted were only were anti-14-3-3 lysates incubated 2–4) Cell with Beads (lanes (C) immunoprecipitated antibody. controls. were anti-Cx43 negative lysates with as Cell immunoblotted served (B) were controls. 2–4) anti-14-3-3 negative (lanes with as immunoprecipitates immunoblotted served were 3) 2–4) (lane (lanes lysates immunoprecipitates and 1) (lane ih1--,nri h cfodn cino 433i the in 14-3-3 of action scaffolding is the tasks is these interact nor that of proteins 14-3-3, the membrane None with in for 2006). understood (scaffolding) well platform Schwappach, sufficiently locations a target 2002; cellular and the providing specific al., of (Mrowiec by at interaction et proteins recruitment the protein other facilitating O’Kelly or with 2003; 2003) protein al., Hauri, et and Yuan ta (Nufer of membrane transport intracellular ARTICLE RESEARCH 138 14-3-3 or 1) (lane GST either with 14-3-3 1. Fig. Conversely, 1A). (Fig. cells MLO-Y4 osteocytic 14-3-3 of co-immunoprecipitate lysates could from Cx43 that showed antibody 14-3-3 to bind 3 integrin directly that found can We Cx43 2006). that previous anabolic showed A of 2012a). release al. al., the et for (Batra important process factors a FFSS, to response and Cx43 between association integrin the that demonstrated we Recently, 14-3-3 RESULTS response in of osteocytes opening of the loading. function for mechanical anabolic essential to the is mediate membrane to of plasma HC Assembly membrane. the plasma on the Cx43 to apparatus 14-3-3 Golgi the of from Our action assembly. scaffolding or a trafficking suggests protein study membrane of regulation h idn ie muorcptto xeietuigCx43 using experiment Immunoprecipitation site. binding h neat ihC4 n integrin and Cx43 with interacts a lydacuilrl o h pnn fC4 Cin HC Cx43 of opening the for role crucial a played 5 h neat ihC4 n integrin and Cx43 with interacts a ,adasssteetopoen omigrate to proteins two these assists and 5, a n Cx43 and 5 h a gtdpoen oteplasma the to proteins rgeted lns2ad3 antibody. 3) and 2 (lanes locnan osnu 14-3- consensus a contains also 5 a ,adi eurdfrthe for required is and 5, nvitro in h nioy ed nuae nywt integrin with only incubated Beads antibody. a 5. A yae fMOY el eeimnpeiiae ihat-x3atbd ln ) ellysates Cell 4). (lane antibody anti-Cx43 with immunoprecipitated were cells MLO-Y4 of Lysates (A) td yPr et Park by study h h h lns2ad3 a ulddw sn etd otiigteCtriu fintegrin of C-terminus the containing peptide a using down pulled was 3) and 2 (lanes h ihmgei ed ln evda eaiecnrl(ae2.Eue uprpnl rmtepull- the from panel) (upper Elutes 2). (lane control negative as served alone beads magnetic with htbnsto binds that Pr tal., et (Park h x3wspeeti h oimnpeiiae sn anti-14-3- using co-immunoprecipitates the 3 in present was Cx43 FSmdae Coeigi lce n h elsurface cell the and blocked is opening HC FFSS-mediated pnn fC4 Ci sects(ar ta. 02) To 2012a). al., et (Batra osteocytes 14-3-3 of in influence HC the determine Cx43 integrin of 14-3-3 and of opening absence Cx43 the between in reduced Association is Cx43 of expression ih14-3-3 with nioy(i.1) h ietbnigasyuigapeptide a using assay binding integrin direct The anti-integrin containing 1C). using (Fig. lysates antibody MLO-Y4 of immunoprecipitates 3 14-3-3 14-3-3 His-tagged ewe x3adintegrin and Cx43 between xrsinlvl fC4 Fg A ih pe ae)o integrin integrin or using panel) upper lower right 2A, 2A, a (Fig. (Fig. Cx43 nM of 14-3-3 75 levels expression with and treatment 60 The with panel). occurred reduction further 3-3 ewe x3adintegrin and Cx43 between 3 integrin 14-3-3 with 3-3 h h h h Fg ,rgtlwrpnl.C-muorcptto studies Co-immunoprecipitation panel). lower right 2, (Fig. 5 nioy ed nuae nywt ihr14-3-3 either with only incubated Beads antibody. odtrieteiprac f14-3-3 of importance the determine To h h nioy(i.1) hs eut ugs htC4 interacts Cx43 that suggest results These 1B). (Fig. antibody h a ucina cfodi aiiaigteinteraction the facilitating in scaffold a as function may a lodmntae.14-3-3 demonstrated. also was nioy(ae2ad3.Teipt lwrpnl eeimmunoblotted were panel) (lower inputs The 3). and 2 (lane antibody a sn pcfcsRA(i.2) prxmtl 0 f14- of 80% Approximately 2A). (Fig. siRNA specific using xrsinwsaoihdwt 0n 14-3-3 nM 30 with abolished was expression nioy(ae4,adcl yae ln )adimmunoprecipitates and 1) (lane lysates cell and 4), (lane antibody 5 h a n integrin and ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal irpe Fg B.Teerslsidct ht14-3- that indicate results These 2B). (Fig. disrupted 5 a h h nioy(ae2 rlsts(ae3 evda negative as served 3) (lane lysates or 2) (lane antibody 5 h neato ewe integrin between interaction The . a iN a h neato ewe x3and Cx43 between interaction the was siRNA nioysoe htol nteclstreated cells the in only that showed antibody 5 h h nioy(ae4,adcl yae ln )and 1) (lane lysates cell and 4), (lane antibody ofre h pcfcitrcinbetween interaction specific the confirmed a a Fg 1D). (Fig. 5 -emnst uldw purified down pull to C-terminus 5 a a ,w eue h xrsino 14- of expression the reduced we 5, 5. h nioy(ae2 rlsts(ae3) (lane lysates or 2) (lane antibody h h nH ucin el treated cells function, HC on iN a oefc nthe on effect no had siRNA h a eetdi the in detected was a srqie o the for required is 5 h h nioy(ae2 or 2) (lane antibody nteinteraction the in a h a h n 14-3- and 5 5( iN and siRNA a peptide) 5 a 5

Journal of Cell Science EERHARTICLE RESEARCH ih14-3-3 with otos u euto fH ciiywsosre ncells in vehicle observed or was activity siRNA was HC scrambled FFSS 14-3-3 of to with with reduction treated response a treated in but uptake, cells controls, dye in LY Lucifer of by observed opening by indicated The uptake. monitored as dye Da) was HC, 457 opening mass molecular HC (LY; and Yellow FFSS, to subjected h iN,srmldsRAadvhcecnrlwere control vehicle and siRNA scrambled siRNA, h iN Fg A.Teqatfcto results quantification The 3A). (Fig. siRNA rti tblt n sititaellrtafcigo proteins a 14- of of knockdown that trafficking Given 3-3 2006). intracellular Schwappach, assist and (Mrowiec and stability protein a 14-3-3 suggesting thereby of 3B), role (Fig. observation this confirmed further rtis h elto f14-3-3 of depletion the proteins, 5 14-3-3 h xrsinhdn feto eeso oa x3adintegrin and Cx43 total of levels on effect no had expression h ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal sasafligpoen a ensont maintain to shown been has protein, scaffolding a as , h nH ciiy(i.3). (Fig. activity HC in iN 3 M eesbetdt ttc()o 16 or (C) static to subjected dynes/cm were 14-3-3 nM) or (30 (SC) siRNA siRNA scrambled transfection (vehicle), with reagent treated cells MLO-Y4 opening. coupling. junctional 14-3-3 gap (A) not but FFSS, by 14-3-3 induced of Knockdown 3. Fig. stertoo el eevn Yt RD. to LY receiving quantified cells was of transfer ratio dye the of as degree the and vehicle Scale panel). (lower quantified 50 level was bar: the uptake and dye panel) LY (upper of FFSS to of subjected taken detection cells were for of control images a Fluorescence integrity. as used membrane was uptake (RD; kDa) dextran 10 Rhodamine assayed. was uptake L-4clstetdwt 14-3-3 with on treated performed scrape- cells was A MLO-Y4 experiment coupling. transfer junction dye gap LY the loading on effect no had SC; and vehicle versus ae) x3(ih pe ae)o integrin or panel) upper (right Cx43 panel), with immunoblotted were cells anti-14-3-3 MLO-Y4 14-3-3 or high 1) of have (lane Lysates to 2). known (lane cells, (C) PC12 left control were the or as 3) untreated the lane only (Vehicle; 4), reagent lane transfection (SC; sequence scrambled a 14-3-3 with treated 14-3-3 integrin and Cx43 between 14-3-3 2. Fig. nioy(ae1 rlsts(ae2 evdas served 2) (lane controls. lysates negative or 1) integrin (lane with antibody only incubated Beads antibody. anti-integrin with immunoblotted with immunoprecipitated was 4) anti-integrin and 3 (lanes siRNA integrin and a Cx43 14-3-3 between of association Knockdown the (B) disrupted panel). lower (left SC quantified. was *** GAPDH to panel) lower 14-3-3 of intensity band the of ratio The .Lstso L-4clstasetdwt 14-3-3 with transfected cells MLO-Y4 of Lysates 5. P , .0 o 14-3-3 for 0.001 m h .*** m. 2 xrsinb iN.MOY el were cells MLO-Y4 siRNA. a by expression h fFS F)fr2husadL dye LY and hours 2 for (FF) FFSS of ncdw niisFS-nue HC FFSS-induced inhibits knockdown h x3 integrin Cx43, , a P nioy lae rmbaswere beads from Eluates antibody. 5 h , srqie o h interaction the for required is .0 o 14-3-3 for 0.001 h h iN lns57,sRAfrom siRNA 5–7), (lanes siRNA a aea feto the on effect an have may h n 5 iN essvhceand vehicle versus siRNA .()14-3-3 (B) 3. a h rGPHantibody. GAPDH or 5 a niisH opening HC inhibits 5. h a h A ncdw of Knockdown (A) iN,S or SC siRNA, rCx43 or 5 -siRNA-treated h n h 5 h knockdown n expression 5 3. lf lower (left 3. a (right 5 a 5 h 139 h h

Journal of Cell Science EERHARTICLE RESEARCH o-eegn emal el hwdta h ufc presence 140 surface the that showed cells permeable biotinylation non-detergent right 4A, surface (Fig. integrin ( cell Cx43 significantly or biotinylated A of panel) surface. level upper the the that on showed assay HC integrin of and Cx43 formation of expression surface were 4) and 3 lanes (biotinylated, integrin beads anti-Cx43, avidin with to 1 immunoblotted lanes bound (input, and beads 2) avidin-conjugated on and loading to prior lysates Cell assay. integrin and Cx43 of expression integrin and Cx43 integrin 14-3-3 4. Fig. estmti esrmnso h aditniy(ih,upradlower and control; upper (right, * intensity respectively). integrin band panel, or the Cx43 of (Input) measurements total densitometric to biotinylated of ratio 14-3-3 nM 30 with treated were IClbldat-he g,rsetvl.Saebr 10 bar: Scale respectively. IgG, anti-sheep FITC-labeled and anti-Cx43 with staining integrin dual-immunofluorescence by detected were 14-3-3 with integrin and Cx43 of labeling integrin and Cx43 n a a 5 nioisfloe yRoaielbldat-abtIGor IgG anti-rabbit Rhodamine-labeled by followed antibodies 5 5. h .()14-3-3 (B) 3. A 14-3-3 (A) iN,srmldsRA(C rtaseto egn (vehicle) reagent transfection or (SC) siRNA scrambled siRNA, h sivle ntecl ufc xrsino x3and Cx43 of expression surface cell the in involved is P , a a sdtce ycl ufc itnlto.MOY cells MLO-Y4 biotinylation. surface cell by detected as 5 sdtce yimnfursec.Cl surface Cell immunofluorescence. by detected as 5 .5 eue.Imnfursec tiigof staining Immunofluorescence reduced. 0.05) P h , h ncdw eue h elsraeepeso of expression surface cell the reduces knockdown .5fr14-3-3 for 0.05 ncdw eue h elsraeepeso of expression surface cell the reduces knockdown a rmnndtretprebeclstreated cells permeable non-detergent from 5 a h a Fg A ih oe ae)was panel) lower right 4A, (Fig. 5 a eemndb ufc biotinylation surface a by determined was 5 iN rvhcecnrl Surface control. vehicle or siRNA a h 5or sRAtetdvru vehicle versus -siRNA-treated b atnatbd.Terelative The antibody. -actin a ,laigt reduced to leading 5, a a uniidusing quantified was 5 m m. o oeeto x3fo og otecl surface. cell the to Golgi from Cx43 of movement for rsneo FSa niae yasgiiat(** significant a the by of the extent indicated in the as reduced FFSS, considerably FFSS of was of absence Cx43 presence of the performed colocalized staining in which were Cx43, perinuclear protein FFSS of majority of 58K (Siller- the to presence with FFSS contrast and In of 6A). absence (Fig. 58K hours the Golgi 2 and in Cx43 of after protein Co-immunostaining 2008). increases al., et Cx43 Jackson of expression ufc nrsos oFFSS to response in surface fbt x3adintegrin and Cx43 both of orlto ofiin R-au)frhrcnimdthe 14-3-3 Pearson’s that confirmed suggests data using This further panel). lower 5, co-localization (Fig. (Rr-value) upper ( 5, of significant coefficient co- (Fig. protein and with marker correlation Quantification staining treated Golgi perinuclear were 58K panel). cells by the when indicated with than localization as surface cells cell control, in the vehicle on that less showed and protein marker 14-3-3 with with 58K immunostaining treated Dual Golgi HC. form and to anti-Cx43 surface and the 1993), to Goodenough, moves oligomerizes and then it (Musil where hexamers Golgi to the monomers in localized from be to reported is Cx43 14-3-3 h ero’ orlto ofiin R)fo h mgJsfwr (lower software ImageJ the from * (Rr) panel). merged coefficient the correlation in Pearson’s shown the is 10 colocalization bar: The Scale DAPI. images. with The stained respectively. were antibodies, nuclei by secondary followed Rhodamine-conjugated antibodies, marker and Golgi FITC 58K and anti-Cx43 with immunostained 14-3-3 with treated cells 14-3-3 5. Fig. eeso x3adintegrin and Cx43 of 14-3-3 levels 14-3-3 of although that knockdown suggest and the Cx43 with of decreased 4B). colocalization Fig. the (SC; showed siRNA integrin scrambled further or images vehicle Merged with treated cells of xrsino ohproteins. both of expression norpeiu ulse td,w hwdta h surface the that showed we study, published previous our In h P scuilfrtedlvr fC4 rmteGlit h cell the to Golgi the from Cx43 of delivery the for crucial is , a ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal ntecl ufc n h inl o ohproteins both for signals the and surface cell the on 5 .5fr14-3-3 for 0.05 P h , sivle nteei fC4 rmteGolgi. the from Cx43 of exit the in involved is .5 nraeo x3acmltdi h Golgi the in accumulated Cx43 of increase 0.05) h m .Tedge fclclzto a uniidusing quantified was colocalization of degree The m. h iN,mr x3acmltdi h Golgi the in accumulated Cx43 more siRNA, iN rtaseto egn vhce eedual- were (vehicle) reagent transfection or siRNA h sRAtetdvru eil control; vehicle versus -siRNA-treated a a erae oprdwt that with compared decreased was 5 h a a iia feto oa protein total on effect minimal has ,i srqie o elsurface cell for required is it 5, h ec,orresults our Hence, . h srequired is P MLO-Y4 n , 5 0.01) 3.

Journal of Cell Science EERHARTICLE RESEARCH fC4 n integrin and Cx43 integrin 14-3-3 of of and depletion Cx43 above, of shown association As the for site 14-3-3 major with a is Golgi The interaction the Correspondingly, previously observed 2008). we 14-3-3 as between al., FFSS hours of et 14-3-3 24 hours of 24 after (Siller-Jackson after level declined HC but The of hours, closure 7A). 2 (Fig. of after FFSS expression more of even surface and 14-3-3 increased of FFSS level the protein with The 14-3-3 Cx43. correlation of level in the FFSS, whether examined then We 14-3-3 enhanced FFSS 14-3-3 with S1). antibody Fig. integrin Cx43, an material 3 of with (supplementary colocalization minimal TGN38 co-immunolabeling observed marker, We by TGN TGN Goodenough, a the against and in (Musil located integrin Cx43, are connexons whether assessed hexameric therefore We 1993). form to (TGN) 6B). (Fig. 58K Golgi significant and a staining by observed cell as the 14-3-3 Cx43 (** to of of Cx43 transport forward of absence promoted transport forward However, right the Cx43 6A, surface. to of (Fig. exit leading the protein Golgi promotes FFSS from 58K that suggest with results These colocalization panel). its in decrease x3adintegrin and Cx43 FSehne h neato ewe 14-3-3 between that interaction indicated results the similar These enhanced 14-3-3 7C). A (Fig. 7B). between FFSS observed (Fig. was interaction FFSS FFSS the upon to subjected in cells increase MLO-Y4 in fold h og otepam ebaet ommr ucinlHC. functional more form to integrin membrane and plasma Cx43 the more to Golgi of the delivery the to led integrin h x3i eotdt easmldi h rn-og network trans-Golgi the in assembled be to reported is Cx43 ihTN8 niaigta x3asml n t association its and assembly Cx43 that indicating TGN38, with P , .1 nraei h ooaiaino x3perinuclear Cx43 of colocalization the in increase 0.01) a ,psil sarsl feeae 14-3-3 elevated of result a as possibly 5, h h n integrin and h a n x3wssbtnilyicesdb 2.5- by increased substantially was Cx43 and 5 h a xrsin n h neato f14-3-3 of interaction the and expression, nteGli odtriewehrthe whether determine To Golgi. the in 5 a a o cu nTNi osteocytes. in TGN in occur not may h nrae fe 0mntsof minutes 30 after increased h eut nteaccumulation the in results h h teutdteFFSS- the attenuated screae ihthe with correlated is h h a n integrin and rtisfrom proteins 5 sehne by enhanced is h a h n 14-3-3 and 5 ees which levels, a n Cx43/ and n 14-3- and 5 h with a a 5 h 5 h nrclua cuuaino x3 n olclzto with co-localization caused and BFA Cx43, with to 14-3-3 of treatment leading accumulation The apparatus stacks. intracellular Golgi Golgi the the the to of ER collapse the the from interferes that transport inhibitor the used commonly with a (BFA), A brefeldin with 14-3-3 with association ercnl hwdta FSidcdteconformational of the induced conformations FFSS the 2010). that al., integrin et of showed alter Jacobs activation 2009; recently Weinbaum, to and We Fritton (Batra 2012b; capacity channels al., mechanosensitive et activate network, the the canalicular and in osteocytic macromolecules results has the bone surrounds the that which to of force fluid of of conversion Application flow 2006). the mechanotransduction al., upon called et process (Orr activated a through rely signals forces mechanical biochemical processes the pathological on and physiological Developmental, DISCUSSION aaaepeetdi h ih aeso h corresponding the of Cx43 panels between colocalization right the integrin the treatment, and BFA in quantification Upon The presented images. conditions. 8B) are (Fig. flow data fluid and 8A) (Fig. Cmdae h ees fsalfcosincluding we factors 9, Fig. 14-3-3 in bone small conditions, illustrated the as static study, of under of this that In function show 2005). release al., anabolic et of for (Cherian the opening The important 2012a). al., prostaglandins, mediates et (Batra HC Cx43 HC of opening the in ntecl.Tgte,teerslssgetta h soito of association the apparatus. that suggest expressed integrin results proteins these Cx43, the Together, of cell. state signals the oligomeric fluorescence in the by and not caused level be was protein could increase total an between such treatment differences but 14-3-3 the BFA protein, with Cx43 that that observed weaker. total be Note showed the to result increased 8C,D). appear (Fig. immunoblotting cells conditions BFA-treated However, in FFSS signals and fluorescence static both neato ewe x3adintegrin and Cx43 between interaction h a infcnl (* significantly was ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal nrae h xto x3fo og.MOY el subjected dynes/cm cells 16 MLO-Y4 (FF; Golgi. from FFSS Cx43 to Golgi. of the exit from the Cx43 increases of exit the 14-3-3 promoting of Loss 6. Fig. sn mgJsfwr n orlto ofiin R)value 10 (Rr) bar: coefficient Scale correlation obtained. a was calculated and was software signals ImageJ merged fluorescence using the of in overlap shown The is images. Golgi colocalization 58K The and protein. Cx43 marker against antibodies with immunostained iN rtaseto egn vhce eesbetdt FFSS to 14-3-3 dynes/cm subjected nM were (16 30 (vehicle) reagent with transfection treated or cells siRNA MLO-Y4 FFSS. by induced two the 14-3-3 of (B) overlap 10 of bar: extent Scale value the colors. (Rr) describes coefficient that correlation obtained a was calculated and were software signals ImageJ merged fluorescence using the of in overlap shown The is images. Golgi colocalization 58K The and protein. Cx43 marker against antibodies with immunostained ess14-3-3 versus a a losgiiaty(** significantly also was 5 a n 433i ieyt cu nteGolgi the in occur to likely is 14-3-3 and 5 h ncdw lcsteicesdGliei fCx43 of exit Golgi increased the blocks knockdown a h 2 h 5 o or n ie el eedual- were cells fixed and hours 2 for ) h b n the and iN treatment; siRNA cusi h og,w rae h cells the treated we Golgi, the in occurs n t neato ihC4 resulting Cx43 with interaction its and 1 P m , .** m. h .5 erae ne ohstatic both under decreased 0.05) teutsteefc fFS on FFSS of effect the attenuates b 2 atncnrl(i.8) These 8E). (Fig. control -actin P o or eefxdaddual- and fixed were hours 2 for ) , m .** m. .1frF esscontrol; versus FF for 0.01 n 5 P P a , 3. , h ,admdae the mediates and 5, .1frF,vehicle FF, for 0.01 .1 eue under reduced 0.01) srqie o the for required is A FFSS (A) n 141 5 h 3.

Journal of Cell Science EERHARTICLE RESEARCH 142 several with interact to shown including dynamic been integrins the has 14-3-3 Herve as 2007; Although, al., well 2007). et Cx43 (Chanson as opening of channel disassembly of regulation modulation the and in assembly role trafficking, their for importance FFSS. considerable the to accommodate response to in surface HC cell functional the of surface. formation to delivered cell are the molecules to integrin Golgi 14-3-3 the and of level from the increases Cx43 FFSS of transport forward ta. 01 um ta. 06,orsuyi h is oso that show to first the is study our 2006), al., 14-3-3 et Nurmi 2001; al., et a 14-3-3 pnn nrsos oFS.14-3-3 FFSS. to response in opening integrin and Cx43 between interaction facilitated be to integrin 14-3-3 likely and of is Cx43 action interaction scaffolding weak, This the 2012a). a through al., showed integrin and et study Cx43 of (Batra recent C-terminus the Our between interaction proteins. direct two these between eesb oeta af u nrgigy esrrdcinwas reduction lesser a intriguingly, but half, a than more by levels HC. of functioning correct and co-migration and integrin and association Cx43 the of mediates and protein chaperone we 14-3-3 Consistently, 2012a). of al., absence et that (Batra observed cells further these in opening HC integrin and Cx43 between that binding demonstrated the disrupting we Recently, interaction. for proximity physical eas euto f14-3-3 of reduction because 5 h neato fC4 ihohrpoen sgaining is proteins other with Cx43 of interaction The ncdw f14-3-3 of Knockdown h h neat ihintegrin with interacts sncsayfrteitrcinbtenC4 n integrin and Cx43 between interaction the for necessary is a .Cneunl,mr x3adintegrin and Cx43 more Consequently, 5. b a a 1, nodrt rn hs w rtisto proteins two these bring to order in 5 otepam ebaefrteformation the for membrane plasma the to 5 a h L b xrsinrdcdtesraeCx43 surface the reduced expression and 2 a h .Mr motnl,w hwthat show we importantly, More 5. ysRAipisteassociation the impairs siRNA by h a n t soito ihCx43 with association its and Dai ta. 09 Han 2009; al., et (Deakin 4 a h u lobokdteHC the blocked also but 5 h evsa scaffolding/ a as serves h o nyaoihdthe abolished only not ybnigwt both with binding by a lce Cx43 blocked 5 ´ tal., et a a 5 5 og,a eosre ee n osbeepaainfrthis for explanation possible of One instead here. ER, observed the from we exit as suggested the Golgi, have facilitates GPR15 reports primarily of Previous 14-3-3 stability that 2011). the Shikano, increasing and and (Okamoto motif suppressing recognition promote by ER GPR15 to phosphorylation- co-receptor the demonstrated HIV example, of been expression For has surface 2003). cell 14-3-3 al., of Shikano et binding 2002; Yuan al., dependent et 2005; (O’Kelly surface al., cell et the at levels expression proteins with these correlate of to their shown was mediating proteins the multimeric 14-3-3 and et by is Shikano 2006). proteins 2006; proteins years Schwappach, al., membrane and membrane recent (Mrowiec target of trafficking in forward the transport roles to surface emerging binding cell its of of promotion One processes. utfntoa rti nw ob novdi eea cellular several in involved be to known integrin protein and Cx43 multifunctional resulting of apparatus, expression an Golgi surface but the reduced level from in be protein exit Cx43 inefficient total to the in of change likely outcome the of is consequence proportionate a which not a surface, with cell 50% the correlated integrin over on with is associated Thus, Cx43 function of connexins. HC reduction of the of of rate because decrease turnover Cx43 with protein. faster associate this relatively necessarily of not life-span may longer integrin surface long-lived much these of the all to However, owing affected be 14-3-3 integrin of of integrin al., absence migration et the (Liu the Therefore, hours disrupts 20 connexins. [over than half-lives 2011)] longer much have integrin integrins for observed eso htls fH ciiyi h bec f14-3-3 of absence the in activity HC of loss that show We a ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal oeue led ntepam ebaewudnot would membrane plasma the on already molecules 5 rlae ipt a acltd(oe ae) nB,C, In panel). to (lower * (IP) calculated beads was from (input) eluates preloaded and of quantified ratio was normalized Cx43 the of intensity Band anti-integrin antibody. or (C) were (B) beads anti-Cx43 from with eluates immunoblotted were or 2) (input) and lysates 1 Preloaded (lanes flow anti-14-3-3 non-fluid with for or immunoprecipitated FFSS 3) to (lane subjected hours cells 2 MLO-Y4 of Lysates (B,C) rgtpnl.** panel). (right 14-3-3 the of 24 and ratio and quantified normalized 4), was intensity (lane anti-14-3-3 Band 2 with antibody. 3), immunoblotted GAPDH (lane were 0.5 hours to 2), 5) subjected (lane (lane cells 0 MLO-Y4 for of flow lysates fluid or 1) (lane integrin lysates and Cx43 with interaction i.7 FSicess14-3-3 increases FFSS 7. Fig. P h , .5frF essnnF (C); non-FF versus FF for 0.05 idn hog ifrn oist several to motifs different through binding a a .Oepsil xlnto sthat is explanation possible One 5. 5. P , .1fr2husvru hours; 0 versus hours 2 for 0.01 a ocl ufc,adthose and surface, cell to 5 h oGPHwscalculated was GAPDH to a oeue ntecell the on molecules 5 h xrsinadits and expression h n a 5 antibody. 5. 3. a A C2cell PC12 (A) .1-- sa is 14-3-3 5. h a 5 n only h 5 h or 3. is

Journal of Cell Science olclzto fteepoen.Itrsigy F treatment BFA Interestingly, proteins. these of a co-localization be 14-3-3 of to association appears a the Golgi for 14-3-3 The site surface of Cx43. FFSS major cell levels with the Moreover, protein association to increased strengthened Golgi membrane. by the accompanied from plasma was Cx43 to the of prior migration (HC) to promoted connexon delivered hexameric where 14-3-3 site form being the of also to Golgi, absence oligomerizes the in that Cx43 Cx43 of observed accumulation the we caused Interestingly, 2004). al., EERHARTICLE RESEARCH R(ui n odnuh 93.Ide,alrefato of fraction large a unique the Indeed, a of 1993). is instead Goodenough, 14-3-3 Golgi Cx43 ER and the 2006). the (Musil in al., ER in oligomerizes et that retention Shikano protein their 2005; membrane prevents al., and localization et ER proteins (Shikano the these masks in C-terminus oligomeric signal the on of 14-3-3 motif Golgi. majority RXRSWTY the of of instead the ER, the Binding Cx43, in assembled unlike are proteins that membrane is discrepancy integrin and Cx43 of association the reduces Golgi of Disruption 8. Fig. integrin ramn nrae oa x3lvl L-4clswr rae ih2 with treated were cells MLO-Y4 level. Cx43 total increases treatment ooaiaini hw ntemre mgs h vra ffursec inl bandwr acltduigIae otaeadacorrelation a and 10 software bars: ImageJ Scale colors. using 14-3-3 two calculated the anti-Cx43, were of 14-3-3 with obtained overlap and immunoblotted of signals Cx43 extent fluorescence the against of describes antibodies overlap that with The attained dual-immunostained was images. value and merged (Rr) fixed the coefficient were in Cells shown hours. is 2 colocalization for A,C) (C; untreated .Dsuto fteGlib F infcnl erae the decreases significantly BFA by Golgi the of Disruption 5. h a n 14-3-3 and 5 sas eotdt elclzdi h og Pesne et (Preisinger Golgi the in localized be to reported also is h L-4clswr rtetdwt rwtot2 without or with pretreated were cells MLO-Y4 . h ommrn rtiscnann the containing proteins membrane to h or b atnantibody. -actin h ihC4 n integrin and Cx43 with h n a and m m F rlf nrae o pt or n ellstswr olce and collected were lysates cell and hours 5 to up for untreated left or BFA M h F o or n eesbetdt FS(F 6dynes/cm 16 (FF; FFSS to subjected were and hours 5 for BFA M a ih14-3-3 with 5 nipnal oei iutnosytasotn integrin transporting simultaneously in role indispensable C pnn fH eitsterlaeo ml bone small of release mechanical of the action anabolic the mediates in loading. mechanosensitive step HC crucial form a of to modulators, membrane Opening plasma the HC. to Cx43 and monomers. from of signals form fluorescence the treatment. as decreased in BFA ER upon the exists Cx43 the explain intracellular treatment in would BFA retained This of Cx43 result proteins. than a stronger monomeric much from are fluorescence proteins that those known oligomeric which presence is from It the emitted stress, network. increases signals trans-Golgi shear also the HC, in flow Cx43 Cx43 Cx43 fluid of of of and assembly colocalization the marker, the enhances trans-Golgi observed a have form oligomerized we with the Indeed, of formation Cx43. the collapse is of affect the Cx43 would network, that Golgi trans-Golgi the is the of in explanation assembled possible total be One to the reported protein. increased Cx43 but of Cx43, level for signals fluorescence decreased oehrterslsrpre eeso ht14-3-3 that show here reported results the Together ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal h . AD F eue h ooaiainbtenCx43, between colocalization the reduces BFA (A–D) m .* m. P , .5 ** 0.05, h AB rC4 and Cx43 or (A,B) P , .1ad*** and 0.01 P , a CD.The (C,D). 5 0.001; 2 ,)o left or B,D) ; n 5 h .()BFA (E) 3. ly an plays 143 a 5

Journal of Cell Science 4 hnicbtdwt anti-integrin with incubated then lrc,S oi,M,UA eeue o immunostaining. for used were USA) MO, Louis, St CA, Aldrich, Cruz, Santa Biotechnology, 14-3-3 Cruz USA), (Santa Cx43 monoclonal USA), NY, and MN, Island, assays, (RD; Grand integrin transfer Technologies, against dye dextran Antibodies (Life and USA). Invitrogen Rhodamine uptake from dye obtained and for were used BFA LY kDa), 10 matrices. mass, appropriate molecular with assays, coated uptake dye and immunostaining EERHARTICLE RESEARCH fdfndtikeswt rvt-rvnfudfo sn peristaltic a using 144 flow fluid gasket a gravity-driven by with separated chambers thickness flow defined plate parallel of by created was FFSS FFSS were lysates Cell centrifugation by ml. pelleted 1 and 1800 USA) to at NJ, Piscataway, volume GE, final Amersham 4B; the 20 make with a to pre-cleared by lysates followed minutes to 30 added for ice was 10,000 on at X-100 incubated spin Triton were lysates 26 0.25% cell of and a used concentration ruptured final then through a and culture buffer immunoprecipitation, RIPA 10,000 triturating tissue in at dissolved was mm spun by pellet 150 and The pellet. PBS from cell in the collected lysed were and plates cells MLO-Y4 Cultured immunoblotting and Immunoprecipitation in and USA), CA, grown Jose, San Biosciences, BD were rat mg/ml; with (0.15 coated I on type USA) collagen cultured TX, tail Antonio, were San bones Plastics, long (Regal murine surfaces polyester from derived cells osteocytic MLO-Y4 reagents and culture Cell METHODS AND MATERIALS ecie rvosy(aoe l,19) -E Lf ehoois Grand Technologies, Ca (Life USA) S-MEM NY, 1997). Island, al., et (Kato previously described eeoe yehne hmlmnsec Aesa Pharmacia (Amersham chemiluminescence USA). NJ, Piscataway, enhanced dilution), and Biotech, 1:300 by gels, (E2; developed SDS-PAGE anti-Cx43 10% purified anti-integrin affinity on with separated immunoblotted were Immunoprecipitates t4 at atesug D S)splmne ih25 B n .%BCS 2.5% and FBS 2.5% with supplemented USA) MD, Gaithersburg, Hcoe oa,U,UA n nuae na5 CO 5% a in incubated and USA) UT, Logan, (Hyclone, ˚ ih20 with C ˚ ,floe ypeiiaino h opee fe nuainat incubation after complexes the of precipitation by followed C, g t4 at h a 110)o anti-14-3-3 or (1:1000) 5 ˚ Eapa hre,M,UA n og akr5K(Sigma- 58K marker Golgi and USA) MA, Shirley, (Exalpha, m g o iue.Spraat pecerdlsts were lysates) (pre-cleared Supernatants minutes. 2 for C rti ed rmueIGaaoebeads. agarose IgG mouse or beads G protein l o 0mntst eoetedbi.RP ufrwas buffer RIPA debris. the remove to minutes 10 for a mdfe seta eim( medium essential -modified 2+ m rti ed poenGSpaoefs flow fast (protein-G–Sepharose beads G protein l remdu,wsue o h y paeasy For assay. uptake dye the for used was medium, free K a a RDSses D9,Minneapolis, CD49e, Systems, (R&D 5 ranti-14-3-3 or 5 ag ede2 ie.Frco- For times. 20 needle gauge el eeclue ngasslides glass on cultured were cells h 110)atbd.Boswere Blots antibody. (1:1000) g o iue ocollect to minutes 2 for h a MM Gibco-BRL, -MEM; 2 nioisovernight antibodies nuao t37 at incubator ˚ Cas up h hcns ftegse eemnstecanlhih.By height. channel the determines gasket the of thickness The pump. dutn h hne egtadfo ae teslvl f1 dynes/cm 16 of levels stress rate, flow and height channel the adjusting el eete nuae o ora 4 The at (RT). temperature hour room 1 at for hour incubated 1 then for were PBS and cells in PBS) BSA in 3% (prepared The X-100 with temperature. Triton blocked room 0.25% at fixed with minutes permeabilized were 20 were collagen for cells with PBS in coated paraformaldehyde slides 4% microscope in on cultured cells The analysis colocalization and microscopy fluorescence confocal labeling, Immunofluorescence cm 5 of area surface medium cell A SMEM flow. in fluid cells to MLO-Y4 subjected of not consisted Controls generated. were nuae ihC4 nioy(:0 iue n3 S)fr1hour 1 for BSA) 3% in diluted (1:300 4 antibody at Cx43 with incubated ubr sa ne fclclzto,teJCPpui fIae was correlation ImageJ Pearson of plugin the JACoP obtain the to colocalization, of order No used. index In image. an each deleted. as of or number, and clarity Brightness added the pixels). improve were 1.5 of to of data off-line iterations diameter adjusted 30 filter were for pass of low contrast (applied software a function with 0.5 spread deconvolution image point of each iterative known thickness a a using with at ImageJ deconvoluted conducted were was scanning Images Optical, The Olympus fluorescence (Fluoview; Japan). microscope performed Tokyo, Confocal scanning was laser confocal microscopy. scanning Laboratories, a mounted z-stacking using for secondary were Vector three-dimensional sealed appropriate Slides with (H-1000, microscopy and RT. the washed medium at CA) were with each mounting Burlingame, cells hour labeled the Vectashield 1 Again, and for using RT. succession PBS at in minutes with antibodies 20 times for PBS three in PFA 4% CO 5% at %PSfr1hu t4 at hour 1 for PBS 3% 1 anti-integrin cold 3% sheep in in washed antibody then secondary and appropriate PBS with the times with three hour rinsed BSA. antibody 1 were monoclonal for (1:300 Cells or 2008) (1:50). incubated 58K al., (1:100) Golgi Cx43 et Cx43 against of (Siller-Jackson antibody developed domain monoclonal we extracellular dilution), which second (E2)], the [Cx43 against antibody polyclonal h niefo ytmwsecsdwti ag aki CO walk-in large a within SMEM. encased was was medium system circulating conducted flow The entire was indicated. The as test time Each respective the microscopy. for immunofluorescence and uptake o ufc aeig h el eeclue sdsrbdabove, described as cultured were cells the labeling, surface For ˚ .Teclswr hnwse he ie ihPSadfxdin fixed and PBS with times three washed then were cells The C. 2 ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal n 37 and eoeigudrmcaia stimulation. and mechanical formation under bone remodeling and/or for autocrine important small factors of paracrine release plasma the the Functional mediate on hemichannels. hemichannels assembly functional for form Golgi to the membrane of out proteins two 14-3-3 integrin 14-3-3 more of to levels leading the increased, FFSS, Under integrin membrane. and plasma Cx43 14-3-3 to of bind levels that low are there FFSS. conditions, to response in membrane 14-3-3 integrin and of Cx43 role of the delivery of Illustration 9. Fig. ˚ a C. nioyaanta xrclua ptp iue in diluted epitope extracellular an against antibody 5 a 6 5 b soitdwt nrae oeeto these of movement increased with associated 1 B olwdb nuainwt polyclonal with incubation by followed PBS ˚ .Clswr ahdoc ihPSand PBS with once washed were Cells C. ˚ ihafnt-uiidrabbit affinity-purified with C a a 5 rmGlit h plasma the to Golgi from 5 b h n oete othe to them move and 1 ne o-FSstatic non-FFSS Under idn oC4 and Cx43 to binding h h 2 nfclttn the facilitating in bu crescents) (blue a sdfrdye for used was 2 incubator h are m m. 2

Journal of Cell Science ahdwt BStretms hnwt B wc,adfnlyfxdin fixed finally and twice, PBS with then times, three HBSS gently with scraped washed subsequently 26 dissolved were RD which a washed 1% cells, and were the with LY to Cells modified 1% applied RD. Then were BSA. the and PBS 1% in LY plus on of HBSS with presence 1987). times based the al., three in et performed scratched (El-Fouly co-workers were was and Cells El-Fouly assay by described to transfer procedure grown dye were loading’ cells MLO-Y4 assay transfer dye Scrape-loading ARTICLE RESEARCH aa ua h colo eiie mr nvriyfrknl rvdn the providing kindly for University Emory Medicine, integrin of 14-3-3 biotinylated School His-tagged of the generation at for Fu Field Haian Gregg Dr thank We Acknowledgements differences significant (* controls of the degree means with the compared the indicate as Asterisks presented software figure determinations. are the data 5.04 in specified the otherwise Student’s Prism legends, Unless paired groups. a GraphPad two and between groups, comparison two were using than test more analyzed Student–Newman–Keul’s for used and were ANOVA One-way data (GraphPad). the All analysis Statistical 14-3-3 transiently nM with 75 hours were 48 or Cells for slides 60 USA). collagen-coated on 30, CA, suspensions as Carlsbad, OPTI transfected antibiotic-free (Invitrogen, in resuspended and medium trypsinized were cells MLO-Y4 treatment BFA and siRNA 14-3-3 Ca a the S-MEM, of another. majority one the with which contact at in density physically a not at were cultured cells were cells experiments inhibition MLO-Y4 and assay uptake Dye were Rhodamine. RD receiving for cells assay. set 500 each filter than for the more counted using significance, RD using statistical and detected reach fluorescein, be To for could (Zeiss set LY microscope which filter dye fluorescence in the The Germany), a minutes. Jena, using Zeiss, 20 examined Axioscope, for were stock) results 16% transfer (from paraformaldehyde 2% fresh snraie y pae(eintda )oe h respective the over 1) as (designated expressed are uptake Results group. image. control dye per the un-stimulated cells as normalized total determined to was as cells uptake fluorescent Dye observed of were microscope. ratio fields PBS fluorescence Similar with the once microscopy. washed for under and slides minutes the 20 mounting for PFA before 2% using fixed then uptake were dye mass for (molecular ( LY incubated 0.2% RD were of cells mixture a FF, with After analysis S-MEM. with times three remdu,wsue o h Feprmnsa lwrt f1 dynes/ 16 of rate flow a at experiments FF the cm for used was medium, free niois ellstswr ujce oimnbotn ihC4,14- Cx43, with secondary immunoblotting fluorescence-labeled to 3-3 subjected respective were were lysates cells the Cell Fixed antibodies. minutes. by 10 14-3-3 followed for and FF antibodies, to Cx43 subjected with were 2 and dual-immunolabeled with hours pre-treated 5 were to 2005). cells up al., MLO-Y4 et avidin- treatment, (Cherian BFA to SDS-PAGE For using binding avidin-conjugated separated of after and purified protein to beads were total conjugated applied samples of were biotin-labeled dye amounts The samples and equal beads. siRNA-treated RD, and were and cells lysed, and the were and control LY assay, biotin biotinylation with cells with surface incubated the incubated SDS-PAGE siRNA-transfected minutes, For examined. 10 to with was for uptake subjected FF slides to was 14-3-3 The subjected which with antibodies. protein immunoblotting GAPDH isolate by Cells antibiotics. to followed without medium lysed fresh with medium were replaced the siRNA transfection, and a the removed after using was hours USA), Three TX, (Ambion). kit (Austin, transfection Ambion by produced commercially 2 h , o 0mnts oto el ntsbetdt F eewashed were FF) to subjected (not cells Control minutes. 10 for or 0ka o iue.R a sda eaiecnrl Cells control. negative a as used was RD minutes. 5 for kDa) 10 b atnantibody. -actin K guenel.Atricbto o iue,clswere cells minutes, 5 for incubation After needle. -gauge h osrc,TmyNue o ehia sitneadthe and assistance technical for Nguyen Tommy construct, h P , iN r6 MsrmldsiRNA scrambled nM 60 or siRNA .5 ** 0.05, , 58%cnlec.Te‘scrape- The confluency. 75–85% P , h h .1 *** 0.01, x3 integrin Cx43, , 6 rC4 n integrin and Cx43 or ...o tlatthree least at of s.e.m. , t 4 a n 0.2% and Da) 547 P ts a sdfor used was -test , a etd,Dr peptide, 5 0.001). m F for BFA M a and 5 2+ a 5 - aos .R,Tmysti,S n atlo .B. A. Castillo, and S. Temiyasathit, R., C. Jacobs, M. Hughes-Fulford, Herve a,D . orge,L .adGa,J L. J. Guan, and G. L. Rodriguez, L. C., D. D. Han, Paul, and A. J. D. H. Donahue, Goodenough, and E. C. Yellowley, Y., Zhang, J., C. Kephart, C., D. Genetos, adn,A . mro,S .adYfe .B. M. Yaffe, and J. S. Smerdon, K., A. Gardino, S. Weinbaum, and P. S. Fritton, rwe,T n cwpah B. Schwappach, and T. Mrowiec, hno,M,Ktis .A,Prcha .adOGay .M. S. O’Grady, and C. Peracchia, A., B. Kotsias, M., Chanson, F., X. G. J. Weber, Jiang, and X., R. Kar, Xia, N., S., Batra, Gu, J., A. Siller-Jackson, S., Burra, N., Batra, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.133553/-/DC1 online available material Supplementary months. material 12 Supplementary after release for PMC number in [grant numbers Deposited Foundation [grant Welch J.X.J]. Health the to and of AQ-1507 J.X.J.]; Institutes to EY012085 National J.X.J., the to by AR46798 supported was work The Funding paper. prepared the and wrote data J.X.J. the and performed analyzed M.A.R. S.B. J.X.J. N.B., M.A.R., and figures; N.B., S.B. project; M.A.R., the N.B., planned experiments; J.X.J. S.B., M.A.R., N.B., contributions Author interests. competing no declare authors The interests Competing lFuy .H,Tok,J .adCag .C. C. Chang, and E. J. Trosko, H., M. el-Fouly, Mostafavi-Pour, J., Schoelermann, S., Warwood, D., M. Bass, O., N. Deakin, F., L. Bonewald, X., R. Civitelli, Wang, S., Gu, J., A. Siller-Jackson, P., P. Cherian, epei rJagslbrtr o rtclraigo h aucitadtheir and manuscript the of reading critical for comments. laboratory valuable Jiang’s Dr in people ue,O n ar,H P. H. Hauri, and S. O. Nufer, A. Shaw, and M. P. Allen, W., J. Tanner, J., A. Muslin, A. D. Goodenough, and S. L. Musil, A., S. Corbett, R., D. Critchley, J., S. Monkley, X., Tian, Y., R. Qi, Liddington, X., and He, H. J., Fu, Liu, J., R. Collier, C., Petosa, J., F. Bienkowska, L. D., Bonewald, and Liu, R. G. Mundy, A., B. Koop, J., J. Windle, Y., Kato, ehnbooyadprclua mechanics. pericellular and mechanobiology STKE functions. to partners from complexes: neato ewe nernbt1ad14-3-3beta. and beta1 integrin between interaction channels. induces connexon hemichannels unpaired junction gap osteocytes. of MLO-Y4 activation from release flow ATP fluid Oscillating (2007). iadcmlxs oprsno h -a rsa tutrso l ua 14- human all of structures crystal X-ray isoforms. the 14-3-3- of 3-3 of comparison localization a subcellular of complexes: regulation ligand and specificities binding 14-3-3 of mechanotransduction. induced neatoso onxn ihohrmmrn hnesadtransporters. and channels membrane other with connexins of Interactions bone. of development 1818 and al. function transmission, et signal E. Sprague, M., E. Lafer, integrin hemichannels. F., stress-activated L. Mechanical Bonewald, D., DeSimone, rnfr ai n ipetcnqet td a ucinlintercellular junctional gap study to technique simple and communication. rapid A transfer. J. activity M. Cdc42 migration. Humphries, localised cell accelerates mediates and and complex C. ternary Ballestrem, alpha4-14-3-3zeta-paxillin D., Knight, Z., Biophys. Biochem. Arch. prostaglandin. of release Cell the Biol. for Mol. X. mechanism novel J. a osteocytes: Jiang, in hemichannels and E. Sprague, Biol. Mol. Biophys. Prog. 433wt inln rtisi eitdb h eonto fphosphoserine. of recognition the by mediated is Cell proteins signaling with 14-3-3 exit after occurs connexin43, junction ER. gap the from protein, channel membrane plasma transport. morphogenesis. epithelial S. embryonic Li, promote and to M. A. Graham, F., S. protein. Lowry, 14-3-3 the of isoform zeta the 191-194. of structure Crystal (1995). 2014-2023. 369-400. salsmn fa sect-iecl ie MLO-Y4. line, cell osteocyte-like an of Establishment ,J . orese,N,Sroih,D n uf,H S. H. Duffy, and D. Sarrouilhe, Bourmeyster, N., C., J. ´, 84 1909-1918. , 2004 889-897. , ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal il Chem. Biol. 20) elcl omncto nteotols/sect lineage. osteoblast/osteocyte the in communication Cell-cell (2008). RE12. , Cell ei.Cne Biol. Cancer Semin. 16 rc al cd c.USA Sci. Acad. Natl. Proc. x.Cl Res. Cell Exp. 3100-3106. , 74 20) inltasuto n ehnclstress. mechanical and transduction Signal (2004). 1065-1077. , 387 20) Repr:cl 14-3-3. call export: ER (2003). 473 94 1227-1236. , 233-244. , 188-192. , a.Rv o.Cl Biol. Cell Mol. Rev. Nat. .Cl Sci. Cell J. nu e.FudMech. Fluid Rev. Annu. 20) li n ouetasoti oe flow- bone: in transport solute and Fluid (2009). 168 20) ehnclsri pn onxn43 connexin opens strain Mechanical (2005). 20) 433poen nmmrn protein membrane in proteins 14-3-3 (2006). 21b.Gpjntosadhmcanl in hemichannels and junctions Gap (2012b). 16 19) utsbntasml fa integral an of assembly Multisubunit (1993). 422-430. , 173-182. , rg ipy.Ml Biol. Mol. Biophys. Prog. a 21) ai1rgltsitgi turnover integrin regulates Talin1 (2011). 20) eodtegp ucin of functions gap: the Beyond (2003). 5 .Cl.Physiol. Cell. J. b nue pnn fcnei 43 connexin of opening induces 1 122 109 1654-1664. , 3359-3364. , 20) dniiaino novel a of Identification (2001). 18) caelaigaddye and Scrape-loading (1987). nu e.Boe.Eng. Biomed. Rev. Annu. 20) tutrldeterminants Structural (2006). o.Cl.Biol. Cell. Mol. Oncogene ur Biol. Curr. .Bn ie.Res. Miner. Bone J. 4 ici.Bohs Acta Biophys. Biochim. 212 41 285-295. , 20) a junctional Gap (2007). 19) neato of Interaction (1996). 347-374. , 20) nintegrin- An (2009). 21) Osteocyte (2010). 207-214. , 94 20 13 31 ,29-65. 346-357. , R391-R393. , 3366-3377. , Nature (2012a). (2007). (1997). 145 376 Sci. 12 12 , , ,

Journal of Cell Science r,A . eme .P,Baka,B .adShat,M A. J. B. M. Warn-Cramer, and Schwartz, V. C. and Guyette, J., R. C. Wallick, B. A., T. Blackman, Freitas, J., P., D. Park, B. Helmke, W., A. Orr, S. Shikano, and Y. A. Okamoto, S. Goldstein, and N. Zilberberg, H., M. Butler, I., C. O’Kelly, S. Fagerholm, and G. C. Gahmberg, M., S. Nurmi, ARTICLE RESEARCH 146 M. Li, and H. Sun, B., Coblitz, S., Shikano, R., Kopajtich, A., Haas, E., Bruyneel, V., Corte, De B., Short, C., Preisinger, rti M3 n ly oei elmgaintruhissbtae14-3- substrate its through migration cell in role a plays 3zeta. and A. GM130 F. Barr, protein and J. 14-3-3 Gettemans, new A Connexin43: of analysis protein. vitro interacting in mode-1 and dynamics Molecular (2006). mechanotransduction. of Mechanisms co-receptor HIV of expression surface cell promotes GPR15. by proteins 14-3-3 reticulum of binding endoplasmic in retention overcomes signals. dibasic binding 14-3-3 transport. cells. T activated in integrin alphaLbeta2 Sci. and filamin both bind inl ietn elsraeexpression. surface cell directing signals 1090 .Cl Biol. Cell J. .Bo.Chem. Biol. J. 318-325. , Cell 164 111 1009-1020. , 286 577-588. , 7171-7181. , rti Sci. Protein 21) hshrlto-eedn C-terminal Phosphorylation-dependent (2011). 20) S1i ciae yteGlimatrix Golgi the by activated is YSK1 (2004). e.Cell Dev. 15 a.Cl Biol. Cell Nat. 20) eei slto ftransport of isolation Genetic (2005). 2344-2355. , 10 11-20. , 20) 433proteins 14-3-3 (2006). 7 985-992. , n.N .Acad. Y. N. Ann. 20) Forward (2002). (2006). un . ihle,K n cwpah B. Schwappach, H., and Leffers, K. A., Michelsen, Aitken, R., H., P. Yuan, Caron, S., Volinia, K., Rittinger, B., M. Yaffe, A. Aitken, Y., Soneji, B. G., M. Yaffe, G. Dodson, H., D. Jones, J., S. Smerdon, B., Xiao, M. L. Siperko, and H. F. Silver, ilrJcsn .J,Bra . u . i,X,Bnwl,L . pau,E. Sprague, F., L. Bonewald, X., Xia, S., Gu, S., Burra, J., A. Siller-Jackson, M. Li, and M. Wu, B., Coblitz, S., Shikano, h sebysau fmliei ebaeproteins. membrane multimeric of 646. status assembly the C. L. specificity. Cantley, binding and 14-3-3:phosphopeptide J. for S. Smerdon, J., S. Gamblin, hypothesis. anvil molecular the and pathways. signalling multiple of J. coordination S. chemical Gamblin, into and converted and matrix? sensed extracellular an energy in mechanical energy is how transduction: ees omcaia loading. mechanical membrane to release of X. J. Jiang, expression and surface and localization proteins. reticulum endoplasmic rnsCl Biol. Cell Trends ora fCl cec 21)17 3–4 doi:10.1242/jcs.133553 137–146 127, (2014) Science Cell of Journal 20) o o1-- rtiswr?–Gtkee phosphorylation Gatekeeper – work? proteins 14-3-3 do How (2002). 20) dpaino onxn4-eihne prostaglandin 43-hemichannel connexin of Adaptation (2008). 19) tutr fa1-- rti n mlctosfor implications and protein 14-3-3 a of Structure (1995). 16 370-375. , 20) ehnsnigadmechanochemical and Mechanosensing (2003). .Bo.Chem. Biol. J. rt e.Boe.Eng. Biomed. Rev. Crit. ESLett. FEBS 20) 433poen:rglto of regulation proteins: 14-3-3 (2006). Nature Cell 283 513 20) 433dmr probe dimers 14-3-3 (2003). 26374-26382. , 91 19) h tutrlbasis structural The (1997). 53-57. , 376 961-971. , 188-191. , ur Biol. Curr. 31 255-331. , 13 638- ,

Journal of Cell Science