6 otWsigo vne e ok Y102 USA. 10032, NY York, New Center, Medical Avenue, University Washington Columbia Fort USA. Medicine, 161 10065, of NY Department York, address: New *Present Avenue, York 1300 College, Medical Cornell Weill el onl eia olg,10 okAeu,NwYr,N 06,USA. 10065, NY York, New Avenue, York 1300 College, Medical Cornell Weill 30Yr vne e ok Y105 USA. 10065, NY York, New Avenue, York 1300 -53,Pdv,Italy. Padova, I-35131, USA. 10065, NY York, New Avenue, York 1300 ayn Dassah cells MaryAnn epithelial bronchial elasticity of pulmonary apoptosis VI, collagen and of secretion mediates A2 Annexin ARTICLE RESEARCH ß 828 2013 November 19 Accepted 2013; July 1 Received { the remodel 3 continuously epithelial lung, Polarized the 2011). mechanotransduction in 1 al., those and et as Lu signaling such 2009; for also cells, al., they pathogens, et conduit and (Bateman toxins cell a to barriers (LeBleu structural provide epithelial as provide tissue serve membranes basement and separate connective do support only Not underlying 2007). that al., assembled their et proteins intricately from of 100-nm- monolayers matrix to composed 50- structure extracellular electron-dense, an sheet-like is thick, membrane basement The INTRODUCTION Secretion VI, Collagen A2, Annexin WORDS: KEY of family conserved We function. evolutionarily unknown the largely an that observed of are proteins phospholipid-binding annexins The ABSTRACT neatosta nelenra umnr ucinadepithelial and function survival. pulmonary cell secretion normal COL6 cell–matrix underlie the establishes that pathway in interactions this ANXA2 that to for show compartment. further role leads VAMP2-positive and new pathway, ANXA2 late-Golgi, a define of a results in absence These SNARE COL6 that the of and of retention and membranes cells, vesicle COL6 epithelial secretory at bronchial with VAMP2 and associates SNAP-23 proteins ANXA2 that revealed microscopy immunoelectron and Immunoprecipitation disadhesion. xrietlrnewt mardln iseeatct,wihwas which elasticity, tissue in lung phenocopied impaired with tolerance exercise utmrcpoen O6sre oaco el obasement to that show cells we large Here, anchor secretion. multimerization to unusually undergoes prior to collagens, directs an other serves unlike ANXA2 COL6, and, membranes COL6 of that protein. secretion postulated multimeric cell and epithelial (COL6), bronchial VI collagen lacks otermti nesAX2epeso a restored. was expression ANXA2 poorly unless adhered and matrix vivo vesicles their intracellular to within COL6 retained ahrn .Hajjar A. Katherine uhrfrcrepnec ([email protected]) correspondence for Author eateto oeua eiie nvriyo aoa il .Bsi58/B Bassi U. Viale Padova, of University Medicine, Molecular of Department College, Medical Cornell Weill Biology, Developmental and Cell of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,8884doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. , AnxA2 2 / 2 rnha pteilclsudretaotssand apoptosis underwent cells epithelial bronchial Col6a1 AnxA2 4 eateto eitis el onl eia College, Medical Cornell Weill Pediatrics, of Department 1 eaAlmeida Dena , 1, 2 2 { / 2 / 2 ugbsmn ebaespecifically membrane basement lung mice. nvitro In AnxA2 5 eateto eei Medicine, Genetic of Department 2 eateto Medicine, of Department 1 eec Hahn Rebecca , , 2 AnxA2 / 2 iehv reduced have mice 2 / 2 fibroblasts 2, ,PooBonaldo Paolo *, In 07.I h ug h aeetmmrn cfodn is scaffolding membrane al., basement et the (LeBleu lung, are proteins the additional compliance of In and expression 2007). stability by whose refined superstructure, laminin Hammad, chronic and 2011). Lambrecht Leigh, asthma, 2006; and and al., Proud including (Cox et 2012; fibrosis Kranenburg disorders, interstitial 2011; and Erler, lung disease pulmonary an human obstructive accompany of composition membrane array basement 2011). in Zent, and Alterations epithelial (Pozzi support differentiation and proteins survival membrane cell-adhesion, basement component turn, its in proteolyzing proteins; and secreting by membrane basement erto fsratn-otiiglmla oisi h yeII type the in bodies lamellar surfactant-containing regulates of ANXA2 secretion cells, calcium-regulated cultured a In have function. to membrane-bridging appears S100A10 which and heterotetramer, ANXA2 a Intracellularly, form 2005). al., et and membrane-fusion (Gerke intracellular events in Flood implicated been 2009; has membrane-binding that conserved, al., proteins of family et a to belongs (Dassah 2011), Hajjar, complex al., receptor et Engvall fibrinolytic 1995; network al., microfibrillar et a Colombatti 2009; into 1986). al., associate et secretion, (Bateman ( classical upon form complexes then, COL6 crosslinked contrast, that 12-subunit highly sharp of collagens megadalton-scale in assembly However, other intracellular 2010). extensive many undergoes al., et to 2003). (Tooley al., similar fibrils et (Gelse is network stable This a into assembly basement extracellular ( most kilodalton- protomers of helical, triple 50% scale into about assembles mass, 2007). for protein al., accounts are membrane et perlecan which (LeBleu and IV, formation of Nidogen Collagen complex 2007). prior intracellular consists al., without et secreted example, form (LeBleu secretion for after that Laminin, ( 2007). chains assembly extracellular al., polypeptide by et followed (LeBleu maturation, and in synthesis dystrophy muscular Bo 2005; congenital al., 2010), Bethlem et al., et (Baker and (Telfer humans zebrafish Ullrich and fibers to 1988). 2003) (Bonaldo al., al., and mouse et collagen et the Irwin 1998; Keene in al., myopathy other 1986; et to al., lead to et COL6 in Engvall filaments Abnormalities 1990; membranes forms al., et basement collagen, (Bonaldo microfibrillar Specks anchor 2003; a al., COL6, et that Gelse 1995). 2010; al., al., to et et together capability (Bober which wall signaling (COL6), bronchial and the VI support collagen mechanical and elasticity, impart 2008) al., tropoelastin et perlecan, nidogen, (Manabe netrin, fibulin, agrin, by buttressed , otbsmn ebae oss faclae Vand IV collagen a of consist membranes basement Most nei 2(NA) opnn facl surface cell a of component a (ANXA2), A2 Annexin intracellular undergo polypeptides membrane basement Many 0 D) hc grgt noahxgnlplmronly polymer hexagonal a into aggregate which kDa), 800 3 tfnWorgall Stefan , , 0 D) hc r loscee before secreted also are which kDa), 500 nean 2011). ¨nnemann, 4,5 and , 00ka,which kDa), 2000 abc trimers

Journal of Cell Science hsooia eeac.W oe that secretion noted membrane We relevance. basement physiological in ANXA2 events membrane-fusion 2007). al., et Wang 2010; al., Umbrecht- et 1990; Jenck al., the et of (Nakata components complex (SNARE) both associated receptor are vesicle SNAP which as (VAMP2), well 2 as protein SNAP-25), membrane and (SNAP-23 25 proteins and acceptor 23 factor N-ethylmaleimide-sensitive soluble interacting by with membrane plasma the to granules secretory crosslink xmnto fln isefo etn ierevealed the mice within COL6 resting of and absence from apoptosis Specific high-level dropout. tissue cells, cell epithelial stiffness. lung bronchial pulmonary dysmorphic abnormal of and Examination tolerance exercise reduced ta. 04.I hs rcse,AX2appears ANXA2 processes, (Knop these bodies In Weibel–Palade 2004). factor al., cell Willebrand et endothelial von ultra-large from of exocytosis (Umbrecht-Jenckmultimers and cells 2010), chromaffin al., from adrenal et catecholamines in of vesicles release core 2007), dense al., et (Wang pneumocyte ARTICLE RESEARCH n O6wr dniidt eascae ihVM2and VAMP2 with associated be to identified were was ANXA2 COL6 cultured COL6 immunoprecipitation, and By and and compartment. In microscopy VAMP2-positive impaired, immunoelectron late-Golgi, was a vesicles. COL6 within retained of secretory secretion fibroblasts, cell bronchial COL6 of epithelial retention with correlated membrane basement ntebsso rvosfnig htipiaeAX2in ANXA2 implicate that findings previous of basis the On nvitro in esuh odfn h oeof role the define to sought we , AnxA2 nvivo in 2 / 2 ieexhibit mice nvitro in AnxA2 AnxA2 n its and , 2 2 / / to 2 2 NP2 ihnasceoyvscemmrn complex. membrane from vesicle muscle secretory skeletal a Interestingly, within SNAP-23 ewe h w eoye ntson.Uo vlainof evaluation differ Upon not shown). however, did (not exercise biomechanics, genotypes heart-to- post pulmonary and two and output, the baseline cardiac between at or rate ratios significant heart no body-mass resting revealed cardiac in echocardiography of difference Evaluation M-mode 1B). distinctly by (Fig. drying and function complete activity to times grooming longer post-exercise reduced showed (49.2 times recovery etitv umnr etltr defect. ventilatory to significant physiologically pulmonary leads a ANXA2 restrictive and of apoptosis absence the dropout, that cell enables and epithelial ANXA2 COL6, that site of show its secretion data specific interstitium, these the Together, in synthesis. of COL6 of accumulation demonstrated ossigo al 0mnt wm vra3we period, week mice, 3 a protocol over exercise swims an minute in 90 AnxA2 enrolled daily were of consisting mice littermate in When identified tolerance exercise Impaired RESULTS infcn erae nbt yai n ttccompliance static and dynamic both in decreases significant Fg 1C,D; (Fig. n 5 e group, per 5 AnxA2 2 / 2 ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal ieehbtdsgiiatylne post-swim longer significantly exhibited mice P 2 , / 2 .01,adcrepnigicessi both in increases corresponding and 0.0001), P iefo iesprt eeoyosmatings heterozygous separate five from mice 5 6 .1;Fg A.Cmae with Compared 1A). Fig. 0.018; . ess70.5 versus 3.5 opeedyesfrtandlittermate AnxA2 trained to for time dryness as complete measured times, recovery swim defect. a ventilatory and restrictive tolerance exercise associated reduced is with deficiency ANXA2 1. Fig. AnxA2 imcaia tde,ecp ,where P H, except studies, biomechanical (J). inspiratory capacity and (I) dampening resistance, tissue (H) airway and (G) elastance, dynamic (F) total tissue and compliance, (E) (D) whole-lung static and (C) dynamic single a for days 21 over means are Shown 6 days. 21 over averaged wmig CI ugboehnc in AnxA2 biomechanics Lung (C–I) swimming. 5 s.e., .3 ( 0.732 +/+ 2 +/+ n / 2 5 n itraepi ieafter mice pair littermate .()Ps-wmrcvr times recovery Post-swim (B) 5. and and 6 5 P AnxA2 0fralgroups). all for 10 AnxA2 . iue;mean minutes; 7.8 , AnxA2 AnxA2 .01frall for 0.0001 AnxA2 2 2 / 2 2 2 / 2 / / 2 2 2 mice AnxA2 / ie including mice, pairs mouse 2 ieshowed mice iealso mice A Post- (A) +/+ AnxA2 6 and s.e., 829 +/+

Journal of Cell Science n o o-iitdbocilrsceoycl)porsosada in and protrusions cilia cell) of secretory cell Clara paucity bronchiolar apical of non-ciliated lack (or membrane, basement compact discrete, 1 Toluidine-Blue-stained uniiaino tiigitniyoe eiso stained membrane of basement series peribronchiolar a in that COL6 over of noted staining intensity we staining sections, Upon of autofluorescence. quantification basement-membrane-associated of loss nt;mean units; aeilFg 1,) crn fclslnn rnhoe 65–85 within bronchioles cells lining cells of m Scoring S1B,C). Fig. material rnhoa pteilclsadterbsmn membranes. basement their and cells epithelial bronchiolar P hnta of that than irsoyfrhrdmntae h nitntntr fthe of nature indistinct the demonstrated AnxA2 further microscopy yai eitnewsas nrae nthe in increased 1E,F; also (Fig. was elastance resistance tissue dynamic and lung whole ARTICLE RESEARCH 830 and level in proteins expression membrane the basement determine bronchiolar al., the To of et 2H). (Vishwanatha distribution (Fig. to membrane proximity the close basement 1995) within in autofluorescent cells, mainly epithelial the localized bronchial was of portions ANXA2 basal that confirmed from mice bronchioles of Immunostaining in distribution COL6 Altered of staining eosin the and in Hematoxylin morphology cell epithelial Abnormal AnxA2 lung. matdb olgn n lsi Ease l,20) was 2000), al., et is (Evans which elastin and autofluorescence, in indistinct collagens membrane by the basement At imparted 2A,B). dense, time, (Fig. and and (BECs) same cells basophilia epithelial cellularity increased bronchiolar of flattened revealed levels normal other at ramification, sections as showed airway well cross-sections as However, bronchioles, mice terminal alveoli. through and bronchioles resting of arborization from tissue 1G; (Fig. o O6wsidsic nthe in staining pattern indistinct membrane was staining basement COL6 in S2), for difference Fig. no material showed (supplementary proteins six Although toolsi,krtn5 olgnI olgnI,lmnnA, proteins laminin seven IV, resting collagen these from tissue I, lung of in collagen COL6) then and pattern We 5, fibrillin-1 2008). keratin expression al., (11 (tropoelastin, had et the lung available (Manabe mouse proteins) adult compared were (16 (7 the in documented antibodies expression demonstrated been basement which staining for which and been which proteins), for for had those proteins), selected mouse expression of we database membrane-specific proteins, a matrix From S1). lung Table material (supplementary aeetmembranes basement nprtr aaiywssgiiatyrdcd(i.1J; (Fig. reduced the in restriction significantly ventilatory and stiffness tissue was P capacity 1I; (Fig. inspiratory increased Tissue was S1A). finally, Fig. dampening, material (supplementary methacholine of doses 1H; (Fig. change significant 5 ndaee ofre euto ntepooto fciliated of proportion the in reduction a confirmed diameter in m , , )cmae with compared 6) .0)(i.2) hs aasgetanraiisi both in abnormalities suggest data These 2G). (Fig. 0.001) .01.Tgte,teedt niae infcnl increased significantly indicated data these Together, 0.0001). AnxA2 2 2 / / 2 2 P 2 rnhoa aeetmmrn (supplementary membrane basement bronchiolar AnxA2 , splmnaymtra i.S;Fg 3A–C). Fig. S2; Fig. material (supplementary / AnxA2 AnxA2 2 6 .01,weesara eitnesoe no showed resistance airway whereas 0.0001), ug irrhclapoc a adopted was approach hierarchical a lung, s.e., 2 2 2 / AnxA2 2 / / 2 2 n ise(18.4 tissue AnxA2 5 AnxA2 ugtsu Fg CD.Eaiainof Examination 2C,D). (Fig. tissue lung rnhoe (8.3 bronchioles 6, m AnxA2 +/+ etosrvae h bec fa of absence the revealed sections m P P 2 5 +/+ , a,o vrg,33tmsgreater 3.3-times average, on was, / 2 AnxA2 .3) vnfloigincreasing following even 0.732), .1 Fg AC.Teedata These 3A–C). (Fig. 0.01) 2 rnhoe (47.5 bronchioles 6 / 2 Es(i.2,) Electron 2E,F). (Fig. BECs AnxA2 . ess5.6 versus 2.4 bronchiolar 2 AnxA2 / 2 AnxA2 6 +/+ ugadmroe the mirrored and lung .4,mean 0.34%, P and , +/+ P 2 AnxA2 .01,whereas 0.0001), , / 2 AnxA2 AnxA2 AnxA2 and .01.Total 0.0001). 6 os lung mouse 6 . arbitrary 1.6 2 3.7, AnxA2 +/+ 2 / 2 2 / / 6 2 2 mouse versus n lung lung s.e.; 5 2 / 8; 2 s16.5 vs O62wr iiihdin whole-lung and COL6a1 diminished reduced of levels were of protein total COL6a2 immunoblots that isoforms, revealed However, alpha3 homogenates and 4C). levels alpha2 transcript (Fig. in alpha1, differences significant no COL6 revealed respectively, the encode which ihn2010 mpntt tutrsta ee naeae 4- average, in on abundant were, more directed that times structures identified antibody punctate was nm material with 200–1000 Immunoreactive within confluence 4B). (Fig. 90% COL6 against at (mEFs) fibroblasts rbdsbellrmcooa n yooi rcin by fractions cytosolic and In microsomal immunoblotting. subcellular probed infcnl fetd(i.3K; (Fig. affected significantly 3J; (Fig. 3I; (Fig. P rsaiiywti h aeetmembrane. basement the deposition within processing, stability post-transcriptional or its in defect a reflected AnxaA2 lhuhsm O6wspeetin However, present 4F). was (Fig. COL6 compartments some both although in expected, as present, h euto nttlCL rti in protein COL6 total in reduction the O6i eandwti rpi-rtce irsmlcompartment microsomal trypsin-protected a within retained is COL6 the the a for in in explanation to increase potential dysfunction leads a pulmonary an COL6 providing and thus mature compliance recoil, of hyperelastic dynamic loss in that reduction al., indicate significant et data (Bonaldo these COL6 helical 1998), triple immunodetectable lack mice in biomechanics pulmonary of Evaluation the in stiffness Lung lcrndnesceoygaue eeams wc sabundant as mice, twice resting almost in from were tissue granules lung secretory in electron-dense BECs nonciliated evaluating In in eitnewsicesdi the in increased was resistance ltigfrclei CX,amcooa akr n lactate and marker, microsomal a (CNX), calnexin for Blotting ossetwt h nrae bnac felectron-dense of abundance in increased granules the secretory with consistent AnxA2 of analyses RT-PCR time we COL6, of secretion in ANXA2 stained of role potential the determine ihyerce in enriched highly crosslinked highly whereas in reduced (Engvall detected, markedly X-100 was Triton 1986), in was al., insoluble (Engvall et is protomeric, X-100 1986), which Triton COL6, of extracellular in al., abundance soluble is the et which in COL6, difference intracellular no Interestingly, ugs O6seii eeti ytei,poesn or processing synthesis, in defect in COL6-specific secretion a suggest Col6a1 epciey.A nthe in As were respectively). 3F,G; elastance tissue (Fig. and increased lung correspondingly whole both 3D,E; whereas (Fig. respectively), decreased significantly irsoy(i.4A). (Fig. microscopy the AnxA2 , AnxaA2 oietf h oaino O6within COL6 of location the identify To easse taysaesnhsso O6in COL6 of synthesis state steady assessed We AnxA2 .01,btara eitnesoe osgiiatchange significant no showed resistance airway but 0.0001), AnxA2 2 +/+ +/ 6 / 2 2 AnxA2 2 2 / .,mean 3.2, ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal 2 2 mouse ise Fg E.Bsdo hs aa ecnlddthat concluded we data, these on Based 4E). (Fig. tissues P P 2 / / iervae rfl eysmlrt htosre in observed that to similar very profile a revealed mice 2 2 , 5 / ugtsu.Sm-uniaieadqatttv real- quantitative and Semi-quantitative tissue. lung AnxA2 2 cells Escmae with compared BECs .01,weesisiaoycpct a not was capacity inspiratory whereas 0.0001), .7) isedmeigwsas increased also was dampening Tissue 0.276). os.Dnmc u o ttc opinewas compliance static, not but Dynamic, mouse. +/+ 2 and Col6a1 AnxA2 6 / 2 AnxA2 s.e., rnha pteilcells. epithelial bronchial AnxA2 AnxA2 AnxA2 Col6a1 2 2 +/+ / n 2 / AnxA2 2 5 os hncpe the phenocopies mouse 2 2 u not but , AnxA2 6–8, 2 / / ughmgnt,AX2was ANXA2 homogenate, lung P 2 2 , / 2 5 Col6a2 than AnxA2 AnxA2 Esosre yelectron by observed BECs 2 .69.Because 0.0639). P rmr os embryonic mouse primary Col6a1 / 2 2 P , / P AnxA2 2 AnxA2 AnxA2 , 5 .0) hs aawere data These 0.001). AnxA2 os,ttldynamic total mouse, mouse. 2 .01and 0.0001 +/+ and .06and 0.0076 AnxA2 / 2 2 AnxA2 Es(i.4) To 4A). (Fig. BECs 2 / 2 +/+ 2 Col6a3 Col6a1 / / 2 ug Fg 4D). (Fig. lungs +/+ 2 2 ug(i.3H; (Fig. lung el (62.7 cells AnxaA2 yoo,i was it cytosol, oprdwith compared / microsomes. , 2 2 / 2 2 ugtissue lung P Col6a1 transcripts, / P , 2 el,we cells, 5 0.0001, +/+ versus 0.578, 6 2 and 2.9 / 2

Journal of Cell Science EERHARTICLE RESEARCH D rnhoe atrduigaFT loecnefle ihU xiain(re;wiearwed niaebsmn ebae.(,)Toluidin (E,F) membrane). basement indicate arrowheads white (green; lung. excitation mouse UV AnxA2-deficient with the filter of in fluorescence sections proteins FITC thin matrix a stained extracellular using of captured distribution bronchioles in abnormal (D) staining and eosin morphology and Hematoxylin epithelial (A,B) bronchial Aberrant 2. Fig. G ifrnilcut fclae n o-iitdeihla el in cells epithelial non-ciliated and ciliated of counts Differential (G) etoso ugtsu mgdfrTF(re) NA rd n AI(le.Ist hwmgiidveso oe ra.Arwed niaebasement indicate Arrowheads areas. boxed of views magnified show Insets (blue). DAPI and (red) ANXA2 (green), 50 TAF bars: for Scale imaged membrane. tissue lung of sections AnxA2 m AD,10 (A–D), m +/+ E and (E) AnxA2 m AnxA2 EF,40 (E,F), m +/+ 2 A and (A) / 2 F rnhoa pteim lc n ryarwed niaeCaaadclae el,respectively. cells, ciliated and Clara indicate arrowheads gray and black epithelium; bronchiolar (F) m (H). m AnxA2 2 / 2 AnxA2 B rnhoe.(,)Tsu uoloecne(A,gen in green) (TAF, autofluorescence Tissue (C,D) bronchioles. (B) +/+ and AnxA2 2 / 2 rnhoe (means bronchioles ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal 6 s.e., n 5 6–8; P AnxA2 , .0) H Frozen (H) 0.001). +/+ C and (C) AnxA2 e-Blue- 831 2 / 2

Journal of Cell Science cl as 50 bars: Scale opine(E; compliance EERHARTICLE RESEARCH ihn6 iue.Fo hs aa ecnld htCOL6 that 832 However, conclude we hydrolysis. environment, data, microsomal trypsin-protected, tryptic these a within From accumulates by completely minutes. 60 almost affected within disappeared microsomal ANXA2 not shown), (not microsome-associated was digestion subjected tryptic native to COL6 full-length, next sensitive Whereas is 4G). using investigate COL6 we (Fig. proteolysis assay limited To trypsin-protection compartments, to a occupied homogenates fractions. lung extravesicular ANXA2 from demonstrated microsomes two or and marker, COL6 the intra- microsome-associated cytosolic of whether separation a complete (LDH), dehydrogenase in deficient is COL6 3. Fig. H n iwy()rssac,tsu apnn J n nprtr aaiy() aarpeetmeans represent Data (K). capacity inspiratory and (J) dampening tissue resistance, (I) airway and (H) defect. imcaisin biomechanics ugtsu from tissue lung A rznscin from sections Frozen (A) m P AnxA2 m. Col6a1 5 .7) hl-ugeatne(F; elastance whole-lung 0.578), +/+ +/ and 2 and AnxA2 AnxA2 AnxA2 Col6a1 2 2 / / 2 2 +/+ 2 iewsicbtdwt niCL g n hnlbldwt A.Arw niaebsmn ebae DK Lung (D–K) membrane. basement indicate Arrows DAB. with labeled then and IgG anti-COL6 with incubated was mice rnhoa pteilbsmn ebae n O6dfcec sascae iharsrcieventilatory restrictive a with associated is deficiency COL6 and membranes basement epithelial bronchiolar / 2 A and (A) ie hw r yai D n ttc()cmlac,woeln F n ise()eatne oa dynamic total elastance, (G) tissue and (F) whole-lung compliance, (E) static and (D) dynamic are Shown mice. AnxA2 P 5 .06,ara eitne(I; resistance airway 0.0076), 2 / 2 B os ugtsu tie o NA rd,CL gen n uli(AI le.()Mouse (C) blue). (DAPI, nuclei and (green) COL6 (red), ANXA2 for stained tissue lung mouse (B) hra NA cuisa nrtce,btneighboring but unprotected, an occupies location. ANXA2 whereas whole n NA rmwhole COL6 from both Anti-COL6 ANXA2 precipitated and IgG, assays. control isotype-matched not immunoprecipitation but IgG, conducted we and ANXA2 between COL6, association physical potential the address in cells To granules epithelial electron-dense bronchiolar on secretory associate nonciliated closely COL6 and ANXA2 g muorcpttdbt NA n O6fo whole from COL6 and ANXA2 both immunoprecipitated IgG P 5 .7)adisiaoycpct (K; capacity inspiratory and 0.276) AnxA2 ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal 2 / 2 6 ugetat Fg A.Smlry anti-ANXA2 Similarly, 5A). (Fig. extracts lung s.e. P , AnxA2 .01fralgntp ar,ecp o static for except pairs, genotype all for 0.0001 +/+ ugetat,adCL from COL6 and extracts, lung P 5 0.0639). n 5 o l groups. all for 9

Journal of Cell Science EERHARTICLE RESEARCH i.4. Fig. e etpg o legend. for page next See ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal 833

Journal of Cell Science infcn ifrnebtenCL in COL6 between difference significant mean respectively, 0.6 versus Fg D.I diin hra 1 fCL ed in beads COL6 of 71% whereas addition, In 5D). (Fig. O6basprAX2CL lse,weescutr in clusters whereas cluster, ANXA2–COL6 AnxA2 per beads COL6 F–NA uincntut(d-2 Fg F.A 72 expressed At was 5F). COL6 (Fig. that revealed (AdV-A2) within analysis construct or immunoblot (AdV-null) fusion hours, alone GFP–ANXA2 GFP either a encoding constructs adenoviral dense diameter, in electron analyses enclosing nm Parallel 5C). membrane vesicles anti-COL6- (Fig. 6 the intracellular material at large, and and located of nm clusters anti-ANXA2- borders in (10 mice, co-associated beads respectively) resting immunogold from of BECs conjugated cell In tissue epithelial studies. lung bronchiolar microscopy the immunoelectron conducted within COL6 indirect. and or direct either undergo be COL6 could and that ANXA2 association that physical suggest a results These 5B). (Fig. rndcin(i.5,lf ae) O6wsas eetdin detected also was COL6 panel). by secreted left matrix 5F, (Fig. transduction a,o vrg,7tmsmr O6in COL6 more 7-times average, on was, AnxA2 ape,cmae with compared samples, with compared samples i.4 O6i soitdwt lcrndnegaue n retained and granules electron-dense in with microsomes associated within is COL6 4. Fig. ARTICLE RESEARCH 834 of absence primary and presence transduced the in we secretion ANXA2, COL6 analyze mEFs by To secretion COL6 modulates association ANXA2 vesicles. physical secretory a within suggest COL6 also and in data ANXA2 associated between These vesicle 5E). were (Fig. 31% BECs vesicle- only secretory structures, membrane-bound, like with associated were BECs AnxA2 O6cmae with compared COL6 AnxA2 oa ubro lsee niCL-ojgtdbas Within beads. anti-COL6-conjugated the clustered AnxA2 in of reduction a number and total beads, anti-ANXA2-labeled of absence the uniaieR-C o O61(3 p,CLa 21b)adCOL6a3 and bp) Semi- (241 in (C) COL6a2 transcripts area. bp), bp) boxed (139 (865 of COL6a1 view for magnified RT-PCR shows quantitative Inset blue). (DAPI, nuclei P ihAVA,btnti arxpoue by produced matrix in not but AdV-A2, with ih ae) iial,primary Similarly, panel). right olmtdtytcdgsin N stelaigcnrl cl as 5 bars: Scale control. loading the subjected is and 25 CNX F, digestion. in tryptic as limited isolated fractions, to microsomes, cytosolic of and Immunoblot microsomal (G) for respectively. represent markers (LDH) and dehydrogenase controls microsomal lactate loading and and cytosolic (CNX) in and COL6 Calnexin fractions (F) fractions. soluble fractions. insoluble for for controls (FN) loading fibronectin represent (TE) in tropoelastin COL6 fractions. and of whole-lung Immunoblot -insoluble (E) and and controls. Triton-X-100-soluble Actin loading conditions. represent reducing (TE) under tropoelastin immunoblot normalization by for analyzed control was internal bars) an (light as qPCR served (means by bp) obtained (530 those GAPDH RT-PCR with (bottom). semi-quantitative compared by were obtained bars) genes (dark COL6 for ratios expression , ofrhreaut h hsclpoiiybtenANXA2 between proximity physical the evaluate further To m .0) B Eswr osandfrAX2(e) O6(re)and (green) COL6 (red), ANXA2 for co-stained were mEFs (B) 0.001). (B). m +/+ +/+ 2 2 +/+ 6 / / AnxA2 lf)and (left) 2 2 s.e., u ete rti from protein neither but , rnha pteilcls efuda vrg fsix of average an found we cells, epithelial bronchial 6 Escnanda vrg fol w mbeads nm 6 two only of average an contained BECs el rndcdwt d-ul ntemti,there matrix, the In AdV-null. with transduced cells . n 4.2 and 0.3 n 5 +/+ elcts.()CL rmwoeln homogenates whole-lung from COL6 (D) replicates). 3 AnxAa2 AnxA2 AnxA2 and AnxAa2 6 2 AnxA2 / AnxA2 2 +/+ AnxA2 s.e., 6 +/+ AnxA2 rgt Es(means BECs (right) 2 . ess0.3 versus 0.9 and / 2 el or cells BECs. 2 n 2 2 AnxA2 AnxA2 5 / 2 +/+ / / 2 2 3, d-ulsmls(4.2 samples AdV-null ape,ad1-ie more 14-times and samples, A ubro es rnlsin granules dense of Number (A) el,rgrls fviral of regardless cells, ape ( samples 2 AnxA2 P +/+ / 2 AnxA2 , AnxA2 AnxA2 eoye tp.Relative (top). genotypes .5.Teewsno was There 0.05). Estasue with transduced mEFs 6 AnxA2 AnxA2 2 . rirr units, arbitrary 0.1 6 2 / 2 2 AnxA2 2 / P s.e., / 2 / 2 2 el transduced cells . b tbln( -tubulin Esrevealed BECs 2 2 .)(i.5F, (Fig. 0.1) ugextracts lung n / Eswith mEFs / nvivo in 2 5 2 2 –1cells; 9–11 / AnxA2 2 AnxA2 AdV.rA2 AdV.rA2 el or cells b m -TBLN) (A), m 6 ,we 2 0.9 +/+ / 2 ooaiainin colocalization hwdCL ob rsn nVM2pstv electron-dense VAMP2-positive in present BECs in be structures of to microscopy immunoelectron COL6 time, showed same the At eosrt htAX2ascae ihSAEcomplexes, SNARE COL6. of with secretion associates enabling thereby ANXA2 that demonstrate iemtra n iorce neither Fig. in immunoreac- MitoTracker material anti-COL6 and of supplementary material colocalization tive no and found 6C We (Fig. S3C,D). (mEFs) fibroblasts aeilFg 4,) oehr hs aaidct htCL is COL6 compartment. VAMP2-positive that late-Golgi indicate a data within these retained Together, S4A,B). Fig. material 3 AP n ytxn a needn fANXA2. of independent that was revealed furthermore, syntaxin2 studies, and microscopy Immunoelectron SNAP- and VAMP2 COL6, between VAMP2 from association 23, the SNAP-23, samples that indicating precipitated parallel 2, syntaxin In anti-COL6 6A). extracts, IgG (Fig. VAMP2 lung SNAP-23, 2 anti-COL6 as syntaxin that well and as found ANXA2, the in we precipitated assays specifically of pull-down similarly, In members extracts, 2007). (Umbrecht-Jenck al., proteins whole-lung with et Wang fusion 2010; interact al., membrane et to pneumocytes, of alveolar reported family II SNARE been type has and ANXA2 neuroendocrine cultured in proteins In complex SNARE cells with epithelial interact bronchiolar COL6 and ANXA2 Both yimnfursec irsoy iN ncdw of of accumulation knockdown to led ANXA2 siRNA with interact to microscopy, known SNAREs immunofluorescence late-Golgi By a within COL6 compartment arrests VAMP2-positive proteins SNARE of Knockdown the differ not did with this between but immunoreactivity GRP94, marker found anti-COL6 (ER) reticulum we endoplasmic of addition, In colocalization S3C). slight Fig. material (supplementary mEFs the along points multiple at COL6 of in pathway location secretory the compartments analyze intracellular specific to for markers standard used We late-Golgi a in compartment COL6 VAMP2-positive arrests ANXA2 of Absence and SNAP-23 both with from BECs colocalized in VAMP2 specifically ANXA2 o ae) ycnrs,etaellrGPtgwsntdtce in detected not was with tag GFP association extracellular 5G, contrast, By (Fig. panel). conditions top nonpermeabilizing anti-GFP under Rhodamine-conjugated fixation by following detected extracellular was the it into where GFP space, secreted construct fusion GFP–COL6 a 3adVM2rltdbas(52 beads VAMP2-related and 23 splmnaymtra i.SD.Hwvr tiigfrthe colocalization within for significant reactivity anti-Col6 staining revealed with (TGN38) However, marker S3D). trans-Golgi Fig. anti-calnexin marker, material ER second (supplementary a for staining upon obtained were xrclua iiu(o hw) oehr hs data these Together, efficient mEFs. the for primary required into by shown). is COL6 GFP a ANXA2 of secretion of release (not with expression not that transduced demonstrate did milieu Cells alone panel). extracellular GFP encoding bottom construct 5G, (Fig. construct incmatet,clclzdwt niCL namne that in manner prominent a more in much anti-COL6 secre-was with post-Golgi colocalized within compartments, reside tion to considered generally VAMP2, tutrsin structures n 5 6 means 16; AnxA2 ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal AnxA2 AnxA2 6 +/+ .. eeascae ihsecretory-vesicle-like with associated were s.e.) AnxA2 AnxA2 and +/+ 2 AnxA2 / 2 u not but , AnxA2 +/+ AnxA2 2 Es(i.6) oehr hs data these Together, 6B). (Fig. BECs / 2 +/+ el Fg C.Fnly tiigfor staining Finally, 6C). (Fig. cells 2 AnxA2 Estasetdwt h same the with transfected mEFs / +/+ AnxA2 2 and AnxA2 Es(i.6) iia results Similar 6C). (Fig. mEFs 6 ug,weesfwrSNAP- fewer whereas lungs, AnxA2 2 1%, / 2 2 2 / 2 / hnin than AnxA2 2 n el,btol minimal only but cells, el (supplementary cells , 2 5 / 2 0vru 14 versus 10 os embryonic mouse +/+ AnxA2 and AnxA2 AnxA2 +/+ AnxA2 nsitu in mEFs. 6 1%, 2 2 +/+ / / 2 2

Journal of Cell Science EERHARTICLE RESEARCH evda h Pcnrl C ooaiaino NA 1 mgl atce n O6( m in nm) (6 IgG COL6 Non-immune and a2. particle) COL6a1, gold and ANXA2 nm for (10 immunoblotted ANXA2 were of IPs Colocalization anti-ANXA2 (C) Reciprocal control. (B) IP ANXA2. secretion. the and COL6 as a2 served mediates COL6a1, for and immunoblotted COL6 were with extracts interacts ANXA2 5. Fig. ih2 F.Scee O6wslbldwt niGP(e) nrni F loecnewsqece ih5 MNH mM 50 with quenched was fluorescence GFP index Intrinsic an (red). as anti-GFP served with expression labeled GFP was conditions. COL6 reducing Secreted under PFA. immunoblot 2% by with analyzed (means vectors infection. granules AdV-A2 cell dense-core or of with AdV-null associated GFP-encoding COL6) with and transduced ANXA2 (both beads of xaddi nes D ubro O6rltdbasprcutrin cluster per beads COL6-related of Number (D) insets. in expanded nrclua gen essetaellr(e)dsrbto fCL.Saebr:20n C,75 (C), nm 200 bars: Scale COL6. of distribution (red) extracellular versus (green) intracellular b TL niae h elfato n N h arx G Eswr rnfce ihaCLA–F uincntutadfxda 8hours 48 at fixed and construct fusion COL6A1–GFP a with transfected were mEFs (G) matrix. the FN, and fraction cell the indicates -TBLN AnxA2 A niCLa n O62imnpeiiae Is rmwoeln lysate whole-lung from (IPs) immunoprecipitates COL6a2 and Anti-COL6a1 (A) +/+ and 6 s.e., AnxA2 n 5 2 8, ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal / 2 P m Es(means BECs 5 (G). m AnxA2 .00) F ellstsadmti xrcsfo mEFs from extracts matrix and lysates Cell (F) 0.00008). +/+ and 6 AnxA2 s.e., n 2 5 / 2 8, Es rohasidct areas indicate Arrowheads BECs. 4 l egdiae show images Merged Cl. P 5 .3) E Proportion (E) 0.038). 835

Journal of Cell Science EERHARTICLE RESEARCH 836 ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal oprmns including compartments, intracellular specific for markers standard with stained mEFs (C) insets. in expanded areas indicate Arrowheads nm). (6 ANXA2 and particles) gold nm (10 VAMP2 panels, Bottom particles). gold nm (10 ANXA2 and particles) gold nm (6 SNAP-23 panels, Top labeling. Immunogold (B) syntaxin-2. and VAMP2 SNAP-23, ANXA2, COL6a1,a2, against directed antibodies with immunoblotted were lysates whole-lung from immunoprecipitates COL6a1,a2 compartment. VAMP2-positive late-Golgi a from COL6 of release enables and SNAREs with interacts ANXA2 6. Fig. 10 (B), nm 200 bars: Scale VAMP2. as well as S3D), S3C, Fig. material (supplementary (ER) CNX and MitoTracker and (trans-Golgi) TGN38 (ER), GRP94 m (C). m A Anti- (A)

Journal of Cell Science EERHARTICLE RESEARCH i.7. Fig. e etpg o legend. for page next See ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal 837

Journal of Cell Science ecridotbocolelrlvg BL Fg A.BAL 8A). (Fig. (BAL) lavage from bronchoalveolar fluid out carried we with arpae (62 macrophages 10 ciae aps--oiieeihla el than cells epithelial caspase-3-positive activated pteilclsi li eoee from of recovered fluid excess in an cells revealed epithelial however, counts, cell Differential retrieved. mean (152 cells of concentrations htAX2mdae erto fCL sntlmtdt the fibroblasts. to tissue limited in occurs not also is but cell, COL6 epithelial of suggestbronchial data secretion These 5F,G). ANXA2-mediated Fig. and and that 4B accumulated (Fig. also COL6 which is secrete to mEFs, finding failed precise on This studies the located. our with are cross-section, consistent fibroblasts in interstitial where cut location myofibers COL6 individual of between accumulation an revealed source staining main immunofluorescence (Bo the are COL6 fibroblasts interstitial of tissue, this In 8E). (Fig. rnfce ihmc iN eesandfrVM2 O6adDAPI and COL6 VAMP2, mEFs for A. stained in were as siRNA analyzed mock mEFs siRNA with VAMP2 from transfected Lysates and (ER), (C) control CNX DAPI. with and with transfected COL6 VAMP2 mEFs for or (B) stained (trans-Golgi) (bottom). were TGN38 were blue) siRNA siRNA (DAPI, SNAP-23 mock nuclei with with and transfected transfected COL6 mEFs SNAP-23, (top). for control stained immunoblotting. loading by a analyzed as were served siRNA late-Golgi a SNAP-23 and in COL6 control arrests proteins compartment. SNARE VAMP2-positive of Knockdown 7. Fig. ARTICLE RESEARCH 838 a and conducted matrix we COL6-dependent, extracellular be to might adhesion membrane cell basement whether cells determine ANXA2-deficient in To adhesion cell–matrix defective rescues COL6 we tissues, apoptosis other from in muscle seen or of skeletal cell examined epithelial bronchial rate the to unique increased an 8C,D). (Fig. confirming bronchioles, ciliated basement of their proportion and reduced the a in BECs cells with both association in in membranes, abnormalities observed and We apoptosis dropout, BEC defects in secretion results ANXA2 COL6. of of Loss secretion the in and role SNAP-23 functional both that a 7A,B) indicate play data (Fig. VAMP2 These SNAP-23 7C,D). either (Fig. VAMP2 of or depleted anti-TGN38 cells and in anti-COL6 of immunoreactivity association increased in found seen we as cells, and Specifically, 7). (Fig. COL6 intracellular bars: Scale DAPI. and COL6 SNAP-23 for or stained (trans-Golgi) 20 were TGN38 siRNA (ER), VAMP2 GRP94 with and transfected mEFs (D) (bottom). AnxA2 pteilcl deinado uvvli the in survival and/or adhesion compromises cell membrane basement epithelial bronchiolar the in alteration an 02.W,teeoe odce UE tiigo lung acid of nucleic staining increased dramatically TUNEL al., revealed et conducted Taddei which therefore, 2001; sections, Screaton, We, and 2012). (Frisch (anoikis) apoptosis in procedure. disadhesion lavage cell the epithelial to bronchial of response rate higher a suggested raaewti rnha pteilclsof cells epithelial bronchial within breakage 6 odtriewehrtesceinealn oeo NA is ANXA2 of role secretion-enabling the whether determine To oso elmti deinmgtidc pteilcell epithelial induce might adhesion cell–matrix of Loss m m. 4%, AnxA2 2 6 / 2 s.e., n 5 rnhoa pteilclscnandtiea many as twice contained cells epithelial bronchiolar AnxA2 AnxA2 +/+ 6, nean21;Zue l,20) Interestingly, 2008). al., et Zou 2011; ¨nnemann n 5 ug Fg B.Frhrsann eeldthat revealed staining Further 8B). (Fig. lungs P 6 2 5 +/+ )wt iia oa oue fBLfluid BAL of volumes total similar with 6) 0v 87 vs 10 / 2 .0) n orsodn euto in reduction corresponding a and 0.001), and os Fg AG.T eemn whether determine To 2A–G). (Fig. mouse AnxA2 6 6 A yae rmmF rnfce with transfected mEFs from Lysates (A) 4v 158 vs 14 3%, 2 AnxA2 / n 2 5 iecnandequivalent contained mice AnxA2 6, 6 +/+ 4cells/ 14 P 5 and AnxA2 2 .02.Ti result This 0.0002). / AnxA2 2 AnxA2 ie(34 mice m ,respectively; l, 2 / 2 2 / AnxA2 b compared AnxA2 2 2 -tubulin / 2 6 mouse, 2vs 12 mice 2 +/+ / 2 ise efuddmnse vrl O6poenlevels, protein within COL6 in COL6 fraction of overall microsomal retention trypsin-protected, and a diminished COL6 matrix-associated found reduced we tissue, ymrhlg n ppoi,tgte iha altered equivalent cell in an Despite COL6 epithelial COL6. with for lacked levels together bronchial that mRNA apoptosis, membrane revealed basement and studies dysmorphology histological eemr eeeyprubdin perturbed severely more were respiration. to of muscles due of compromise disorders to in addition earlier the insufficiency that suggest respiratory COL6 studies a These of 2006). value had predicted al., development the ambulatory, et of Kooi still 70% der than were less (van was whom that capacity of vital with forced of patients all of loss myopathy, 16% muscular Similarly, follows respiratory Bethlem 2009). typically Duchenne al., et failure from (Nadeau from respiratory ambulation which suffered difference in nearly distinct patients dystrophy study, recent a of still-ambulatory a out insufficiency, In of frequently myopathy. of is 50% degree that the defect to ventilatory proportion restrictive intolerance. a can the exhibit exercise which in dystrophy, mutations from muscular and result congenital Ullrich dysfunction with Humans respiratory observed erto nthe in secretion secretion unique COL6. a macromolecular normal mediating for that or by similar and pathway lung, indicate survival SNAP-23 mouse a the data cell either in epithelial these biomechanics found which bronchial Together, promotes in depleted. We ANXA2 cells been of membrane. had in of VAMP2 absence COL6 plasma of components secretory the the accumulation of with all fusion VAMP2-positive In impaired late-Golgi, vesicles are suggesting a machinery. strongly in which compartment, accumulated by exocytotic COL6 2, ANXA2, reversed epithelial SNAP-23, syntaxin proteins be SNARE the and the could with complex VAMP2 a that ANXA2 form that COL6 revealed and secretion and experiments Co-immunoprecipitation microscopy COL6 expression. immunoelectron ANXA2 to of restoration block a rBAcae oytrn.Itrsigy however, Interestingly, in defect the corrected polystyrene. completely AnxA2 COL6 of BSA-coated supplementation or by produced matrices to well 60–70% adhered Whereas genotypes AnxA2 8F). both of (Fig. cells using of assays cell-adhesion of series ncultured In osceeCL sascae ihatnae cell–matrix attenuated the In pulmonary tolerance. with exercise significant reduced associated physiologically with stiffness and is apoptosis collagens, COL6 adhesion, other Failure ANXA2 secrete not membranes. of basement but into to deficiency COL6, cells that epithelial of bronchial time secretion from first aberrant the in for results reveal data of These deficiency a DISCUSSION with associated is cells COL6. epithelial bronchial to superior were mEFs uprscl deint xrclua arxsbtae,and substrates, by displayed matrix adhesion matrix extracellular reduced the to that suggest adhesion cell supports Col6a1 arcspoue by produced matrices norploayfnto tde,eatneadcompliance and elastance studies, function pulmonary our In nipratqeto swehrteipimn fCOL6 of impairment the whether is question important An 2 +/+ uain ih elc oso umnr opinein compliance pulmonary of loss reflect might mutations 2 / 2 / 2 ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal E deint S.Teedt niaeta COL6 that indicate data These BSA. to adhesion mEF Es ny2–0 fec eoyeahrdto adhered genotype each of 20–30% only mEFs, ie hsrsl ugssta NA ih lya play might ANXA2 that suggests result This mice. AnxA2 AnxA2 2 / 2 AnxA2 AnxA2 2 irbat,mroe,w uncovered we moreover, fibroblasts, / 2 COL6A1 os ssfiin oepanthe explain to sufficient is mouse 2 2 / / 2 AnxA2 2 AnxA2 Esi deigt uncoated to adhering in mEFs Es naddition, In mEFs. , 2 COL6A2 2 / 2 / 2 AnxA2 and iecmae with compared mice or AnxA2 AnxA2 2 COL6A3 / 2 ugcells. lung AnxA2 AnxA2 2 +/+ / 2 lung, lung loci, 2 +/+ / 2

Journal of Cell Science EERHARTICLE RESEARCH i.8. Fig. e etpg o legend. for page next See ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal 839

Journal of Cell Science arwed niaeeihla el) B UE tiigof staining TUNEL (B) cells). epithelial indicate (arrowheads pteilcls(P n arpae M)i A li from fluid (Ly), BAL lymphocytes in of (MP) macrophages counts and cell (EP) differential cells epithelial and muscle. cell Blue) skeletal reduced interstitial (Trypan Total in apoptosis, COL6 (A) with of associated secretion is impaired and ANXA2 adhesion of Absence 8. Fig. ARTICLE RESEARCH 840 Ca a in SNAP-23 to binds ANXA2 In 2012). process, al., et (Dietl this membrane plasma the with bodies fusion lamellar upon al., of SP-D, and et SP-A proteins and Pulido phospholipids a surfactant, of annexin-A2– release complex Rojo pneumocytes alveolar the II 2004; type lung, as al., the In well 2011). et as (Knop VAMP3, heterotetramer S100A10 and 4 of including proteins, syntaxin cultures SNARE or primary SNAP-23, requires In process histamine 2009). this cells, Sadler, thrombin, endothelial 2002; membrane Hogg, as plasma the and with such (Pimanda fusion trigger stimuli, that factors specific complement specialized to within in exocytosis response by pre-packaged released and bodies, are Weibel–Palade the structures, (vWF) Factor Willebrand (Lamande myopathy, secretion and Bethlem its in and prevent as 1986) (Colombatti also assembly, al., COL6 et vesicles perturb Engvall that 1995; secretory al., mutations et within Colombatti 1987; secretion Bonaldo, occurs then polymerization to extensive which prior This dimers, complexes. of disulfide-stabilized 12-chain pairing to form Antiparallel rise 1983). gives al., higher- monomers et form COL6a3), (Furthmayr that be polymers and monomers order into genetically must COL6a2 three intracellularly (COL6a1, of COL6 assemble (Bateman often which chains most that vesicles composed polypeptide is surprising distinct COL6 secretory not 2009). matrix not al., is specialized other et perhaps most that within or COL6 is pre-assembly prepackaged collagens membrane of it other Because basement of level proteins, secreted. secretion and high additional for abnormally possibility, a required one involves Our is this least secretion assembly. with fibulin-2, at consistent intracellular protein, that are high-level indicate proteins data matrix undergo additional unpublished of must secretion regulating that in role larger Rt h og n6–0n otpoencmlx(COP)II- with complex fusion vesicle membrane protein to plasma prior coat the vesicles, to nm COPI-coated Golgi similar-sized the 60–90 in from then in and vesicles, Golgi coated the to ER Su and Rizo 2012; al., et SNARE-related support (Kasai exocytosis or regulate be that might proteins ANXA2 diverse that appears the among it Thus, 2007). al., et (Wang manner ciiy(C,gen in caspase- green) Cleaved (CC3, (C,D) activity respectively. 3 controls, negative and positive are TdT rd n O6(re) F deinof Adhesion (F) (green). COL6 and (red) and ebaesceoygauecnat(ruze l,1978; ultralarge al., cell, endothelial et the complex, In (Creutz 2010). ( protein plasma- al., contact of et points SNARE Umbrecht-Jenck at granule syntaxin, a and through membrane-secretory VAMP2 SNAP-25, to molecules of consisting of manner classes S100A10-dependent catechol three protein granule-associated least SNARE with at interactions of exocytosis cross- of images Confocal (E) blue). of (DAPI, sections nuclei and (red) E-cadherin AnxA2 nBAaoevru S O6.Saebr:50 bars: Scale COL6). + BSA versus alone BSA on arcs tahdclswr eoe n xrse sapretg of percentage a as (means expressed plated and cells removed total were cells Attached matrices. , nteboytei-ertr aha,poen htl rmthe from shuttle proteins pathway, biosynthetic-secretory the In NA a enfudt atcpt nteregulated the in participate to found been has ANXA2 000ka utmr ftepoogln rti von protein procoagulant the of multimers kDa) 20,000 ANXA2 2 / 2 Es(le.Ednces-rae etosadsmlslacking samples and sections Endonuclease-treated (blue). BECs AnxA2 2 / 2 bto)mc.Dt ersn means represent Data mice. (bottom) +/+ and 6 AnxA2 AnxA2 s.e.; mns NA rnlctsi an in translocates ANXA2 amines, +/+ 2 P / , 2 .3 o oprsnof comparison for 0.039 C and (C) udiesmsl aee o ANXA2 for labeled muscle quadriceps .Drn ees fchromaffin of release During s. ´ AnxA2 ta. 1999). al., et AnxA2 do,2012). ¨dhof, +/+ 2 and / m 2 m. 6 D Eslbldfor labeled BECs (D) AnxA2 s.e., AnxA2 AnxA2 AnxA2 2+ n 2 5 -dependent / 2 6) Esto mEFs +/+ 2 +/+ / 2 (top) cells and xeiet ihebyncfbolssrvae impaired and to revealed genotype lavage, either fibroblasts bronchoalveolar of cells embryonic to of airway adhesion response with of Clara-like dropout in experiments by confirmed cells replacement studies epithelial Further indicating of cells. pulmonary proportion possibly progenitor the dramatic in a cells, reduction appearance, striking most flattened ciliated a and dense, the index a apoptotic displayed high among cells These were findings. cells epithelial tde,idctn htclua deint O6i dependent is COL6 previous with to consistent adhesion finding cellular upon a that is indicating This studies, COL6. of addition by usrt ol ersoe otelvlse for seen level the to restored be could substrate xgnseiso eetv euaino uohg (Grumati 2003). autophagy al., et reactive of Irwin of regulation 2010; al., defective induction et or integrin-mediated species by which oxygen triggered pore, cyclosporine- be transition the might of permeability opening reflect mitochondrial to A-sensitive, thought process a in death arx mardahso of adhesion Impaired matrix. O4apa ob omlylclzdi h bronchial the in localized and This normally COL1 the export. of be because membrane COL6, basement to defying epithelial to appear unique thus ANXA2, COL6 COL4 be of of vesicles, might absence step mechanism the late-Golgi VAMP2- final In within a accumulates positive 2012). COL6 the and fails al., in fusion membrane et VAMP2, SNAREs (Kasai fusion with secretion and/or which interacts vesicles interacting of with appears docking turn in by ANXA2 membrane ANXA2. plasma in enable model and higher-level, to interacts which SNAP-23 extravesicular a protein, even with vesicular propose COL6 undergoes transmembrane We which intravesicular assembly. COL6, tetrameric of Our pos export 2011). additional an Erlmann, that suggest in and data resulting (Malhotra coating, TANGO vesicles membrane at COPII larger-diameter assemble delay as to (cutaneous and appear sites cTAGE5 ER-exit such 5) triple- and antigen lymphoma-associated organization) proteins nm T-cell Golgi 300–400 ‘monomers’, and form (transport rod-like which and collagens, helical COL7, other accommodate to possibly However, membrane. plasma the iahso nue ppoi eldahi anchorage- whereby in anoikis, death to due cell is apoptotic that loss induces cell disadhesion suggested cells h neato ewe arxascae O6adcell- and COL6 matrix-associated between membrane-associated on specifically interaction depends viability soluble the cell fibroblasts, Interestingly, corneal (Ru and avian Bax fibroblasts In 2012). protein, serum-starved pro-apoptotic Frisch of the al., apoptosis suppressing 2008; by prevent et to Giannoni, appears Taddei COL6 and 2001; (Chiarugi Screaton, cells dependent rvnino ppoi eldootadnra pulmonary normal membrane, and basement dropout to cell adhesion apoptotic function. cell of subsequent secretion prevention could to fundamental COL6, is proteins membrane ANXA2 of that In matrix show elastance. data basement and our normal compliance summary, pulmonary secreting normal additional to contribute of cells that deposition of compromise loss related lrvoe raito rery u o ae iseinfarction, al., et tissue Peters 2012; inducers, late, al., In et not chemical Luther 2011). 2011; but to al., et and early, (Cheng due neurons respectively or apoptosis chondrocytes, irradiation protect from ultraviolet to myocytes appears cardiac also COL6 and norsuisi the in studies our In h ihlvlo ppoi in apoptosis of level high The b 1 nern Pafe l,19) utemr,teCOL6- the Furthermore, 1993). al., et (Pfaff integrins ora fCl cec 21)17 2–4 doi:10.1242/jcs.137802 828–844 127, (2014) Science Cell of Journal Col6a1 2 / 2 b ie yfbr neg ppoi cell apoptotic undergo myofibers mice, AnxA2 nern Hwl n on,1998), Doane, and (Howell integrins 1 AnxA2 2 / 2 os,cagsi bronchiolar in changes mouse, AnxA2 -og dpainfacilitates adaptation t-Golgi 2 / 2 AnxA2 Est albumin–matrix to mEFs AnxA2 2 / 2 rnha epithelial bronchial 2 2 / h ta. 1999). al., et ¨hl AnxA2 2 / 2 mouse. mEF-derived +/+ mEFs

Journal of Cell Science aswscluae sn h rlt lis oml Ln ta. 2005). al., et (Lang formula ellipse prolate the using formula calculated the was from mass derived was output Cardiac ifrnebtenVl n oS jcinfato ( fraction Ejection VolS. formula: and the using VolD calculated between difference eepae ncgscnann omlbdigadmitie t21 at maintained and bedding normal containing cages in placed were AnxA2 Mice METHODS AND MATERIALS ARTICLE RESEARCH hne n Vectto.Frimnhsooy %paraformaldehyde 4% FITC-filter immunohistology, green Unstained For a excitation. eosin. using UV autofluorescence and and by channel hematoxylin imaged also with were stained sections initially were Sections histology and processing Tissue ( determine resistance to served (airway) oscillations resistance Newtonian forced tissue Broadband Single- obtained model. determine capacity. compartment to measurements residual used functional were pressure ( oscillations and plateau forced capacity Flexivent frequency lung the using total Salazar– to the between assess analyzed using equation to determined were was Knowles used compliance and Static were (Scireq). mechanics perturbations software respiratory Three ventilator and Canada). baseline animal tracheostomized computer-controlled Montreal, were a (Sireq, that using i.p.) ventilated xylazine; mechanically mg/kg mg/kg (125 15 mice anaesthetized pentobarbital, on evaluated were mechanics Respiratory biomechanics Pulmonary seven of chest anterior depilated the was gel ultrasound to prewarmed handling, applied to acclimatization of days 3 After evaluation Cardiac room- stationary containing tank plexiglass a (23 in temperature place took testing Swim testing Exercise itraepiso iecosbe xesvl oteC57BL/6 the to Either extensively 2004). cross-bred al., mice et used. 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Journal of Cell Science ig .S,Nln .J,Go . aaae,A . a,Z,Rsnas Z., Rosenwaks, Z., Cao, O., A. Babazadeh, P., Guo, J., D. Nolan, S., B. Ding, T. J. Erler, and R. T. P. Cox, Bonaldo, and T. M. Mucignat, A., Colombatti, P. Bonaldo, and A. Colombatti, E. Giannoni, and and L. P. Y. Chiarugi, Liu, H., W. Chang, T., H. Huang, E., Hwang, C., Y. Lin, H., I. Cheng, it,P,Hle,T n rc,M. Frick, and T. Haller, P., A. Dietl, K. Hajjar, and K. He, B., A. Deora, M., Dassah, B. H. Pollard, and J. C. Pazoles, E., C. Creutz, Bressan, and D. Bo Volpin, S., Piccolo, M., Zanetti, P., Braghetta, P., A. Bonaldo, Colombatti, and R. Doliana, F., Bucciotti, V., Russo, P., Bonaldo, Mo and M. Collin, C., Enochsson, M., Bober, Lamande and P. R. Boot-Handford, F., J. Bateman, L443 L443adH009 oKAH;T2H072-0t ..;the M.D.]; to numbers T32-HL007423-30 [grant K.A.H.; Health to of HL090895 Institutes and National the HL046403 by HL042493, supported was research This Funding interests. competing no declare authors Thea stages. interests all at Competing manuscript the on discussed commented authors and All the implications, manuscript. conceived results, the jointly co-wrote the and K.A.H. project, tests. the function supervised pulmonary study, the analyzed and planned ARTICLE RESEARCH ae,N . Mo L., N. Baker, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.137802/-/DC1 online available material Supplementary months. 12 material after release Supplementary for PMC Foundation in Hartwell Deposited a M.D. and K.A.H.]; to to Fellowship 6-FY12-356 Postdoctoral number [grant Dimes of March ooa,M . i,M . ig,P,Rnset . ree,R,Hh,R,Wang, R., Hahn, R., Kraemer, T., Ringstedt, P., Wiegn, I., M. Lin, J., M. Donovan, es,K,Po K., J. 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Physiological American MD: Bethesda, Kregel). uc,T . ’er,D . ak,C . ol,D . aaa,A .e al. et W. A. Ra’anan, C., D. Poole, M., C. Parks, obstructive S., (2006). D. chronic O’Leary, I., S. T. in Musch, H. proteins Sharma, matrix and I. extracellular disease. W. of pulmonary Boer, expression cells. de T., endothelial bronchial from K. tPA, V. not Alagappan, but vWF, of secretion regulated J. in this EMBO role for a function play anchoring an suggests cartilage and network. skin filamentous human in collagen n noddtil ei fclae yeVI. type collagen of helix triple unfolded and R. dystrophy. muscular congenital Ullrich of ofgrtosudryn h iest fexocytosis. of diversity the underlying configurations dynamics. membrane to iseEg atA Part Eng. 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