Journal of Cell Science 111, 3045-3057 (1998) 3045 Printed in Great Britain © The Company of Biologists Limited 1998 JCS4604

VE-cadherin and desmoplakin are assembled into dermal microvascular endothelial intercellular junctions: a pivotal role for plakoglobin in the recruitment of desmoplakin to intercellular junctions

Andrew P. Kowalczyk1,*, Pilar Navarro2, Elisabetta Dejana2, Elayne A. Bornslaeger1, Kathleen J. Green1, Daniel S. Kopp1 and Jeffrey E. Borgwardt1 1Departments of Dermatology, Pathology, and The Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, USA 2The Laboratory of Vascular Biology, Mario Negri Institute for Pharmacological Research, Milan, Italy *Author for correspondence at present address: Department of Dermatology, Emory University School of Medicine, 5007 Woodruff Memorial Building, 1639 Pierce Drive, Atlanta, GA 30322, USA (e-mail: [email protected])

Accepted 6 August; published on WWW 23 September 1998

SUMMARY

Vascular endothelial cells assemble adhesive intercellular and DP-NTP were recruited to cell-cell borders. junctions comprising a unique cadherin, VE-cadherin, which Interestingly, β-catenin could not substitute for plakoglobin is coupled to the actin cytoskeleton through cytoplasmic in the recruitment of DP-NTP to cell borders, and DP-NTP interactions with plakoglobin, β-catenin and α-catenin. bound to plakoglobin but not β-catenin in the yeast two- However, the potential linkage between VE-cadherin and the hybrid system. In addition, DP-NTP colocalized at cell-cell vimentin intermediate filament cytoskeleton is not well borders with α-catenin in the L-cell lines, and endogenous characterized. Recent evidence indicates that lymphatic and desmoplakin and α-catenin colocalized in cultured dermal vascular endothelial cells express desmoplakin, a cytoplasmic microvascular endothelial cells. This is in striking contrast to desmosomal protein that attaches intermediate filaments to epithelial cells, where desmoplakin and α-catenin are the plasma membrane in epithelial cells. In the present study, restricted to desmosomes and adherens junctions, desmoplakin was localized to intercellular junctions in respectively. These results suggest that endothelial cells human dermal microvascular endothelial cells. To determine assemble unique junctional complexes that couple VE- if VE-cadherin could associate with desmoplakin, VE- cadherin to both the actin and intermediate filament cadherin, plakoglobin, and a desmoplakin amino-terminal cytoskeleton. polypeptide (DP-NTP) were co-expressed in L-cell fibroblasts. In the presence of VE-cadherin, both plakoglobin Key words: Vimentin, Cytoskeleton, Adhesion, Catenin

INTRODUCTION transduction pathways that ultimately control gene expression and cell behavior (Klymkowsky and Parr, 1995; Barth et al., Endothelial cells form a non-thrombogenic lining that 1997). In this manner, adhesive intercellular junctions appear functions as a semipermeable barrier between the plasma and to be plasma membrane sites that integrate mechanical and tissue extracellular compartments (van Hinsbergh, 1997; Lum chemical signaling pathways. and Malik, 1996; Dejana, 1996). Individual cells that comprise The mechanisms by which adhesive interactions are the endothelial monolayer are connected to one another by established between vascular endothelial cells have been adhesive contacts, termed intercellular junctions. In addition to studied extensively (Dejana et al., 1995; Dejana, 1996). mediating cell-cell adhesion, intercellular junctions also Endothelial cells express a unique cadherin, termed VE- function as plasma membrane attachment sites for cytoskeletal cadherin or cadherin-5 (Breviario et al., 1995; Tanihara et al., networks, such as actin and intermediate filaments (Cowin and 1994), which mediates calcium-dependent, homophilic Mechanic, 1994; Cowin and Burke, 1996; Green and Jones, adhesion (Breviario et al., 1995; Ali et al., 1997). A second 1996; Garrod et al., 1996). This architectural arrangement endothelial cadherin termed VE-cadherin-2 has recently been functions to couple adhesive forces through the transmembrane described (Telo et al., 1998). VE-cadherin-2 also mediates glycoproteins to the cytoskeleton, thereby influencing cell calcium-dependent adhesion, but this cadherin includes a shape and tissue integrity (Fuchs and Cleveland, 1998). Recent unique cytoplasmic domain, and the role of this protein in evidence indicates that in addition to mediating adhesive endothelial function remains to be determined. However, the interactions between cells, intercellular junctions also function molecular components that couple the originally described VE- as macromolecular complexes that participate in signal cadherin to the actin cytoskeleton have been studied in detail 3046 A. P. Kowalczyk and others

(Lampugnani and Dejana, 1997). Like the classical cadherins, between VE-cadherin and the vimentin intermediate filament such as E-cadherin, VE-cadherin associates with cytoplasmic network in vascular endothelial cells. Interestingly, several proteins termed catenins. Originally identified as cadherin recent reports have demonstrated that the intermediate filament associated proteins and termed α, β, and γ-catenin (Ozawa et binding protein desmoplakin is expressed in endothelial cells al., 1989; Ozawa and Kemler, 1992), these cytoplasmic and colocalizes with VE-cadherin. In lymphatic endothelial proteins have been found to be critical in the association of cells in vivo, desmoplakin was found to be expressed and cadherins with the actin cytoskeleton and the adhesive function assembled into intercellular junctions termed complexus of the cadherins. γ-Catenin is now known to be identical to adhaerentes (Schmelz and Franke, 1993; Schmelz et al., 1994). plakoglobin (Cowin et al., 1986; Knudsen and Wheelock, In these junctions, desmoplakin colocalized with both 1992). Both β-catenin and plakoglobin bind directly to the plakoglobin and VE-cadherin. These investigators did not cytoplasmic domain of the cadherins. In addition, β-catenin observe desmoplakin in tissue sections of blood vessels. and plakoglobin also bind directly to α-catenin (Aberle et al., However, analysis of human umbilical vein endothelial cells 1994; Jou et al., 1995; Nieset et al., 1997; Obama and Ozawa, (HUVEC) demonstrated that these cells express desmoplakin 1997; Huber et al., 1997), a vinculin related protein that mRNA and the protein was assembled into endothelial promotes association of the cadherin-catenin complex with the junctions and colocalized with VE-cadherin (Valiron et al., actin network (Nagafuchi et al., 1991; Herrenknecht et al., 1996). Together, these studies demonstrate that endothelial 1991). α-Catenin, in turn, binds directly to actin (Rimm et al., cells express desmoplakin and that desmoplakin is 1995), and to the actin binding protein α-actinin (Knudsen et incorporated into intercellular junctions and colocalizes with al., 1995; Nieset et al., 1997). The cadherins, including VE- VE-cadherin. cadherin, are thought to be specifically attached to the actin In the present study, desmoplakin was found to assemble microfilament network through these interactions. into intercellular junctions of postconfluent cultures of human In epithelial cells, classical cadherins assemble into actin- dermal microvascular endothelial cells. In addition, vimentin associated adhesive contacts termed adherens junctions. In intermediate filaments were found to colocalize with addition to adherens junctions, epithelial cells assemble desmoplakin at cell-cell borders. Using several model systems separate junctions termed desmosomes, which function as to analyze interactions between junctional proteins, VE- plasma membrane attachment sites for the intermediate cadherin was found to recruit desmoplakin to cell-cell borders. filament network (Kowalczyk and Green, 1996; Cowin and Furthermore, the recruitment of desmoplakin to cell borders by Burke, 1996). The adhesive interface of the desmosome is VE-cadherin required plakoglobin. Interestingly, β-catenin, thought to be formed by a distinct class of cadherins, termed although closely related to plakoglobin, could not substitute for desmosomal cadherins. The cytoplasmic domains of the plakoglobin in the association of desmoplakin with VE- desmosomal cadherins bind directly to plakoglobin, but not β- cadherin. In addition, using the yeast two-hybrid system, catenin or α-catenin (Mathur et al., 1994; Plott et al., 1994; desmoplakin was found to bind to plakoglobin but not β- Roh and Stanley, 1995). Instead, the desmosomal cadherins catenin. These results suggest that VE-cadherin is coupled to and plakoglobin associate with desmoplakin, a large desmoplakin and that this complex may play a role in cytoplasmic protein that binds directly to intermediate filament anchoring VE-cadherin to the vimentin intermediate filament polypeptides, including keratins, vimentin, and desmin (Meng network in endothelial cells. In addition, plakoglobin appears et al., 1997; Kouklis et al., 1994). Recent studies have to play a specific and critical role in the association of VE- demonstrated that the amino-terminal domain of desmoplakin cadherin with desmoplakin, suggesting that one function of binds directly to plakoglobin (Kowalczyk et al., 1997; Smith plakoglobin in endothelial cells may be to mediate VE- and Fuchs, 1998) and that desmoplakin is required for cadherin association with the vimentin cytoskeleton. intermediate filament networks to associate with the cytoplasmic plaque of the desmosome (Bornslaeger et al., MATERIALS AND METHODS 1996). Endothelial cells do not express any of the known Cell culture desmosomal cadherins, and the adhesive intercellular junctions that form in endothelial cells are similar to the actin based Human dermal microvascular endothelial cells (HDMEC) were purchased from Clonetics (San Diego, CA) and cultured according to adherens junctions that are assembled in epithelial cells. In vendor recommendations. Mouse L-cell fibroblasts were used to addition to VE-cadherin, vascular endothelial cells also express establish cell lines expressing full length VE-cadherin, plakoglobin and N-cadherin. However, N-cadherin is localized in a diffuse either full length desmoplakin or an amino-terminal desmoplakin pattern on the endothelial plasma membrane and VE-cadherin polypeptide (DP-NTP). Parental L-cells were transfected using competitively inhibits the assembly of N-cadherin into calcium-phosphate precipitation and selected in an active endothelial junctions (Navarro et al., 1998). In addition, concentration of 400 µg/ml G418 (Gibco BRL, Grand Island, NY). endothelial cells also express PECAM, a member of the Drug resistant colonies were isolated using cloning cylinders and calcium-independent immunoglobulin family of adhesion expression of each exogenously expressed protein was verified using both immunoblot and immunofluorescence analysis. After initial molecules. PECAM has been implicated in endothelial cell µ adhesion (Albelda et al., 1991; Newman et al., 1990; Newman, selection, cell lines were routinely passaged in 200 g/ml G418 in β DMEM supplemented with 10% fetal bovine serum and 1997) and has been reported to associate with -catenin penicillin/streptomycin. At least three independently derived clones (Matsumura et al., 1997). The majority of studies focusing on were characterized for each type of cell line that was established. For endothelial intercellular junctions to date have analyzed the transient transfection experiments, a subclone of COS 7 cells (COS 7- association of VE-cadherin with the actin cytoskeleton. 20) was transfected by the calcium phosphate precipitation method, However, much less is known about the potential interactions rinsed after 18 hours, and processed for immunofluorescence analysis Endothelial junction assembly 3047 after 48 hours. COS cells were routinely cultured in DMEM to centrifugation at 14,000 g. An antibody directed against the supplemented with 10% FBS and penicillin/streptomycin. All media desmoplakin amino terminus (NW161) (Bornslaeger et al., 1996) was and serum were obtained from Gibco BRL and tissue culture incubated with the cell lysate for 2 hours at 4°C. Protein G-beads plasticware was purchased from Becton Dickinson (Lincoln Park, NJ). (Pharmacia) were then added for 2 hours at 4°C, immune complexes were captured by centrifugation, and the beads were washed five times cDNA constructs in Tris buffered saline containing 0.5% Triton X-100 for 10 minutes Full length cDNAs for human VE-cadherin and a truncated VE- with gentle rotation at 4°C. Immune complexes were released by cadherin lacking the catenin binding domain were generated and incubation in reducing SDS-PAGE sample buffer at 95°C and subcloned into the pECE vector using the SV40 promoter as described analyzed by immunoblot using Enhanced Chemiluminescence previously (Breviario et al., 1995; Navarro et al., 1995). Human (Amersham). Plakoglobin was detected using mAb 11E4, and DP- plakoglobin was expressed using the LK444 vector, a gift fom Dr K. NTP was detected using monoclonal antibody M2 directed against the Trevor, which uses the β-actin promoter (Kowalczyk et al., 1994; amino-terminal FLAG epitope tag on the DP-NTP polypeptide. Palka and Green, 1997). A cDNA encoding the first 584 amino acids of desmoplakin, producing a desmoplakin polypeptide termed DP- Yeast two hybrid constructs and assays NTP, was generated as described previously (Bornslaeger et al., 1996; Yeast two hybrid vectors encoding the GAL4 DNA binding (pAS- Kowalczyk et al., 1997). An epitope tagged DP-NTP with an amino- CYH2) (Harper et al., 1993) or transcription activation domain terminal FLAG epitope tag was expressed using a CMV promoter (pACTII) (Bai and Elledge, 1995) were generously provided by Dr S. (Green et al., 1997). Full-length human desmoplakin containing a Elledge. Two hybrid constructs for full-length β-catenin and the carboxyl terminal c-myc epitope tag was constructed as described carboxyl-terminal domain of α-catenin were kindly provided by Drs previously using a CMV promoter (Stappenbeck et al., 1993). Full T.-S. Jou and W. J. Nelson (Jou et al., 1995). The Dsg1 cytoplasmic length human β-catenin was expressed using a CMV promoter and domain and DP-NTP constructs were generated as described was a generous gift from Drs P. McCrea and P. Polakis. previously (Kowalczyk et al., 1997). The plakoglobin construct lacking the amino and carboxyl terminal domains was constructed as Barrier function asssay described previously (Kowalczyk et al., 1997) and subcloned into the To monitor barrier function of various L-cell lines, cells were cultured SalI and EcoRI sites of pACTII. on Transwell polycarbonate filter membranes (24 mm diameter with To assay for interactions between proteins, yeast (strain HF7c) were a 0.4 µm pore size, Corning Costar Corporation, Cambridge, MA.). transformed with the plasmids of interest and grown on synthetic In this culture system, the filter and cell layer separate an apical and defined media lacking either tryptophan (SD−trp) for the pAS1-CYH2 basal chamber. L-cells were grown to confluence and 6×105 CPM of vector or leucine (SD−leu) for the pACTII vector to select for goat 125I-IgG (ICN, Irvine, CA) in 1.5 ml of normal growth medium transformed clones. Dual transformants were grown on medium was added to the apical chamber (approximately 20 ng of IgG/well). lacking both tryptophan and leucine to select for clones that were At various times, 200 µl samples were taken from the basal chamber transformed with both plasmids. Transformations were performed and the media analyzed using a gamma counter. The CPM that entered according to methods published in the MatchmakerTM Two Hybrid the basal chamber was determined and the results expressed as a product protocol (Clontech Laboratories Inc., Palo Alto, CA) and as percentage of the CPM originally added to the apical chamber. described previously (Kowalczyk et al., 1997). β-Galactosidase activity was monitored using 4-methylumbelliferyl β-D- Antibodies and immunofluorescence galactopyranoside (4-MUG) as a substrate following previously Cells were grown on glass coverslips to the desired stage of confluence, published methods (Meng et al., 1997). Single colonies of yeast were rinsed in phosphate buffered saline, and fixed in methanol at –20°C. VE- grown in 20 ml liquid cultures at 30°C to a density of 1.0 at A600 and cadherin was monitored using mouse monoclonal antibody BV6 100 µl of the yeast cultures were pelleted and frozen in liquid (Bioline, London; Breviario et al., 1995) or a commercially available nitrogen. The yeast were then resuspended in 350 µl of Z buffer cadherin-5 mouse monoclonal antibody (Transduction Laboratories, containing 50 µl of a 0.001 M MUG in 0.01 M phosphate buffer, pH Lexington, KT). DP-NTP was detected using a rabbit polyclonal 7.0. After one hour at 37°C, the reaction was terminated by adding antibody NW161 directed against the desmoplakin amino-terminal 400 µl stop solution (0.1 M glycine, pH 10.3). β-Galactosidase domain (Bornslaeger et al., 1996) or the monoclonal antibody M2 activity was determined as a function of the amount of 4-MU released directed against the FLAG epitope tag (Eastman Kodak, Rochester NY). from 4-MUG using a fluorometric plate reader with excitation at 360 Full-length desmoplakin was monitored using NW6, which is directed nm and emission at 450 nm. As a second reporter for interactions, against the desmoplakin carboxyl terminal domain (Angst et al., 1990). colonies were also tested for growth on SD−leu−trp−his in the presence Plakoglobin was monitored using monoclonal antibody 11E4 of 20 mM 3-aminotriazole (Sigma). Materials for base media and agar (Kowalczyk et al., 1997) which was a gift from Dr M. Wheelock, or with were obtained from Difco Laboratories, Detroit MI, and materials for a rabbit polyclonal antibody that recognizes mouse plakoglobin which synthetic defined media were purchased from Clontech Laboratories was a gift from Dr J. Papkoff (Kowalczyk et al., 1994). α-catenin was Inc. detected using a rabbit polyclonal antibody from Dr P. McCrea or a mouse monoclonal antibody 1G5 from Dr M. Wheelock, and β-catenin was detected using mouse monoclonal antibody 5H10 from Dr M. RESULTS Wheelock. Vimentin was detected using mouse monoclonal antibody V9 (Sigma). Appropriate species specific antibodies conjugated to either Desmoplakin colocalizes at intercellular junctions rhodamine or fluorescein were used for dual label immunofluorescence. with VE-cadherin in primary cultures of dermal Control experiments were carried out to verify that fluorescence was not microvascular endothelial cells due to secondary antibody cross reactivity. Microscopy was carried out As discussed above, desmoplakin was localized in vivo to using a Leica DMZ fluorescence microscope with narrow band-width lymphatic endothelium (Schmelz and Franke, 1993; Schmelz et filters and equipped with a 35mm camera. al., 1994) and was found to be expressed by cultured HUVEC Immunoprecipitation and immunoblot analysis and assembled into intercellular junctions (Valiron et al., 1996). Immunoprecipitation was carried out as described previously To determine the distribution of desmoplakin in cultured (Kowalczyk et al., 1994, 1997). Briefly, cells were scraped into Tris microvascular endothelial cells, primary human dermal buffered saline containing 0.5% Triton X-100, vortexed and subjected microvascular endothelial cells (HDMEC) were processed for 3048 A. P. Kowalczyk and others

Fig. 1. Desmoplakin localization in confluent cultures of human dermal microvascular endothelial cells. Dual label immunofluorescence was carried out to determine the distribution of desmoplakin relative to VE-cadherin, plakoglobin, and α α-catenin. In general, desmoplakin (B,D,F) colocalized with VE-cadherin (A), plakoglobin (C), and α-catenin (D). Note that desmoplakin was not present along some areas of the plasma membrane. Antibodies used were mAb 11E4 directed against plakoglobin, mAb 1G5 against α-catenin, cadherin-5 antibody against VE-cadherin, (Transduction Laboratories), and NW6 against desmoplakin. Bar, 50 µm. immunofluorescence analysis and the distribution of polypeptide comprising the first 584 amino acids of the desmoplakin was compared to VE-cadherin, plakoglobin and α- desmoplakin amino-terminal domain (DP-NTP) was found to catenin (Fig. 1). In non-confluent and newly confluent associate with desmosomal cadherins and plakoglobin and endothelial cells, desmoplakin was not present at intercellular cluster these cadherin-plakoglobin complexes (Kowalczyk et junctions (not shown), consistent with the previous report that al., 1997). To determine if the amino-terminal domain of desmoplakin expression is upregulated in post confluent cultures desmoplakin also associates with VE-cadherin, we co- of human umbilical vein endothelial cells (HUVEC) (Valiron et expressed VE-cadherin, plakoglobin and DP-NTP in stable L- al., 1996). In HDMEC that were 3-5 days postconfluent, cell fibroblast cell lines. Parental L-cells and neomycin resistant desmoplakin (B,D,F) colocalized with VE-cadherin (A), L-cells do not express detectable levels of plakoglobin or plakoglobin (C), and α-catenin (E). Although desmoplakin often desmoplakin (Kowalczyk et al., 1994, 1996). Likewise, VE- colocalized with these junctional proteins, desmoplakin staining cadherin was not detected by either immunofluorescence (not appeared less continuous. To determine the localization of shown) or immunoblot (see Fig. 8A) in neomycin resistant desmoplakin relative to the vimentin intermediate filament control L-cells. In L-cell lines co-expressing VE-cadherin, network, dual label immunofluorescence analysis for plakoglobin and DP-NTP, VE-cadherin was predominantly desmoplakin and vimentin (Fig. 2A) and plakoglobin and concentrated at cell-cell borders and colocalized with both vimentin (Fig. 2B) was performed. The HDMEC assembled an plakoglobin (Fig. 3A,B) and with DP-NTP (Fig. 3C,D). In extensive vimentin intermediate filament network. In addition, addition, DP-NTP colocalized with the actin associated proteins bundles of vimentin were often found to terminate or align α-catenin (Fig. 3E,F) and β-catenin (not shown), which are parallel to the plasma membrane in areas that also stained present endogenously in L-cells (Ozawa et al., 1989). No positive for desmoplakin and plakoglobin. obvious difference in VE-cadherin distribution was observed in L-cells in the presence of DP-NTP compared to L-cells VE-cadherin recruits plakoglobin and desmoplakin expressing only VE-cadherin and plakoglobin (not shown). to cell-cell borders when co-expressed in stable L- In addition to DP-NTP, L-cell lines co-expressing VE- cell fibroblast lines cadherin, plakoglobin and full-length desmoplakin were also The results above suggest that desmoplakin may play a role in established (Fig. 4). Although DP-NTP expression in L-cell linking the vimentin intermediate filament network to cell-cell lines was typically homogeneous, the expression of full-length borders in vascular endothelial cells. In a previous study, a desmoplakin in the L-cell lines tended to be heterogeneous, Endothelial junction assembly 3049

Fig. 2. Vimentin filaments localize along the endothelial plasma membrane at regions containing plakoglobin and desmoplakin. Human dermal microvascular endothelial cells were processed for dual label immunofluorescence using antibodies directed against vimentin (mAb V9, Sigma), plakoglobin (rabbit polyclonal antibody from J. Papkoff), or desmoplakin (rabbit polyclonal antibody NW6). The vimentin antibody was detected using a fluorescein conjugated secondary antibody and the plakoglobin and desmoplakin antibodies were detected using rhodamine conjugated secondary antibodies. Double exposures of vimentin and desmoplakin (A) or vimentin and plakoglobin (B) were taken using color slide film. Note areas of colocalization at cell-cell borders where vimentin filaments terminate at the plasma membrane. Bar, 10 µm. with only some cells within the population expressing full- intermediate filament networks when expressed in cultured length desmoplakin. In these cell lines, full-length cells (Stappenbeck and Green, 1992; Stappenbeck et al., 1993), desmoplakin (A,C) colocalized with VE-cadherin (B) and full-length desmoplakin was often detected in a filamentous plakoglobin (D). Due to the presence of the desmoplakin staining pattern and in perinuclear aggregates. In addition, carboxyl terminal domain, which aligns with and disrupts desmoplakin was also recruited to cell-cell borders in some

Fig. 3. Immunofluorescence analysis of L-cell lines expressing VE- cadherin, plakoglobin, and DP-NTP. To determine if VE-cadherin recruits desmoplakin to cell-cell borders, L- cell lines co-expressing exogenous VE-cadherin, plakoglobin, and the amino-terminal domain of desmoplakin (DP-NTP) were established and characterized by immunofluorescence. In these cell lines, VE-cadherin (A,C) colocalized with both plakoglobin (B) and DP- NTP (D) at cell-cell borders. In addition, endogenous α-catenin (E) and β-catenin (not shown), which are α up-regulated in L-cells expressing classical cadherins (Nagafuchi et al., 1991), also colocalized with DP- NTP (F) at cell-cell borders. There was no detectable VE-cadherin, plakoglobin, or desmoplakin in parental or neomycin control L-cells as determined by immunofluorescence (not shown) or immunoblot analysis (Fig. 8) (see also Kowalczyk et al., 1994, 1996, 1997). Bar, 50 µm. 3050 A. P. Kowalczyk and others

Fig. 4. Analysis of L-cell lines co-expressing VE- cadherin, plakoglobin and full-length desmoplakin. L-cell lines co-expressing VE-cadherin, plakoglobin, and full-length desmoplakin were established and analyzed by immunofluorescence microscopy. Desmoplakin (A,C,E) colocalized with VE-cadherin (B,F) and with plakoglobin (D). In untreated L-cells, desmoplakin was often observed in a filamentous staining pattern and in perinuclear aggregates (A) that colocalized with VE-cadherin (B). These aggregates are similar to those reported previously that contain both desmoplakin and intermediate filament polypeptides (Stappenbeck and Green, 1992). Desmoplakin was also observed at cell borders in some areas (see arrows in C and D). In L-cells treated with 1 µM forskolin for 24 hrs, desmoplakin staining at borders (E) was more prominent and less filamentous staining was detected, consistent with the ability of forskolin to inhibit desmoplakin interactions with intermediate filament networks (Stappenbeck et al., 1994). Bar, 50 µm.

Fig. 5. Analysis of barrier properties of L-cell lines expressing VE-cadherin. The ability of L-cell lines to function as a barrier to protein flux across cell monolayers was tested by growing the cells on polycarbonate filter membranes (Costar) in which the cell layer separates apical and basal chambers containing growth medium. 125I-IgG was then added to the apical chamber and the CPM that entered the basal chamber were monitored by taking samples of the basal media at various times. L-cells expressing full-length VE-cadherin and plakoglobin (L-V/Pg) or VE-cadherin, plakoglobin and DP-NTP (L-V/Pg/DP-NTP) exhibited increased barrier function compared to Neo control cells (A) or L-cells co-expressing plakoglobin and DP-NTP with a truncated VE-cadherin lacking the catenin-binding domain (L-V∆ICS/Pg/DP-NTP) (B). Error bars represent s.e.m. with each point representing at least triplicate wells. Data shown are representative of at least three independently conducted experiments. Endothelial junction assembly 3051

Fig. 6. DP-NTP is not recruited to cell-cell borders in the absence of plakoglobin in α stable L-cell lines. L-cell lines co-expressing VE-cadherin and DP-NTP were established and processed for immunofluorescence microscopy. VE-cadherin (A) was present at cell-cell borders, but DP-NTP (B) was diffuse in the cytoplasm and did not exhibit any co- localization with VE-cadherin. In contrast, VE-cadherin (C) colocalized with α-catenin (D) at cell-cell borders. In addition, β-catenin (E) was present at cell-cell borders and colocalized with α-catenin (F). (G) Immunoblot analysis demonstrating that β α these L-cell lines express VE-cadherin and DP-NTP. Neomycin control cells (lane 1), L-cells expressing VE-cadherin and plakoglobin (lane 2), and L-cells co- expressing VE-cadherin and DP-NTP (lane 3) are shown. Plakoglobin and VE-cadherin were detected by immunoblot of whole cell lysates and DP-NTP was immunoprecipiated from cell lysates with antibody NW161 against the desmoplakin amino-terminal domain and detected by immunoblot using an antibody directed against the FLAG tag (mAb M2). Plakoglobin expression was monitored using a polyclonal antibody that recognizes both mouse and human plakoglobin (Hinck et al., 1994; Kowalczyk et al., 1994). Although endogenous plakoglobin is sometimes detected in L-cells expressing cadherins (Kowalczyk et al., 1994), the levels of endogenous plakoglobin are apparently too low to support the recruitment of DP-NTP to cell-cell borders (B) or clustering of desmosomal cadherins (Kowalczyk et al., 1997). Bar, 50 µm. cells (see arrows). In a previous study, activation of protein capillaries and into the interstitium (van Hinsbergh, 1997; Lum kinase A by forskolin treatment resulted in the phosphorylation and Malik, 1996; Dejana, 1996). To determine if L-cells of a serine residue near the desmoplakin carboxyl terminal expressing VE-cadherin exhibited enhanced barrier function, domain and the disruption of desmoplakin alignment with the cells were cultured on polycarbonate filter membranes and keratin networks (Stappenbeck et al., 1994). Therefore, L-cell the flux of radiolabelled IgG across the monolayers was lines expressing full length desmoplakin were treated with measured over time (Fig. 5). L-cells expressing VE-cadherin forskolin to prevent sequestration of desmoplakin along and plakoglobin or VE-cadherin, plakoglobin and DP-NTP vimentin filaments (E,F). In L-cells treated with forskolin, were compared to neomycin resistant control L-cells (Fig. 5A). filamentous desmoplakin staining was reduced and desmoplakin The flux of IgG across monolayers of L-cells expressing VE- staining at borders was more pronounced (E). Again, full-length cadherin was consistently lower than flux across monolayers desmoplakin often colocalized with VE-cadherin (F) and of neo control L-cells. However, expression of DP-NTP did not plakoglobin (not shown). These data demonstrate that VE- appear to alter the barrier properties of the L-cells. Due to the cadherin recruits both DP-NTP and full-length desmoplakin to heterogeneity of L-cell lines expressing full-length cell-cell borders when expressed in L-cell fibroblasts. desmoplakin, it was not possible test the contribution of full- Endothelial intercellular junctions are thought to play a length desmoplakin expression to the barrier properties of the major role in regulating the flux of fluid and solutes across L-cells. L-cells co-expressing plakoglobin, DP-NTP and a 3052 A. P. Kowalczyk and others

Fig. 7. The recruitment of DP-NTP to COS cell-cell borders by VE-cadherin requires the plakoglobin binding domain of VE-cadherin. VE-cadherin cDNA or a truncated VE- cadherin cDNA were expressed transiently in COS cells with cDNAs encoding DP-NTP and plakoglobin or β-catenin. In the absence of VE-cadherin, plakoglobin is distributed diffusely in the cytoplasm and DP-NTP is often present in aggregates in a perinuclear pattern (not shown) (Kowalczyk et al., 1997). In cells co-transfected with VE-cadherin, plakoglobin, and DP-NTP cDNAs, VE- cadherin (A) recruited DP-NTP (B) and plakoglobin (not shown) to cell-cell borders. In contrast, a mutant VE-cadherin lacking the plakoglobin binding domain, VE- Cad∆ICS (C), did not recruit either plakoglobin (not shown) or DP-NTP (D) to cell junctions. In addition, VE-cadherin (E) does not recruit DP-NTP (F) to cell-cell borders when co-expressed with β-catenin. Although β-catenin is recruited to cell-cell junctions (G), DP-NTP remains predominantly in cytoplasmic aggregates (H). Bar, 10 µm. truncated VE-cadherin lacking the β-catenin/plakoglobin (Lampugnani et al., 1995). Therefore, we sought to determine binding domain were also tested (Fig. 5B). L-cells expressing whether DP-NTP is recruited to sites of VE-cadherin mediated the truncated VE-cadherin mutant did not exhibit increased adhesion by plakoglobin or β-catenin. To test this, stable L-cell barrier function compared to neo control L-cells, similar to lines co-expressing VE-cadherin and DP-NTP were results obtained when this mutant was expressed in CHO cells established (Fig. 6). Although L-cells do express low levels of (Navarro et al., 1995). These data support the idea that VE- endogenous plakoglobin, the levels are not sufficient to support cadherin mediated adhesion plays an important role in the DP-NTP clustering of the desmosomal cadherins (Kowalczyk establishment of endothelial barrier function and that linkage et al., 1997). In contrast, significant levels of α- and β-catenin of VE-cadherin to the cytoskeleton is critical for the accumulate in L-cells co-expressing classical cadherins, which establishment of this barrier. dramatically upregulate catenin protein levels presumably by rescuing the catenins from rapid proteolytic degradation VE-cadherin requires plakoglobin to recruit (Nagafuchi et al., 1991; Kowalczyk et al., 1994). In L-cells co- desmoplakin to cell-cell borders expressing VE-cadherin and DP-NTP, VE-cadherin In contrast to the desmosomal cadherins, which bind accumulated at cell-cell borders (Fig. 6A). In contrast to L- preferentially to plakoglobin (Plott et al., 1994), VE-cadherin cells co-expressing plakoglobin with DP-NTP and VE- has been shown to interact with both plakoglobin and β-catenin cadherin (Fig. 3), in the absence of exogenous plakoglobin DP- Endothelial junction assembly 3053

Fig. 9. DP-NTP binds to plakoglobin but not β-catenin in the yeast two hybrid system. To determine if the amino-terminal domain of desmoplakin binds specifically to plakoglobin, DP-NTP was tested for the ability to interact with plakoglobin and full-length β-catenin using both a β-galactosidase assay (A) and growth in the absence of histidine (B) as reporter assays for protein interactions. For the β- galactosidase assay, the galactosidase substrate MUG was used and enzyme activity was monitored as an indication of protein interactions. As reported previously, the carboxyl terminal domain of α-catenin interacts directly with β-catenin and DP-NTP binds directly to plakoglobin. As a negative control, yeast were β cotransformed with DP-NTP and the desmosomal cadherin Dsg1, which do not interact directly (Kowalczyk et al., 1997). However, no interactions were detected between DP-NTP and β-catenin using either the galactosidase assay or the histidine growth assay. Fig. 8. Plakoglobin, but not β-catenin, co-immunoprecipitates with DP-NTP. Stable L-cell lines co-expressing VE-cadherin, plakoglobin, and DP-NTP were examined for expression of each protein by western blot analysis of whole cell lysates (A). VE- cadherin, plakoglobin and DP-NTP were detected only in L-cell lines transfected with the cDNAs encoding these proteins (VE-cad/Pg/DP- NTP) and were not detected in L-cells expressing only the neomycin resistance marker (Neo). In addition, immunoprecipitation of DP- NTP using NW161 from L-cell lysates co-precipitated plakoglobin, demonstrating that these proteins were in a complex in cell lines co- expressing VE-cadherin (B). Although these cells also express β- catenin, which colocalizes with VE-cadherin (see Fig. 7), no β- catenin was detected in the DP-NTP immunoprecipitations. Fig. 10. Model describing the possible molecular arrangements of both actin and vimentin binding proteins in endothelial intercellular junctions. VE-cadherin binds directly to both plakoglobin and β- α NTP remained diffusely distributed in the cytoplasm (Fig. 6B). catenin, both of which can bind directly to -catenin, which mediates However, VE-cadherin colocalized at intercellular borders with interactions with the actin filament system. Interactions between VE- cadherin and vimentin may be established by plakoglobin, which α-catenin (Fig. 6C and D), and extensive β-catenin staining α specifically associates with desmoplakin. Other proteins that are not also colocalized with -catenin (Fig. 6E and F). Immunoblot shown may also play roles in establishing interactions between VE- analysis confirmed that these these cell lines co-expressed VE- cadherin and these two cytoskeletal network systems. In addition, cadherin and DP-NTP (G). VE-cadherin, plakoglobin, and DP- regions of the plasma membrane may include domains in which VE- NTP were not detected in neo control L-cells (lane 1). cadherin is anchored to only the actin or vimentin network. 3054 A. P. Kowalczyk and others

Plakoglobin was present in L-cells co-transfected with cDNA reporters for protein interactions. As previously demonstrated, constructs encoding plakoglobin and VE-cadherin (lane 2), but β-catenin binds directly to α-catenin (Jou et al., 1995), and plakoglobin was not detected in L-cells co-expressing VE- plakoglobin binds directly to DP-NTP (Kowalczyk et al., cadherin and DP-NTP (lane 3). 1997). However, binding was not detected between DP-NTP In the L-cell lines, the junctional proteins are expressed at and β-catenin in either the fluorescence-based assay or the moderate levels. To verify that DP-NTP recruitment to cell histidine growth assay. Together with the results presented borders could not be mediated by β-catenin when the proteins above, these data indicate that plakoglobin specifically binds were expressed at higher levels, the recruitment of to desmoplakin and couples this intermediate filament binding desmoplakin to intercellular junctions was monitored using protein to VE-cadherin. transient transfection experiments in COS cells (Fig. 7). In each experiment, duplicate transfections and multiple combinations of antibodies were used for dual label DISCUSSION immunofluorescence to verify that each protein was expressed. In the absence of VE-cadherin, plakoglobin was diffusely The results of the present study indicate that the intermediate distributed in the cytoplasm and DP-NTP was found filament binding protein desmoplakin is assembled into the predominantly in punctate aggregates that were often present intercellular junctions of dermal microvascular endothelial in a perinuclear distribution (not shown, see Kowalczyk et al., cells. In addition, VE-cadherin recruits desmoplakin to cell- 1997). However, in the presence of VE-cadherin, both cell borders and the association of desmoplakin with VE- plakoglobin (not shown) and DP-NTP were redistributed to cadherin requires plakoglobin. Furthermore, the amino- cell-cell borders and colocalized with VE-cadherin (Fig. terminal domain of desmoplakin specifically forms complexes 7A,B). To determine if the catenin-binding domain of VE- with plakoglobin and binds directly to plakoglobin but not to cadherin was required for the recruitment of DP-NTP to cell- β-catenin, suggesting that plakoglobin plays a specific role in cell borders, a truncated VE-cadherin lacking the the attachment of an intermediate filament binding protein to plakoglobin/β-catenin binding domain was co-expressed with VE-cadherin. These findings raise the possibility that VE- plakoglobin and DP-NTP (Fig. 7C,D). Although DP-NTP (Fig. cadherin in endothelial junctions may be coupled to both the 7D) and plakoglobin were expressed (not shown), DP-NTP actin microfilament system and the intermediate filament was not recruited to cell-cell borders by the truncated VE- cytoskeleton. cadherin. In addition, DP-NTP was not recruited to cell-cell Several studies have indicated that VE-cadherin plays an borders when VE-cadherin was co-expressed with β-catenin important role in adhesion, endothelial barrier function, and in (Fig. 7E,F). Although exogenously expressed β-catenin (G) angiogenesis (Ali et al., 1997; Bach et al., 1998; Matsumura et was recruited to cell-cell borders with VE-cadherin, DP-NTP al., 1997; Vittet et al., 1997). VE-cadherin is coupled to the (H) remained in cytoplasmic aggregates in the absence of actin cytoskeleton by β-catenin and plakoglobin (Lampugnani exogenously expressed plakoglobin. These data indicate that et al., 1995; Navarro et al., 1995), both of which bind to the plakoglobin is required for DP-NTP to be recruited to cell-cell actin associated protein α-catenin (Aberle et al., 1994; Jou et junctions with VE-cadherin, and that β-catenin is unable to al., 1995). The observations that desmoplakin is expressed in substitute for this function of plakoglobin even when expressed endothelial cells and that VE-cadherin recruits desmoplakin to at high levels in the transient transfection system. cell-cell borders, suggest that VE-cadherin may associate with the vimentin intermediate filament network through specific The desmoplakin amino-terminal domain binds to cytoplasmic interactions. This is supported by the observation plakoglobin but not β-catenin that VE-cadherin is assembled into intercellular junctions of The observation that DP-NTP was recruited to cell-cell borders lymphatic endothelium that have been termed complexus by VE-cadherin and plakoglobin but not by VE-cadherin and adhaerentes, the components of which appear to include VE- β-catenin suggested that DP-NTP may form complexes with cadherin, plakoglobin and desmoplakin (Schmelz and Franke, plakoglobin but not β-catenin. To determine if plakoglobin 1993; Schmelz et al., 1994). Similar observations have been specifically forms complexes with DP-NTP in L-cells co- made in cultured HUVEC (Valiron et al., 1996), and as expressing VE-cadherin, DP-NTP was immunoprecipitated reported here, in cultured HDMEC. It is likely that the from L-cells co-expressing DP-NTP, plakoglobin and VE- vimentin intermediate filament network in endothelial cells cadherin (Fig. 8). As shown in Fig. 8A, VE-cadherin, plays an important role in endothelial cell function. Endothelial plakoglobin, and DP-NTP were specifically detected only in L- cells assemble an extensive vimentin network that appears to cell lines transfected with cDNA constructs encoding these terminate at cell-cell borders (Fig. 2), and alterations in the junctional proteins. In addition, β-catenin was upregulated in vimentin cytoskeleton have been associated with the degree of these cells and was detected in total cell lysates (Fig. 8B, far endothelial cell confluence (Savion et al., 1982) and barrier right lane). As previously reported (Kowalczyk et al., 1997), function (Stasek et al., 1992). plakoglobin consistently co-immunoprecipitated with DP- The results presented here suggest that VE-cadherin is NTP, but β-catenin could not be specifically co- coupled to desmoplakin by plakoglobin. This interpretation is immunoprecipitated with DP-NTP (Fig. 8B). These results based on the observation that DP-NTP binds directly to suggested that DP-NTP may bind to plakoglobin but not β- plakoglobin and that plakoglobin is required for the catenin. To test this possibility, interactions between these recruitment of DP-NTP to cell-cell borders by VE-cadherin in proteins were analyzed in the yeast two hybrid system using both COS cells and L-cells. When expressed in COS cells, full both a semi-quantitative fluorescence based β-galactosidase length VE-cadherin recruited DP-NTP to cell borders but a assay (Fig. 9A) and a histidine growth assay (Fig. 9B) as truncated VE-cadherin lacking the plakoglobin binding domain Endothelial junction assembly 3055 failed to associate with DP-NTP. Similarly, plakoglobin β-catenin, with β-catenin mediating linkage primarily to the mediates the interaction between the desmosomal cadherins actin cytoskeleton and plakoglobin mediating interactions and DP-NTP (Kowalczyk et al., 1997) (E. A. Bornslaeger and primarily with the vimentin cytoskeletal network. Homophilic K. J. Green, unpublished), suggesting that plakoglobin plays a or lateral (Norvell and Green, 1998) interactions between critical role in linking desmoplakin to both the desmosomal cadherin molecules on adjacent cells could recruit both actin cadherins and VE-cadherin. Unlike the desmosomal cadherins, and vimentin associated proteins to the same domains of the VE-cadherin also binds to β-catenin. Although β-catenin and plasma membrane, thereby leading to the assembly of a mixed plakoglobin are closely related, β-catenin could not substitute junction, containing both actin and vimentin binding proteins. for plakoglobin in the recruitment of DP-NTP to cell junctions The data presented in the present study as well as in previous with VE-cadherin. Furthermore, DP-NTP binds directly to papers (Schmelz and Franke, 1993; Schmelz et al., 1994; plakoglobin in the yeast two hybrid system (Fig. 9) and forms Valiron et al., 1996) are consistent with this model. It should complexes with plakoglobin that can be co- be noted that some membrane domains could be pure, rather immunoprecipitated from L-cell lysates (Fig. 8). However, we than mixed, and might consist of only actin or vimentin were unable to detect complex formation between DP-NTP and associated proteins. At present, this model represents an β-catenin or direct binding between these proteins using the hypothesis, and it will be important to define further the nature two hybrid analysis. These results suggest that one function of of these junctions using both ultrastructural and biochemical plakoglobin in endothelial junctions is to promote association approaches to understand precisely how both actin and with the vimentin network, and this function appears to be vimentin associated proteins might co-assemble at a common specific to plakoglobin and not β-catenin. adhesive interface. In keratinocytes and other epithelia, associations between The presence of mixed junctions containing both actin and the keratin intermediate filament cytoskeleton and intermediate filament binding proteins was reported previously desmosomes is thought to play an important role in in A431 epithelial cells expressing the desmoplakin amino- maintaining the structural integrity of epithelial sheets (Fuchs, terminal polypeptide DP-NTP (Bornslaeger et al., 1996). 1994; Coulombe and Fuchs, 1994; Steinert and Bale, 1993; Expression of this protein uncoupled keratin filament Fuchs and Cleveland, 1998). The ability of plakoglobin to attachment to desmosomes and caused mixing of adherens couple VE-cadherin to desmoplakin suggests that these junction and desmosomal components. Interestingly, co- complexes may play an important role in the structural integrity expression of DP-NTP with plakoglobin and the desmosomal of endothelial monolayers. The kinetics of endothelial junction cadherins in L-cells caused the cadherin-plakoglobin complex assembly after switching the cells from low to normal calcium to cluster into punctate regions of staining (Kowalczyk et al., levels to induce junction formation indicates that β-catenin is 1997). In contrast, staining for VE-cadherin and plakoglobin rapidly assembled into the junctions, whereas plakoglobin was not punctate in L-cells co-expressing DP-NTP (Fig. 2). In (Lampugnani et al., 1995) and desmoplakin (Valiron et al., these cells, VE-cadherin, plakoglobin and DP-NTP were 1996) are associated preferentially with mature endothelial present at cell-cell borders in a somewhat continuous staining junctions. Experiments in which antisense oligononucleotides pattern. Unlike desmosomes, which are highly punctate and were used to inhibit plakoglobin expression indicated that densely organized structures, endothelial junctions are more plakoglobin is important for endothelial cells to resist the similar to adherens junctions, and tend to be much more forces of shear stress (Schnittler et al., 1997). One possible continuous along the plasma membrane. This suggests that the explanation for these observations is that initial endothelial associations between desmoplakin and plakoglobin might be junction assembly is driven by VE-cadherin and actin influenced by the type of cadherin to which plakoglobin is associated proteins, whereas mature junctions also incorporate bound. These interactions might influence both the degree of plakoglobin and vimentin binding proteins such as clustering that occurs as well as whether the cadherin desmoplakin, thereby promoting the mechanical strength of the associates with actin, intermediate filaments, or with both types junctions. This remains to be demonstrated directly, and it is of cytoskeletal networks. likely that other intermediate filament associated proteins in In addition to co-expressing DP-NTP with VE-cadherin and addition to desmoplakin are assembled into endothelial plakoglobin, we also co-expressed full-length desmoplakin intercellular junctions. with VE-cadherin and plakoglobin. While the amino-terminal The observation that VE-cadherin associates with both actin domain of desmoplakin binds to plakoglobin, the carboxyl and intermediate filament binding proteins raises the intriguing terminal domain of desmoplakin binds to intermediate filament possibility that endothelial cells assemble junctions that are polypeptides, including keratins, vimentin, and desmin anchored to both the actin and intermediate filament networks. (Kouklis et al., 1994; Meng et al., 1997; Stappenbeck and In epithelial cells, actin associated adherens junctions and Green, 1992). Although VE-cadherin, plakoglobin, and DP- intermediate filament associated desmosomes occupy different NTP were expressed homogeneously in the L-cells, we were plasma membrane domains. For example, in normal epithelial unable to isolate stable L-cell lines that expressed full-length cells, desmoplakin and α-catenin do not colocalize. However, desmoplakin in a homogeneous manner. It was possible to in HDMEC, we observed significant co-localization of clearly demonstrate that full-length desmoplakin was recruited desmoplakin and α-catenin (Fig. 1) and in L-cells, VE- to cell-cell borders with VE-cadherin, but it was not possible cadherin recruited both DP-NTP and α-catenin to the same to do biochemical or functional analyses on these cell lines. regions of the plasma membrane (Fig. 3). These observations Interestingly, forskolin treatment of L-cells expressing full- lead us to propose that mature endothelial intercellular length desmoplakin increased desmoplakin accumulation at junctions might be organized as shown in Fig. 10. In this cell-cell borders and decreased the degree of filamentous model, VE-cadherin could associate with either plakoglobin or staining. Activation of protein kinase A causes 3056 A. P. Kowalczyk and others phosphorylation of a serine residue near the desmoplakin protein desmoplakin from cell-cell interfaces disrupts anchorage of carboxyl terminus and decreased interaction between intermediate filament bundles and alters intercellular junction assembly. J. desmoplakin and intermediate filament networks (Stappenbeck Cell Biol. 134, 985-1001. Breviario, F., Caveda, L., Corada, M., Martin-Padura, I., Navarro, P., et al., 1994) (Fig. 4). Upregulation of cAMP in endothelial cells Golay, J., Introna, M., Gulino, D., Lampugnani, M. G. and Dejana, E. appears to enhance endothelial barrier function in cells treated (1995). Functional properties of human vascular endothelial cadherin with inflammatory mediators or growth factors (Minnear et al., (7B4/Cadherin-5), an endothelium-specific cadherin. Arterioscler. Thromb. 1989). These results are consistant with the hypothesis that a Vasc. Biol. 15, 1229-1239. Coulombe, P. A. and Fuchs, E. (1994). Molecular mechanisms of keratin balance between kinases and phosphatases regulates gene disorders and other bullous diseases of the skin. In Molecular desmoplakin assembly into junctions and may thereby Mechanisms of Epithelial Cell Junctions: from Development to Disease (ed. influence junctional integrity. S. Citi), pp. 259-285. Austin: R. G. Landes Co. It is currently unclear why the vimentin and actin networks Cowin, P., Kapprell, H., Franke, W. W., Tamkun, J. W. and Hynes, R. O. might be co-assembled around a common adhesive interface in (1986). Plakoglobin: A protein common to different kinds of intercellular adhering junctions. Cell 46, 1063-1073. endothelial cells. It is possible that this type of architectural Cowin, P. and Mechanic, S. (1994). Desmosomal cadherins and their arrangement facilitates the ability of endothelial cells to rapidly cytoplasmic interactions. 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Intermediate filaments and disease: mutations that cripple networks with alterations in cell adhesion and motility. Thus, cell strength. J. Cell Biol. 125, 511-516. it will be important to determine what regulates the Fuchs, E. and Cleveland, D. W. (1998). A structural scaffolding of incorporation of plakoglobin into endothelial junctions and intermediate filaments in health and disease. Science 279, 514-519. how these complexes with intermediate filament binding Garrod, D., Chidgey, M. and North, A. (1996). Desmosomes: differentiation, proteins might contribute to endothelial barrier function, development, dynamics and disease. Curr. Opin. Cell Biol. 8, 670-678. Green, K. J. and Jones, J. C. R. (1996). Desmosomes and hemidesmosomes: angiogenesis, and leukocyte diapedesis. structure and function of molecular components. FASEB J. 10, 871-881. Green, K. J., Bornslaeger, E. A., Kowalczyk, A. P., Palka, H. L. and The authors thank Drs M. Wheelock, K. Johnson, J. Papkoff, and Norvell, S. M. (1997). Specificity of desmosomal plaque protein K. Trevor for sharing antibody and cDNA reagents that made this interactions with intermediate filaments: keeping adhesive junctions work possible. Thanks also to Drs S. Elledge, T.-Z. Jo and W. J. segregated. J. Gen. Physiol. 52, 123-139. Nelson for generously providing yeast two hybrid reagents. The Harper, J. W., Adami, G., Wei, N., Keyomarsi, K. and Elledge, S. J. (1993). authors would also like to thank Dr M. Lingen for providing dermal The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin- microvascular endothelial cells and for assistance with culturing dependent kinases. Cell 75, 805-816. Herrenknecht, K., Ozawa, M., Eckerskorn, C., Lottspeich, F., Lenter, M. endothelial cells, Dr N. Clipstone for the use of the Fluorscan plate and Kemler, R. (1991). The uvomorulin-anchorage protein alpha catenin is reader, and L. Bannon and S. Norvell for providing insightful a vinculin homologue. Proc. Nat. Acad. Sci. 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