The VE-cadherin cytoplasmic domain undergoes proteolytic processing during endocytosis

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1 The VE-cadhern cytoplasmc doman undergoes proteolytc processng durng endocytoss Wenj Su, Emory Unversty Andrew Kowalczyk, Emory Unversty Journal Ttle: Molecular Bology of the Cell Volume: Volume 28, Number 1 Publsher: Amercan Socety for Cell Bology , Pages Type of Work: Artcle Fnal Publsher PDF Publsher DOI: /mbc.E Permanent URL: Fnal publshed verson: Copyrght nformaton: 2017 Su and Kowalczyk. Ths s an Open Access work dstrbuted under the terms of the Creatve Commons Attrbuton-NonCommercal-ShareAlke 3.0 Unported Lcense ( Accessed February 12, :17 AM EST

2 MBoC ARTICLE The VE-cadhern cytoplasmc doman undergoes proteolytc processng durng endocytoss Wenj Su a,b and Andrew P. Kowalczyk b,c,d, * a Graduate Program n Bochemstry, Cell and Developmental Bology, b Department of Cell Bology, c Department of Dermatology, and d Wnshp Cancer Insttute, Emory Unversty, Atlanta, GA ABSTRACT VE-cadhern traffckng to and from the plasma membrane has emerged as a crtcal mechansm for regulatng cadhern surface levels and adheson strength. In addton, proteolytc processng of cadhern extracellular and cytoplasmc domans has been reported to regulate cadhern adheson and sgnalng. Here we provde evdence that VE-cadhern s cleaved by calpan upon entry nto clathrn-enrched domans. Ths cleavage event occurs between the β-catenn and p120-bndng domans wthn the cadhern cytoplasmc tal. Of nterest, VE-cadhern mutants that are resstant to endocytoss are smlarly resstant to cleavage. Furthermore, p120-catenn overexpresson blocks cadhern nternalzaton and cleavage, couplng entry nto the endocytc pathway wth proteolytc processng. Of mportance, the cleavage of the VE-cadhern tal alters the postendocytc traffckng tnerary of the cadhern, resultng n a hgher turnover rate due to decreased recyclng and ncreased degradaton. In concluson, ths study dentfes a novel proteolytc event that regulates the traffckng of VE-cadhern after endocytoss. Montorng Edtor Jeffrey D. Hardn Unversty of Wsconsn Receved: Sep 13, 2016 Revsed: Oct 17, 2016 Accepted: Oct 19, 2016 INTRODUCTION Vascular endothelal cells form a lnng on the nteror surface of blood vessels and play mportant roles n thromboss, vascular permeablty, and nflammaton (Boulanger, 2016). Endothelal cell cell adheson s essental for normal endothelal barrer functon and mmune responses (Dejana and Orsengo, 2013; Gavard, 2014). Adherens junctons are the major adhesve cell cell junctons n endothelal cells and are crtcal for endothelal barrer propertes and for angogeness durng development, wound healng, and tumor growth. Vascular endothelal cadhern (VE-cadhern) s the major adheson molecule n endothelal adherens juncton (Dejana and Orsengo, 2013; Lagendjk and Hogan, 2015). As a member of the classcal cadhern famly, VE-cadhern medates homophlc adheson Ths artcle was publshed onlne ahead of prnt n MBoC n Press ( on October 26, *Address correspondence to: Andrew P. Kowalczyk (akowalc@emory.edu). Abbrevatons used: ALLM, N-acetyl-Leu-Leu-methonal; β-me, β-mercaptoethanol; CBD, catenn-bndng doman; E-cadhern, epthelal cadhern; EEA1, early endosome antgen 1; GFP, green fluorescent proten; JMD, juxtamembrane doman; MEC, mcrovascular endothelal cell; N-cadhern, neural cadhern; p120, p120 catenn; RFP, red fluorescent proten; VE-cad and VE-cadhern, vascular endothelal cadhern Su and Kowalczyk. Ths artcle s dstrbuted by The Amercan Socety for Cell Bology under lcense from the author(s). Two months after publcaton t s avalable to the publc under an Attrbuton Noncommercal Share Alke 3.0 Unported Creatve Commons Lcense ( -nc-sa/3.0). ASCB, The Amercan Socety for Cell Bology, and Molecular Bology of the Cell are regstered trademarks of The Amercan Socety for Cell Bology. through cadhern repeats n the extracellular doman, whle the cytoplasmc doman assocates wth lnker molecules that stablze the cadhern and couple the adheson molecule to the actn cytoskeleton (Gavard, 2014; Cadwell et al., 2016b). Cytoplasmc bndng partners for VE-cadhern nclude p120-catenn, whch bnds to the juxtamembrane doman of the cadhern cytoplasmc tal, and β-catenn, whch bnds to the membrane-dstal, carboxyl-termnal doman of VE-cadhern (Gavard, 2014; Cadwell et al., 2016b). Prevous studes showed that p120 bndng to the cadhern tal stablzes the cadhern by preventng endocytoss (Davs et al., 2003; Xao et al., 2003a; Chasson et al., 2009; Nanes et al., 2012; Kourtds et al., 2013), whereas β-catenn bndng functons as a lnker to actn-bndng protens such as α-catenn (Buckley et al., 2014; Banchn et al., 2015). VE-cadhern dynamcs at the plasma membrane s beleved to be essental n modulatng endothelal adheson strength and adherens juncton plastcty (Cadwell et al., 2016b). The level of cell surface VE-cadhern s regulated n part by endocytoss. Prevous work found that VE-cadhern undergoes clathrn-medated endocytoss (Xao et al., 2005; Chasson et al., 2009; Kowalczyk and Nanes, 2012; Semna et al., 2014; Zhang et al., 2014; West and Harrs, 2016). Ths process s nhbted by p120-catenn, whch bnds to the cadhern tal and masks an endocytc motf n the juxtamembrane doman (Chasson et al., 2009; Nanes et al., 2012). Ths regulatory mechansm s beleved to be a major control pont for cadhern expresson levels on the plasma membrane n a varety of mammalan cell types and tssues. In addton to endocytoss, cadhern cell 76 W. Su and A. P. Kowalczyk Molecular Bology of the Cell

3 FIGURE 1: VE-cadhern s cleaved durng endocytoss. (A) Western blot showng that overnght chloroqune treatment leads to the accumulaton of a VE-cadhern cleavage fragment, detected usng antbodes aganst the VE-cadhern extracellular doman. (B) Expermental procedure for an nternalzaton assay to montor VE-cadhern extracellular doman (BV6) and carboxyl-termnal doman (C19). (C) Immunofluorescence followng the protocol n B, suggestng that VE-cadhern s cleaved durng endocytoss, resultng n removal of the VE-cadhern carboxyl-termnal tal. Cell surface VE-cadhern was labeled n MECs, and cells were ncubated for 3 h wth 100 μm chloroqune treatment at 37 C. Scale bar, 20 μm. (D) Quantfcaton of results n C. Colocalzaton between endocytosed VE-cadhern (BV6, acd wash ) and C-termnal end of VE-cadhern (C19) was quantfed. Border regons of MECs wthout acd wash were used for comparson. Data are presented as means ± SEM; 21 cells n each group. Each condton was conducted n trplcate and represents data from three ndependent experments. **p < 0.01, two-taled t test. surface levels can also be regulated by proteolyss. For example, metalloprotenases cleave the VE-cadhern extracellular doman, leadng to reduced cell cell adheson strength (Dreymueller et al., 2012; Flemmng et al., 2015). Both γ-secretase and caspase-3 cleave cadherns to promote dsassembly of adherens junctons and reduce adheson (Hunter et al., 2001; Stenhusen et al., 2001; Marambaud et al., 2002). Calpan, a calcum-dependent cystene protease, also has been shown to cleave several classcal cadherns and thereby down-regulate cell cell adheson (Jang et al., 2009; Myazak et al., 2011; Ye et al., 2013; Kudo-Sakamoto et al., 2014; Trllsch et al., 2016). Despte the growng evdence of a role for calpan n modulatng the actvty of cadherns and other adheson receptors, the subcellular localzaton of cadhern cleavage by calpan and the relatonshp of ths processng to VE-cadhern endocytoss have not been establshed. Here we provde evdence that VE-cadhern s proteolytcally processed upon entry nto clathrn-enrched membrane domans durng the process of endocytoss. Ths cleavage event, whch removes the catenn-bndng doman of the cadhern tal, s medated by calpan and fates the cadhern for a degradatve rather than recyclng pathway. These fndngs reveal a novel mechansm for how proteolytc processng could modulate cadhern surface levels by alterng the tnerary of the cadhern durng endocytoss. RESULTS VE-cadhern s cleaved durng endocytoss Our prevous studes demonstrated that a fragment of VE-cadhern mssng the β-catenn bndng doman accumulates n endothelal cells treated wth chloroqune, whch nhbts lysosomal degradaton (Xao et al., 2003b). Data shown n Fgure 1A confrm that chloroqune treatment results n the accumulaton of an 95-kDa fragment of VE-cadhern. Ths processed form of VE-cadhern can be de- tected usng antbodes to the VE-cadhern extracellular doman but not wth antbodes drected aganst the catenn-bndng doman of the cadhern tal (Xao et al., 2003b). To determne whether ths fragment was generated durng cadhern nternalzaton, we labeled the cell surface pool of VE-cadhern usng an antbody drected aganst the extracellular doman (BV6) and followed the fate of the cadhern durng endocytoss over a 3-h perod n the presence of chloroqune. To dstngush cell surface from nternalzed pools of cadhern, we removed BV6 bound to the surface pool of VE-cadhern usng a low-ph wash. The presence of the cytoplasmc catenn-bndng doman was montored usng C19, an antbody drected aganst the VE-cadhern carboxyl-termnal doman (Fgure 1B). Colocalzaton of the antbodes drected aganst the VE-cadhern extracellular doman (BV6) and carboxyl-termnal tal (C19) was measured at cell cell borders and n endosomal compartments. As expected, BV6 and C19 exhbted extensve colocalzaton at cell cell borders (Fgure 1, C, top, and D). In contrast, the nternalzed pool of VE-cadhern remaned labeled wth BV6, but the majorty of ths nternalzed cadhern faled to label wth C19 (Fgure 1, C, bottom, and D). Smlar results were obtaned n COS7 cells exogenously expressng VE-cadhern labeled wth a carboxyl-termnal red fluorescent proten (RFP) tag (Supplemental Fgure S2). These data were further confrmed by the absence of β- catenn colocalzaton wth nternalzed pools of VE-cadhern. Whereas β-catenn exhbted hgh levels of colocalzaton wth the VE-cadhern extracellular doman at cell cell borders, very lttle colocalzaton was observed at endosomes (Fgure 2). Collectvely these data ndcate that the carboxyl-termnal tal, ncludng the β- catenn bndng doman of VE-cadhern, s removed durng endocytoss or subsequent traffckng of the cadhern through the endosomal system. Endocytoss s requred for VE-cadhern cleavage To determne whether endocytoss s requred for VE-cadhern processng, we used several approaches to nhbt VE-cadhern nternalzaton from the plasma membrane. Frst, we used a VE-cadhern endocytc mutant n whch the DEE amno acd resdues wthn the juxtamembrane doman were mutated to alannes. Mutaton of these DEE resdues results n a dramatc nhbton of VE-cadhern endocytoss and a falure to enter clathrn-enrched membrane domans (Nanes et al., 2012). Cells expressng wld type or the VE-cad DEE endocytc mutant were treated wth chloroqune and analyzed by Western blot. In comparson to the wld-type VE-cadhern, the VE-cad DEE mutant exhbted dramatcally reduced fragmentaton (Fgure 3, A and B). These fndngs suggest that VE-cadhern endocytoss s requred for cleavage. Prevously we found that overexpresson of p120-catenn reduces VE-cadhern endocytoss, whereas loss of p120-catenn accelerates VE-cadhern nternalzaton (Xao et al., 2003a; Nanes et al., 2012). Therefore we manpulated p120- catenn expresson levels as an alternatve approach to alterng VE-cadhern endocytoss rates. Smlar to the results obtaned wth the VE-cad DEE mutaton, overexpresson of p120-gfp dramatcally Volume 28 January 1, 2017 Proteolytc processng of VE-cadhern 77

4 FIGURE 2: β-catenn colocalzes wth junctonal but not nternalzed VE-cadhern. (A) Labelng setup of the experment n B. (B) COS7 cells were nfected wth adenovrus expressng VE-cadhern. Immunofluorescence magng reveals β-catenn does not colocalze wth the VE-cadhern extracellular doman (BV6) after endocytoss ( acd wash ). Scale bar, 20 μm. (C) Quantfcaton of colocalzaton between β-catenn and the VE-cadhern extracellular doman at cell cell borders and n acd wash samples (Internalzed). Data are presented as means ± SEM; 69 cells n each group. Each condton was conducted n trplcate and represents data from three ndependent experments. **p < 0.01, two-taled t test. nhbted VE-cadhern fragment formaton (Fgure 3, C and D). Ths overexpresson system was complemented by a loss-of-functon approach n whch VE-cadhern fragmentaton was montored n a p120-null background n whch cadhern nternalzaton rates are ncreased (Oas et al., 2010). p120-null endothelal cells (Oas et al., 2010) were treated wth chloroqune overnght, and VE-cadhern fragmentaton was montored by Western blot analyss. Increased VE-cadhern fragmentaton was observed n p120-null cells compared to controls (Fgure 3, E and F). Collectvely these observatons ndcate that endocytoss s requred for VE-cadhern cleavage and removal of the catenn-bndng doman. VE-cadhern s cleaved at the plasma membrane upon entry nto clathrn-enrched domans To determne the subcellular locaton and endocytc step n whch the cadhern cytoplasmc tal s processed, we performed threechannel colocalzaton analyss for the VE-cadhern extracellular doman, the VE-cadhern carboxyl-termnal doman, and varous endocytc markers. For these experments, we expressed VE-cadhern wth a C-termnal RFP tag and used the BV6 antbody aganst the cadhern extracellular doman as llustrated n Fgure 4A. Cell surface cadhern was labeled wth BV6 at 4 C, followed by ncubaton at 37 C for varous amounts of tme, dependng on the marker (5 mn for clathrn and 30 mn for EEA1). As expected, BV6 and the carboxyl-termnal RFP tag exhbted extensve colocalzaton at cell cell junctons, ndcatng the presence of ntact, full-length cadhern. However, colocalzaton s reduced as the cadhern enters clathrn-enrched membrane domans (Fgure 4B) and s further reduced by the tme the cadhern enters early endosomes (Fgure 4C). These fndngs, together wth the data demonstratng that endocytoss s requred for cleavage (Fgure 3), suggest that VE-cadhern s proteolytcally processed n clathrn-enrched domans, and the cleavage s completed before VE-cadhern reaches the early endosome. The catenn-bndng doman regulates VE-cadhern turnover rates Prevous studes suggested that the catennbndng doman of classcal cadherns regulates cadhern transport to the plasma membrane (Chen et al., 1999). Therefore we hypotheszed that VE-cadhern cleavage, whch was shown here (Fgures 1 and 2) and prevously to remove the catenn-bndng doman (Xao et al., 2003b), alters the traffckng dynamcs of VE-cadhern and leads to cadhern degradaton nstead of recyclng. To determne how loss of the catennbndng doman alters the traffckng dynamcs of cell surface cadhern, we examned the turnover rate of VE-cadhern and a VE-cadhern truncaton mutant lackng the catenn-bndng doman (VE-cadhern ΔCBD; Fgure 5A). The VE-cadhern ΔCBD comgrates wth the VE-cadhern cleavage fragment observed n chloroqune-treated cells (Xao et al., 2003b) and therefore was used to mmc the N-termnal cleaved fragment of VE-cadhern. Cell surface botnylaton and pulse-chase analyss were used to montor VE-cadhern turnover rates. Of nterest, deleton of the catenn bndng reduced the half-lfe of cell surface VE-cadhern from around 7.5 to 3.8 h (Fgure 5, B and C). The rate constant, k, of VE-cadhern turnover ncreased from 0.09 (wld type) to 0.18 (ΔCBD). To determne whether the ncreased rates of cadhern turnover were due to ncreased endocytoss or alteratons n postendocytc traffckng of the cadhern, we measured endocytoss rates and the subcellular localzaton of nternalzed full-length VE-cadhern and the ΔCBD mutant cadhern. To avod msnterpretaton due to the cleavage of full-length VE-cadhern, we used another VE-cadhern extracellular doman antbody (Cad-5, ; BD TransLab) nstead of C-termnal antbody to vsualze the total pool of VE-cad. We were unable to detect any sgnfcant dfference n endocytoss rates when we compared full-length VE-cadhern to VE-cadhern ΔCBD (Fgure 6, A and B). However, usng both transferrn (Fgure 6, C and D) and Rab11 (Fgure 6, E and F) as markers for recyclng compartments (Lock and Stow, 2005; Mayle et al., 2012; Yan et al., 2016), we observed sgnfcantly less colocalzaton of nternalzed ΔCBD VE-cadhern wth recyclng markers than we dd for the fulllength cadhern. On the other hand, nternalzed VE-cadhern ΔCBD exhbted more colocalzaton wth the lysosomal marker CD63 than the full-length VE-cadhern (Fgure 6, G and I). Together our data suggest that loss of the catenn-bndng doman alters the VE-cadhern postendocytc traffckng tnerary, resultng n a hgher turnover rate due to less recyclng and more degradaton. VE-cadhern s cleaved by calpan Several prevous studes showed that calpan cleaves the cytoplasmc doman of cadherns (Jang et al., 2009; Myazak et al., 2011; Ye et al., 2013; Kudo-Sakamoto et al., 2014; Trllsch et al., 2016). To determne whether calpan actvty s requred for VE-cadhern 78 W. Su and A. P. Kowalczyk Molecular Bology of the Cell

5 FIGURE 3: VE-cadhern endocytoss s requred for cleavage. (A) COS7 cells were transfected wth ndcated constructs and treated wth chloroqune for 6 h before to lyss. Western blot analyss reveals sgnfcantly less cleavage of the VE-cadhern DEE endocytc mutant than for wld-type VE-cadhern. (B) Quantfcaton of the results n A. Data are presented as means ± SEM. Each condton was conducted n trplcate and represents data from three ndependent experments. *p < 0.05, two-taled t test. (C) MECs were nfected wth adenovrus expressng p120-gfp 48 h before to the experment. Cells were treated wth chloroqune overnght. Western blot analyss reveals sgnfcantly reduced fragment accumulaton of VE-cadhern wth p120 overexpresson compared to the control. (D) Quantfcaton of the result n C usng the same method as descrbed n B. **p < 0.01, two-taled t test. (E) p120 knockout results n ncreased VE-cadhern cleavage. Mouse skn endothelal cells wth or wthout p120 were treated wth chloroqune overnght. Western blot shows ncreased fragmentaton n p120-null cells compared to the control. (F) Quantfcaton of the result n E usng the same method as descrbed n B. *p < 0.05, two-taled pared t test. processng and the removal of the catenn-bndng doman, we pretreated cells wth the calpan nhbtor Calpeptn and then analyzed colocalzaton between the cadhern extracellular doman and carboxyl-termnal tal after a 30-mn nternalzaton perod. Immunofluorescence data show that Calpeptn treatment reduced the loss of the cadhern tal durng endocytoss (Fgure 7, A and B). Fnally, we drectly tested whether VE-cadhern can be cleaved by calpan n vtro. Cell lysates from adenovrus-nfected COS7 cells expressng VE-cadhern-RFP were treated wth purfed actve calpan 1 large subunt n the presence of CaCl 2 wth or wthout calpan nhbtors (Calpeptn and ALLM). Calpan treatment resulted n the formaton of a 95-kDa fragment dentcal to that observed n cells treated wth chloroqune (Fgure 7, C and D). Together wth the observaton that Calpeptn nhbts cleavage of the cadhern tal n lvng cells, these fndngs suggest that calpan cleaves VE-cadhern durng endocytoss. DISCUSSION The results of ths study dentfy a proteolytc processng event that occurs durng endocytoss of VE-cadhern. Our fndngs suggest that VE-cadhern s cleaved by calpan upon entry nto clathrn-enrched membrane domans durng endocytoss and that ths cleavage event removes the β-catenn bndng doman of the cadhern. Ths cleavage event appears to fate the cadhern for degradaton rather than recyclng, suggestng that the calpan-medated cleavage of VE-cadhern nfluences the traffckng tnerary of the cadhern after endocytoss. These fndngs reveal a novel means by whch cadhern endocytoss and recyclng are regulated. A number of studes have demonstrated that cadherns are targeted by extracellular and cytoplasmc proteases, ncludng metalloprotenases, elastase, and cathepsn G (Dejana et al., 2008; Dreymueller et al., 2012). One study suggested that the VEcadhern cytoplasmc tal s cleaved by m- calpan n response to nflammatory medators durng atheroscleross (Myazak et al., 2011). In the present study, we found that VE-cadhern s cleaved by calpan durng endocytoss. Ths nterpretaton s based on the fact that nhbtng endocytoss prevents cleavage (Fgure 2) and on magng data suggestng that the cadhern tal s removed as cell surface cadhern s recruted nto clathrn-enrched membrane domans (Fgure 4). Ths s also supported by our data showng that the VE-cadhern DEE mutant, whch fals to enter clathrn-enrched domans (Nanes et al., 2012), does not get cleaved (Fgure 3). Of nterest, bndng to phospholpds at the plasma membrane s known to facltate calpan actvaton (Kubok et al., 1992; Tompa et al., 2001; Shao et al., 2006), and calpan s a component of clathrn-coated vescles (Sato et al., 1995). These fndngs suggest that calpan may cleave the cadhern upon recrutment nto clathrn-coated pts and/or early n endocytc traffckng and before the clathrn coat dssocates from endocytc vescles. Addtonal studes wll be needed to defne wth hgher spatal and temporal resoluton precsely where and when the cadhern tal s cleaved by calpan. The observaton that VE-cadhern s cleaved nto only two fragments s consstent wth the fact that calpan only partally dgests Volume 28 January 1, 2017 Proteolytc processng of VE-cadhern 79

6 FIGURE 4: VE-cadhern cleavage occurs after VE-cadhern enters clathrn-enrched membrane domans but before t reaches the early endosome. (A) Where VE-cadhern cleavage may occur durng endocytoss and the labelng approach used for experments n B and C. (B) Adenovrus-nfected COS7 cells expressng VE-cadhern RFP were labeled wth BV6 and then warmed to 37 C for 5 mn to vsualze surface pools and very early endocytc events. Three-channel colocalzaton reveals that cleavage occurs whle VE-cadhern enters clathrn-enrched domans. Scale bar, 20 μm. (C) COS7 cells were warmed to 37 C for 30 mn to nduce endocytoss, followed by acd wash to remove cell surface antbodes. Three-channel colocalzaton reveals cleavage occurs before VE-cadhern reaches the early endosome. Scale bar, 20 μm. (D) Quantfcaton of the results n B and C. Border regons of COS7 cells expressng VE-cadhern RFP wthout acd wash were used as postve controls. Colocalzaton analyss between BV6 and clathrn or BV6 and EEA1 was used to dentfy cadhern pools at dfferent steps of endocytoss. Colocalzaton analyss between RFP and VE-cadhern n clathrn-enrched domans or early endosomes was then performed to reveal whether the VE-cadhern carboxyl-termnal doman was present at these endocytc steps. Data are presented as means ± SEM; 16 cells n each group. Each condton was conducted n trplcate and represents data from three ndependent experments. **p < 0.01, two-taled t test. ts substrates and s therefore consdered to be a regulatory protease (Goll et al., 2003; Ono and Sormach, 2012). The VE-cadhern ΔCBD polypeptde, whch mmcs the N-termnal fragment of VE-cadhern after cleavage (Xao et al., 2003b), exhbts decreased colocalzaton wth recyclng markers and ncreased colocalzaton wth lysosomal markers compared to full-length VE-cadhern (Fgure 6). These fndngs suggest that the cleavage of VE-cadhern n the cytoplasmc doman results n an altered traffckng and sortng tnerary of VE-cadhern. Thus calpan cleavage appears to be a fate-determnng step durng VE-cadhern endocytc processng. Prevous studes showed that β-catenn facltates E-cadhern transport to the plasma membrane (Chen et al., 1999). Therefore t s lkely that deceased VE-cadhern recyclng after cleavage (Fgure 6) s due to the removal of the β- catenn bndng doman of VE-cadhern. Ths altered traffckng dynamcs s lkely to affect VE-cadhern cell surface levels. Thus calpan actvty could be a potental regulatory target for modulatng VE-cadhern surface levels durng a varety of pathophysologcal crcumstances. Calpan has been shown to cleave a varety of classcal cadherns (Jang et al., 2009; Ye et al., 2013; Kudo-Sakamoto et al., 2014; Trllsch et al., 2016), suggestng that the mechansm of cadhern regulaton reported here may apply to other cadherns n a varety of cell types. For example, t has been shown that E-cadhern cleavage by calpan s nvolved n tumor progresson (Ye et al., 2013; Trllsch et al., 2016) and that calpan cleavage of N-cadhern reduces cell cell adheson (Jang et al., 2009; Kudo-Sakamoto et al., 2014). VE-cadhern cleavage by calpan has also been shown to result n dsorganzaton of adherens junctons and hyperpermeablty of vascular endothelal cells (Myazak et al., 2011). It s possble that the alteraton n cadhern adhesve functon observed n these other model systems and cell types could reflect an underlyng mechansm of calpan-medated regulaton of adherens junctons through altered cadhern traffckng. Calpan s a calcum-dependent protease that s actvated by ntracellular calcum (Campbell and Daves, 2012; Ono and Sormach, 2012). Thus ncreases n ntracellular calcum downstream of nflammatory or angogenc sgnalng could result n ncreased calpan actvty and altered endothelal cell cell 80 W. Su and A. P. Kowalczyk Molecular Bology of the Cell

7 cleavage by calpan durng endocytoss. Fnally, addtonal studes are needed to determne the fate of both β-catenn and the carboxyl-termnal VE-cadhern fragment after cleavage, snce these polypeptdes may exhbt bologcal actvty upon release from the cadhern durng endocytoss. FIGURE 5: Deleton of the VE-cadhern catenn-bndng doman ncreases VE-cadhern turnover rates. (A) VE-cadhern ΔCBD mmcs the cleaved fragment of VE-cad. (B) Surface botnylaton reveals faster turnover of VE-cadhern ΔCBD than for full-length VE-cadhern. COS7 cells were transfected wth ndcated constructs. (C) Quantfcaton of the results n B. Data are presented as means ± SEM. The half-lfe of each cadhern s labeled wth red dotted lnes. Rate constant, k, on the rght n red. Each condton was conducted n trplcate and represents data from three ndependent experments. MATERIALS AND METHODS Cell culture Human dermal mcrovascular endothelal cells (MECs) were cultured n endothelal growth medum 2 (CC-3159; Clonetcs, Walkersvlle, MD) supplemented wth EGM-2 MV SngleQuots (CC-4147; Clonetcs) on gelatn-coated plates. The Afrcan green monkey kdney fbroblast-lke (COS7; Amercan Type Culture Collecton, Manassas, VA) and HEK QBI-293A cell lnes (MP Bomedcals, Santa Ana, CA) were cultured n DMEM ( CV; Cornng, Cornng, NY) supplemented wth 10% fetal bovne serum and 1% antbotc antmycotc soluton ( CI; Cornng). Prmary mouse endothelal cells were cultured as descrbed prevously (Oas et al., 2010). adheson. In fact, ncreases n the ntracellular calcum levels have been shown to dsrupt endothelal adherens juncton (Sandoval et al., 2001; Komarova et al., 2012). Although the exact mechansm of how the adherens juncton s dsrupted n response to ncreased ntracellular calcum levels s not clear, our fndngs provde evdence that supports calpan actvaton as an ntermedate step durng the dsassembly of the adherens juncton n response to ncreased ntracellular calcum. The work presented here and n our prevous studes ndcates that VE-cadhern s cleaved between the juxtamembrane and the catenn-bndng doman (Xao et al., 2003b). It has also been shown that calpan cleaves E-cadhern and N-cadhern n a smlar regon and generates smlar-szed fragments ( 100 kda; Jang et al., 2009; Ye et al., 2013; Kudo-Sakamoto et al., 2014; Trllsch et al., 2016). Calpan s known to act based on proten tertary structures nstead of specfc amno acd sequences and exhbts a preference for nterdoman unstructured regons (Stabach et al., 1997; Goll et al., 2003; Ono and Sormach, 2012). Based on structural studes of the E-cadhern cytoplasmc doman (Ishyama et al., 2010), there s a flexble lnker regon between the juxtamembrane and the catenn-bndng doman that s a lkely target for calpan cleavage (Fgure 8A). A prevous study ndcated that fve amno acds wthn ths lnker regon n VE-cadhern are requred for m-calpan cleavage (Myazak et al., 2011). To determne whether ths regon was also nvolved n VE-cadhern processng durng endocytoss, we generated a smlar VE-cadhern construct lackng these fve amno acds ( ) between the juxtamembrane doman and catenn-bndng doman (VE-cadhern 691Δ5-RFP). Indeed, mmunofluorescence-based assays showed reduced cleavage of the cadhern tal durng endocytoss (Supplemental Fgure S3). However, bochemcal assays ndcated that calpan-1 could ndeed cleave ths VE-cadhern polypeptde n vtro (unpublshed data). Although further studes wll be needed to map the precse ste of calpan cleavage, these studes suggest that the lnker sequence between the juxtamembrane and the catenn-bndng doman s a potentally mportant regulatory ste that s subject to Vrus producton To generate adenovrus for proten expresson n mammalan cells, the gene of nterest was cloned nto the gateway pad/cmv/v5-dest vector (V49320; Invtrogen, Carlsbad, CA). The vector was lnearzed usng PacI and transfected nto HEK QBI-293A cells usng TransFectn ( ; Bo-Rad, Hercules, CA) to produce vrus. After a second round of nfecton, cells were lysed and vrus was harvested. Internalzaton assay and mage analyss Cells were nfected wth adenovrus or transfected wth plasmd expressng the proten of nterest 48 hours before the experment. Transfecton was conducted usng Lpofectamne 2000 ( ; ThermoFsher, Hudson, NH) or 3000 (L ; ThermoFsher) accordng to the protocol provded by the manufacturer. Internalzaton assays were performed as descrbed prevously (Xao et al., 2003a; Chasson et al., 2009; Cadwell et al., 2016a). Brefly, cultured cells on glass coverslps were ncubated wth an antbody aganst the VE-cadhern extracellular doman (BV6; MABT134; Mllpore, Bllerca, MA) n cell culture medum for 30 mn at 4 C. Cells were washed three tmes wth cold phosphate-buffered salne (PBS) contanng calcum and magnesum to remove unbound antbody. To allow nternalzaton, cells were ncubated n prewarmed medum at 37 C. Cells were then returned to cold medum. A low-ph buffer (100 mm glycne, 20mM magnesum acetate, and 50 mm potassum chlorde, ph 2.2) was used to remove any remanng antbody from the cell surface. Cells were then fxed and permeablzed by ncubaton n 4% paraformaldehyde for 10 mn, followed by 0.1% Trton X-100 for 10 mn at room temperature. Goat ant VE-cadhern antbody (C19; sc-6458; Santa Cruz Botechnology, Dallas, TX) or C-termnal RFP tag was used to vsualze the C-termnal doman of VE-cadhern. Mouse ant VE-cadhern (Cad-5, ; BD TransLab, San Jose, CA), an alternatve antbody that recognzes the extracellular doman of VE-cadhern, was used to determne the total amount of VE-cadhern. Addtonal prmary antbodes used ncluded mouse ant clathrn heavy chan (610500; BD TransLab), mouse ant-eea1 (610457; BD TransLab), Volume 28 January 1, 2017 Proteolytc processng of VE-cadhern 81

8 mouse ant-p120 (610134; BD TransLab), rabbt ant β-catenn (C2206; Sgma-Aldrch, St. Lous, MO), rabbt ant-rab11 ( ; Invtrogen), and mouse ant-cd63 (H5C6; IowaLabs). Secondary antbodes conjugated to fluorescent dyes (Alexa Fluor 488, 555, or 647 nm; Lfe Technologes, Carlsbad, CA) were used to vsualze antbody bndng. To quantfy cleavage, colocalzaton between the sgnals from endocytosed VE-cadhern (labeled at the extracellular doman) and C-termnal end of VE-cadhern (labeled at the C-termnal doman) were quantfed usng the Manders correlaton coeffcents: M 1 R,colocal = and M R 2 G,colocal where R denotes the red sgnal, R,colocal the red sgnal that colocalzes wth the green sgnal, G the green sgnal, and G,colocal the green sgnal that colocalzes wth the red sgnal. The Coloc 2 program from ImageJ/ Fj was used for quantfcaton. To quantfy nternalzaton, sgnals from endocytosed VE-cadhern were dvded by sgnals from the total pool of VE-cadhern labeled wth Cad-5, an alternatve extracellular doman antbody. Mcroscopy was performed usng an epfluorescence mcroscope (DMRXA2; Leca, Buffalo Grove, IL) equpped wth a 63 ol mmerson objectve wth apochromatc aberraton and flat-feld correctons, narrowbandpass flters, and a dgtal camera (ORCA-ER C ; Hamamatsu Photoncs, Hamamatsu Cty, Japan). Images were captured usng Smple PCI software (Hamamatsu Photoncs). Quantfcaton was done n ImageJ/Fj. Statstcal analyss was performed n RStudo. = G FIGURE 6: The VE-cadhern catenn-bndng doman regulates cadhern fate after endocytoss. (A) VE-cadhern ΔCBD has an endocytoss rate smlar to that of wld-type cadhern. An nternalzaton assay was performed usng BV6 antbody to label nternalzed VE-cadhern. Total VE-cadhern was detected usng Cad-5 antbody. Scale bar, 20 μm. (B) Quantfcaton of results n A. Data are presented as means ± SEM; 17 cells n each group. Each condton was conducted n trplcate and represents data from three ndependent experments. NS, nonsgnfcant, two-taled t test. (C, E) VE-cadhern ΔCBD exhbts reduced colocalzaton wth recyclng markers compared to full-length VE-cadhern. Internalzaton assays were performed usng BV6 to montor the nternalzed VE-cadhern. Cells were warmed to 37 C for 30 mn n the presence of tetramethylrhodamne-conjugated transferrn at 50 μg/ml n C or for 60 mn and followed by Rab11 stanng n E. Scale bar, 20 μm. (D) Quantfcaton of the results n C. Data are presented as the means ± SEM; 43 cells n each group. Each condton was conducted n trplcate and represents data from three ndependent experments. **p < 0.01, two-taled t test. (F) Quantfcaton of the results n E. Data are presented as means ± SEM; 31 cells n each group. Each condton was conducted n trplcate and represents data from three ndependent experments. **p < 0.01, two-taled t test. (G) VE-cadhern ΔCBD exhbts more colocalzaton wth the lysosomal marker CD63 than observed for wld-type cadhern. Cells were warmed to 37 C for 60 mn and then fxed and staned for CD63. Scale bar, 20 μm. (H) Quantfcaton of the results n G. Data are presented as means ± SEM; 33 cells n each group. Each condton was conducted n trplcate and represents data from three ndependent experments. **p < 0.01, two-taled t test. Western blot analyss Samples were prepared by ether scrapng cells nto Laemml Sample Buffer ( ; Bo-Rad) wth 5% β-mercaptoethanol (β-me) or addng the Laemml Sample Buffer wth 5% β-me drectly nto the reacton mxture. Samples were then heated at 95 C for 5 mn before SDS PAGE and analyzed by mmunoblottng on ntrocellulose membranes (Whatman, Madstone, Unted Kngdom). Quantfcaton was done usng the gel analyss program n ImageJ/Fj, and statstcal analyss was performed n RStudo. Prmary antbodes used were mouse ant VE-cadhern (Cad-5, ; BD TransLab), mouse ant VE-cadhern (BV6, MABT134; Mllpore), rat ant mouse VE-cadhern (E028599; eboscence, San Dego, CA), rat ant-mouse VE-cadhern (550548; BD Pharmngen, San Jose, CA), and rabbt ant-p120 (sc-1101; 82 W. Su and A. P. Kowalczyk Molecular Bology of the Cell

9 Santa Cruz Botechnology). Secondary antbodes conjugated to horseradsh peroxdase (Bo-Rad) and ECL Western blot detecton reagents (RPN2106; GE Healthcare, Chcago, IL) were used to vsualze protens by Western blot. FIGURE 7: VE-cadhern s cleaved by calpan. (A) Immunofluorescence stanng ndcates that nhbtng calpan actvty reduces VE-cadhern cleavage. COS7 cells were pretreated wth 10 μm Calpeptn for 30 mn, followed by a 30-mn nternalzaton assay. Scale bar, 20 μm. (B) Quantfcaton of the results n A. Data are presented as the means ± SEM; 14 cells n each group. Each condton was conducted n trplcate and represents data from three ndependent experments. **p < 0.05, two-taled t test. (C) Western blot showng VE-cadhern s dgested by calpan n vtro. COS7 cells nfected wth adenovrus expressng VE-cadhern RFP were lysed n M-PER lyss buffer. Actve calpan large subunt was added to the lysate n the presence of CaCl 2 wth or wthout calpan nhbtors (Calpeptn and ALLM). (D) Quantfcaton of the results n C. Data are presented as means ± SEM. Each condton was conducted n trplcate and represents data from three ndependent experments. **p < 0.01, one -way analyss of varance. Surface botnylaton pulse-chase assay and VE-cadhern half-lfe determnaton COS7 cells were transfected wth the plasmd of nterest 48 h before to the experment. Cells were pulse-labeled wth botn (21331; ThermoFsher) on ce for 30 mn. Excess botn was quenched by 50 mm ammonum chlorde. Cells were then ncubated at 37 C for varous amounts of tme, returned to ce, and then lysed wth 1% Trton X-100 n PBS wth calcum and magnesum for 30 mn. Cell lysates were centrfuged at 13,200 rpm for 30 mn at 4 C. The supernatants were added to streptavdn beads (20349; ThermoFsher) and ncubated for 1 h at 4 C. Beads were washed four tmes usng ce-cold PBS wth 0.1% Tween-20. Botnylated proten was eluted usng Laemml Sample Buffer wth 5% β-me at 95 C for 5 mn, followed by Western blot analyss. The followng equaton was used calculate VE-cadhern half-lfe: t 1/2 = log t Nt N 1/2 wth the rate constant N ln t N0 k = t 0 where t 1/2 s the half-lfe, t s the gven tme, N t s the amount of proten at the gven tme, and N 0 s the amount of proten at tme 0. In vtro calpan dgeston COS7 cells were nfected wth adenovrus expressng VE-cadhern RFP 48 h before the experment. Cells were lysed n M-PER lyss buffer (78501; ThermoFsher) supplemented wth 1% protease nhbtors (P8849; Sgma-Aldrch) for 10 mn on ce. We added 1 mm CaCl 2, 1 U of actve calpan 1 large subunt (C6108; Sgma-Aldrch), 100 μm Calpeptn ( ; Mllpore), and 250 μm ALLM (CAS ; Santa Cruz Botechnology) separately to the cell lysates. The reacton mxture was ncubated at room temperature for 30 mn. The reacton was then termnated by addng Laemml Sample Buffer wth 5% β-me to the mxture, followed by Western blot analyss. FIGURE 8: Model. (A) Open structure regon between the juxtamembrane doman (JMD) and the catenn-bndng doman (CBD) s lkely the cleavage ste for calpan. (B) Upon entry nto clathrnenrched domans, a porton of the VE-cadhern s cleaved by actve calpan. (C) Cleaved VE-cadhern s more lkely to be degraded rather than recycled back to the cell surface. Volume 28 January 1, 2017 Proteolytc processng of VE-cadhern 83

10 ACKNOWLEDGMENTS We thank Susan Summers for prmary cell solatons and members of the Kowalczyk lab for help and advce. We also acknowledge the Emory Unversty Custom Clonng Core Faclty for help makng the VEcadhern 691Δ5 construct. Ths work was supported by Natonal Insttutes of Health Grants RO1AR and RO1AR to A. P. K. REFERENCES Banchn JM, Ktt KN, Gloerch M, Pokutta S, Wes WI, Nelson WJ (2015). Reevaluatng alphae-catenn monomer and homodmer functons by characterzng E-cadhern/alphaE-catenn chmeras. J Cell Bol 210, Boulanger CM (2016). Endothelum. Arteroscler Thromb Vasc Bol 36, e26 e31. Buckley CD, Tan J, Anderson KL, Hanen D, Volkmann N, Wes WI, Nelson WJ, Dunn AR (2014). Cell adheson. The mnmal cadhern-catenn complex bnds to actn flaments under force. Scence 346, Cadwell CM, Jenkns PM, Bennett V, Kowalczyk AP (2016a). Ankyrn-G nhbts endocytoss of cadhern dmers. J Bol Chem 291, Cadwell CM, Su W, Kowalczyk AP (2016b). 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