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Developmental Biology 319 (2008) 298–308

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Developmental Biology

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Identification of a novel intermediate filament-linked N-cadherin/γ-catenin complex involved in the establishment of the cytoarchitecture of differentiated lens fiber cells

Michelle Leonard, Yim Chan, A. Sue Menko ⁎

Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 571 Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, USA

article info abstract

Article history: Tissue morphogenesis and maintenance of complex tissue architecture requires a variety of cell–cell junctions. Received for publication 17 December 2007 Typically, cells adhere to one another through cadherin junctions, both adherens and desmosomal junctions, Revised 14 April 2008 strengthened by association with cytoskeletal networks during development. Both β-andγ-catenins are Accepted 18 April 2008 reported to link classical cadherins to the actin cytoskeleton, but only γ-catenin binds to the desmosomal Available online 8 May 2008 cadherins, which links them to intermediate filaments through its association with desmoplakin. Here we fi γ Keywords: provide the rst biochemical evidence that, in vivo, -catenin also mediates interactions between classical fi γ-catenin cadherins and the intermediate lament cytoskeleton, linked through desmoplakin. In the developing lens, Cadherin which has no desmosomes, we discovered that vimentin became linked to N-cadherin complexes in a Intermediate filament differentiation-state specific manner. This newly identified junctional complex was tissue specific but not Vimentin unique to the lens. To determine whether in this junction N-cadherin was linked to vimentin through γ- Lens development catenin or β-catenin we developed an innovative “double” immunoprecipitation technique. This approach fi Lens ber cell differentiation made possible, for the first time, the separation of N-cadherin/γ-catenin from N-cadherin/β-catenin complexes and the identification of multiple members of each of these isolated protein complexes. The study revealed that vimentin was associated exclusively with N-cadherin/γ-catenin junctions. Assembly of this novel class of cadherin junctions was coincident with establishment of the unique cytoarchitecture of lens fiber cells. In addition, γ-catenin had a distinctive localization to the vertices of these hexagonally shaped differentiating lens fiber cells, a region devoid of actin; while β-catenin co-localized with actin at lateral cell interfaces. We believe this novel vimentin-linked N-cadherin/γ-catenin junction provides the tensile strength necessary to establish and maintain structural integrity in tissues that lack desmosomes. © 2008 Elsevier Inc. All rights reserved.

Introduction and the organization of actin filaments at the plasma membrane (Cowin and Burke, 1996; Hulsken et al., 1994; Nagafuchi et al., 1991; Peifer et al., A well-recognized function of cadherin junctions is the establish- 1994). In contrast, in desmosomes, cell–cell junctions that provide cells ment and maintenance of the tissue cytoarchitecture (Gumbiner, 1996, with tensile strength, the desmosomal cadherins desmocollin and 2005; Halbleib and Nelson, 2006; Takeichi, 1995; Wheelock and Jensen, desmoglein link to the intermediate filament cytoskeletal network. This 1992). In our own studies of the lens we show that proper morphogen- linkage is mediated by γ- but not β-catenin through its association with esis depends on the ability of lens cells to organize mature N-cadherin intermediate filament-linking proteins such as desmoplakin (Cowin et cell–cell junctions (Ferreira-Cornwell et al., 2000). To date, the function al., 1986; Franke et al., 1994; Fuchs and Cleveland, 1998; Gallicano et al., of classical cadherins like N-cadherin in tissue morphogenesis has been 1998; Getsios et al., 2004; Koch and Franke, 1994; Troyanovsky et al., understood to involve their interaction with the actin cytoskeletal 1994). Thus, γ-catenin is a versatile component of cadherin complexes, network (Cowin and Burke,1996; Geiger,1989; Gumbiner, 2005; Pavalko capable of fostering the interaction of classical cadherins with the actin and Otey, 1994). This linkage to the actin cytoskeleton is mediated cytoskeleton in adherens junctions and desmosomal cadherins with the through α-, β-, and γ-catenins (Cowin and Burke, 1996; Nagafuchi et al., intermediate filament cytoskeleton in desmosomes. While γ-catenin 1991; Peifer et al., 1994), with β-catenin and γ-catenin (plakoglobin) has the potential to link non-desmosomal cadherins to the intermediate binding directly to the cytoplasmic tail of classical cadherins in a filament cytoskeleton, the existence of a cadherin junction with this mutually exclusive manner (Cowin and Burke,1996; Hulsken et al.,1994; property has not yet been shown in vivo. Since the lens has no Nagafuchi et al., 1991; Peifer et al., 1994). α- catenin then binds to these desmosomes to provide tensile strength (Straub et al., 2003), but is catenins where it is believed to play a role in the recruitment of actin to subject to significant forces as it focuses images on the retina, it is an ideal tissue in which to investigate the existence of novel junctional ⁎ Corresponding author. Fax: +1 215 923 3808. structures that could provide tensile strength to tissues without E-mail address: [email protected] (A.S. Menko). desmosomes.

0012-1606/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2008.04.036 M. Leonard et al. / Developmental Biology 319 (2008) 298–308 299

Evidence that non-desmosomal cadherins might be linked to the show that the vimentin intermediate filament cytoskeleton is linked intermediate filament cytoskeleton was first shown in studies of VE- to N-cadherin, the principal cell–cell adhesion molecule of differ- cadherin, a Type II classical cadherin (E-cadherin and N-cadherin are entiating lens fiber cells. Using a novel double immunoprecipitation Type I classical cadherins), and the principal cadherin in cell–cell technique we developed for this study, we separated N-cadherin/ contacts of the vascular endothelium (Lampugnani et al., 1992; γ-catenin junctions from N-cadherin/β-catenin junctions, and de- Navarro et al., 1998; Uccini et al., 1994). While, like the lens, vascular monstrated that the link between N-cadherin and intermediate fila- endothelial cells lack desmosomes and desmosomal cadherins ments occurs only in N-cadherin/γ-catenin junctions. In addition, our (Schmelz and Franke, 1993), in cultured human umbilical vein endo- studies show that these N-cadherin/γ-catenin junctions contain the thelial cells the vimentin intermediate filament protein co-localizes at intermediate filament-linker protein desmoplakin, the classical linker the plasma membrane with both VE-cadherin and desmoplakin, an between γ-catenin and intermediate filament proteins. Our studies intermediate filament binding protein (Valiron et al., 1996). When provide the first evidence of an N-cadherin/γ-catenin/intermediate VE-cadherin and desmoplakin are co-transfected into cultured filament complex in vivo. The existence of this unique intermediate fibroblasts they co-localize only if γ-catenin is present (Kowalczyk filament-linked cadherin junction is particularly exciting as its pro- et al., 1998) and expression of an N-cadherin/estrogen receptor fusion perties are likely to provide the structural stability necessary to protein by fibroblasts results in its association with endogenous establish and maintain complex tissue cytoarchitecture. vimentin (Kim et al., 2005); however, there is of yet no biochemical evidence that γ-catenin links classical cadherins to the intermediate Materials and methods filament cytoskeleton in vivo. Lens microdissection The function of cadherins in embryonic development is likely mediated through their interactions with specific cytoskeletal net- Lenses were isolated from embryonic day 10 (E10) chicken eggs (Truslow Farms, works, as cadherin junctions provide anchoring sites for cytoskeletal Chestertown, MD) and microdissected as previously described (Walker and Menko, 1999) components at the cell membrane (Cowin and Burke, 1996; Pavalko to yield four distinct regions of differentiation: the central anterior epithelium (EC), the fi fi and Otey, 1994). Such cytoskeletal–cell membrane attachments are equatorial epithelium (EQ), cortical ber cells (FP), and the nuclear ber zone (FC) (Fig.1A). central to establishing the relationships between cells that are neces- Protein extraction, standard immunoprecipitation and immunoblotting sary for tissue morphogenesis and for maintaining the structural integrity of tissues following differentiation (Gumbiner, 1996; Tissue samples were extracted in Triton (1% Triton X-100, 100 mM NaCl, 1 mM Halbleib and Nelson, 2006). However, the current repertoire of cell– MgCl2, 5 mM EDTA, 10 mM imidazole, pH 7.4), Triton/Octylglucoside (OG) (44.4 mM n- Octyl β-D glucopyranoside, 1% Triton X-100, 100 mM NaCl, 1 mM MgCl2, 5 mM EDTA, cell junctions is unlikely to mediate all the types of cell interactions 10 mM imidazole, pH 7.4) or RIPA (5 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% Sodium needed to establish and maintain tissue cytoarchitecture. Develop- deoxycholate, 0.1% SDS, 50 mM Tris–HCl, pH 7.4) buffers, as specified in each study. Each ment of the lens involves dramatic morphogenetic changes that begin extraction buffer contained 1 mM sodium vanadate, 0.2 mM H2O2, and Protease after the lens epithelial cells withdraw from the cell cycle and initiate Inhibitor Cocktail (Sigma, St. Louis, MO). Protein concentrations were quantified using μ their differentiation. Much of this morphogenetic differentiation the BCA assay (Pierce, Rockford, IL). For direct immunoblot analysis, 15 g of protein fi extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis occurs in the cortical region of the embryonic lens where the ber (SDS/PAGE) on precast 4–12% Tris–Glycine gels (Novex, San Diego, CA). cells become organized with precise hexagonal packing coincident For sequential extraction studies samples were extracted first in Triton, then Triton/ with their tremendous elongation so that they span from the anterior OG and lastly in 2× sample buffer (125 mM Tris–HCl, 4% SDS, 20% glycerol, 2% β- to the posterior aspects of the lens (Bassnett, 2005; Menko, 2002; Mercaptoethanol, 0.5% Bromophenol Blue). In these studies, in order to determine the relative distribution of proteins between the different detergent compartments Piatigorsky, 1981). Stabilization of this elongated morphology and the proteins in the Triton soluble fractions were loaded at equal protein concentration formation and maintenance of the highly ordered structure of the (15 μg) and their respective Triton/Octylglucoside and SDS fractions were loaded at lens, critical to lens clarity and function, is likely to require the volumes equal to the Triton soluble fractions. For analysis of complexes in the highly structural and tensile strength of cell–cell junctions, yet the junctions insoluble fraction samples were extracted first in Triton/OG and then in RIPA. that perform this function are yet to be identified. For each immunoprecipitation study 100 lenses were microdissected into the four differentiation specific fractions, the fractions extracted as specified in the results, and N-cadherin is the predominant cadherin in both epithelial and then each cell fraction in its entirety subjected to either standard immunoprecipitation fiber cells of the embryonic chicken lens (Hatta and Takeichi, 1986; or the new double immunoprecipitation protocol. This approach allowed for similar Leong et al., 2000), and the formation of N-cadherin cell–cell junctions amounts of N-cadherin to be pulled down from each fraction by immunoprecipitation. is required for proper lens morphogenesis (Ferreira-Cornwell et al., For standard immunoprecipitations, samples were incubated at 4 °C sequentially with primary antibody and then protein G (Sigma, St. Louis, MO) and the immunoprecipi- 2000). In the developing lens, this cadherin forms adhesion complexes tates subjected to SDS/PAGE as described previously (Walker et al., 2002). Proteins were with both β- and γ-catenins (Bagchi et al., 2002; Ferreira-Cornwell electrophoretically transferred from the gels onto Immobilon-P membranes (Millipore, et al., 2000; Straub et al., 2003). Conditional knockouts of β-catenin Bedford, MA) and detected using standard Western Blot techniques as described have demonstrated that while β-catenin is required to maintain the previously (Walker et al., 2002). Antibodies included N-cadherin, β-catenin, γ-catenin lens epithelium, and for the differentiation of epithelial cells into fiber (BD Transduction, San Jose, CA), actin (Sigma, St. Louis, MO), vimentin (Developmental fi Studies Hybridoma Bank, Iowa City, IA), and desmoplakin (Abcam Inc., Cambridge, MA). cells, this catenin is not essential for the maintenance of lens ber cells Secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch (Smith et al., 2005). It is therefore likely that an N-cadherin/γ-catenin Laboratories, West Grove, PA) were detected using ECL reagent from Amersham junction provides the structural and tensile strength necessary to Biosciences (Piscataway, NJ). Immunoblots were scanned and densitometric analysis establish and maintain the highly ordered structure of the lens. was performed using Kodak 1D software (Eastman Kodak Company, Rochester, NY). In co-immunopreciptiation studies the relative, differentiation-specific changes in the Lens cells lack desmosomes, the desmosomal cadherins desmo- association of any specific protein with the immunoprecipitated protein was collin and desmoglein, and the desmosomal catenin plakophilin determined following densitometry analysis. The densitometry data for each protein (Straub et al., 2003), but do express the intermediate filament-linker was first normalized to the EC fraction. This made it possible to control for differences in protein periplakin (Straub et al., 2003) and high levels of intermediate the antibody and HRP/ECL reactions between the multiple studies included in our filament proteins including vimentin, desmin, and the lens specific statistical analysis. The averages of at least three independent studies are calculated. fi fi Then the ratio of the co-immunoprecipitated protein to the immunoprecipitated beaded intermediate lament proteins lensin and CP49 (Bloemendal protein was determined and presented graphically with standard deviations. et al., 1981; Ellis et al., 1984; Merdes et al., 1991; Ramaekers et al., 1982; Ramaekers et al., 1980). Although these intermediate filament Double IP proteins have been shown to be present at the plasma membrane of Antibody directed against N-cadherin (BD Transduction, San Jose, CA) was lens fiber cells (Blankenship et al., 2001; Bloemendal et al., 1981; immobilized using the ProFound Co-Immunoprecipitation Kit from Pierce (Rockford, Merdes et al.,1991; Quinlan et al.,1996; Sandilands et al.,1995), little is IL) which allows for the isolation of intact, native protein complexes from a lysate. known about how they are anchored to the membrane. Here, we Antibody immobilization was performed by coupling antibody to aldehyde activated gel 300 M. Leonard et al. / Developmental Biology 319 (2008) 298–308

Fig. 1. Association of γ-catenin with the N-cadherin complex increases following the initiation of lens cell differentiation. (A) Embryonic day 10 chick lenses were microdissected (A) to yield four distinct regions of differentiation; (EC) undifferentiated epithelial cells, (EQ) differentiation initiation, (FP) fiber cell morphogenetic differentiation, and (FC) fiber cell maturation. (B) Differentiation specific changes in the composition of N-cadherin complexes was determined by co-immunoprecipitation analysis, and graphically represented in panel C, showing the ratio of both β- and γ-catenin to N-cadherin. Ratios were determined following densitometric analysis and normalization to EC. While there was no change in the ratio of β-catenin to N-cadherin, the recruitment of γ-catenin to N-cadherin complexes increased greater than 2.5-fold with lens fiber cell differentiation. Results are representative of at least three independent studies. beads (AminoLink Plus Coupling Gel) as instructed by the manufacturer. The immobilized another cell type. Just as importantly, and unique to this developing antibody was then used to isolate N-cadherin complexes from microdissected, tissue, multiple stages of differentiation are expressed concurrently at differentiation-specific E10 lens fractions extracted in Triton/Octylglucoside or RIPA buffers as indicated for individual experiments. Lysates were incubated with immobilized a single stage of development. This characteristic enabled us to isolate antibody at 4 °C for 2 h. Complexes were eluted from the column using a non-reducing significant quantities of the differentiation-specific zones of the elution buffer provided by the manufacturer, and diluted immediately in Triton buffer. embryonic lens (Fig. 1A) in order to perform biochemical analysis of Isolated, intact N-cadherin complexes, devoid of N-cadherin antibody were then sub- cadherin complexes and investigate the dynamic changes in these jected to a second immunoprecipitation using antibody directed against either γ-or junctions as lens cells differentiate in vivo. For this study lenses were β-catenin, following the standard immunoprecipitation protocol described above. removed at day 10 of chick embryo development and four distinct Immunofluorescence analysis and fluorescence detection of F-actin regions of differentiation were separated by microdissection (Walker and Menko, 1999). These differentiation-specific zones included the Sections: E10 lens cryosections were prepared and immunostained as described anterior epithelium, which contains undifferentiated lens epithelial previously (Walker and Menko, 1999). Briefly, cryostat sections of methanol (N-cadherin) or formaldehyde (γ-catenin, β-catenin, and vimentin) fixed E10 lenses cells (Fig. 1A, EC); the region at the lens equator where lens epithelial were prepared and immunostained with antibodies to N-cadherin, γ-catenin, β-catenin cells initiate their differentiation (Fig. 1A, EQ); cortical fiber cells in the (BD Transduction, San Jose, CA) or vimentin (Developmental Studies Hybridoma Bank, peripheral fiber zone, both the principal region of lens fiber cell Iowa City, IA) followed by rhodamine-conjugated secondary antibody (Jackson morphogenetic differentiation and the zone where lens-specific ImmunoResearch Laboratories, West Grove, PA). Formaldehyde fixed sections were cytoarchitecture is first established (Fig. 1A, FP); and the central, nuc- co-stained for F-actin using Alexa448-conjugated phalloidin (Invitrogen-Molecular fi fi Probes Eugene, OR). N-cadherin immunostaining studies used 8 μm sections examined lear ber cell zone, the region of lens ber cell maturation (Fig. 1A, FC). with a Nikon fluorescent microscope (Nikon Eclipse 80i); for γ-catenin, β-catenin, vimentin and F-actin, localization was determined using 20 μm sections examined by Differentiation-specific assembly of the N-cadherin/γ-catenin junctional confocal microscopy using a Zeiss LSM510 META confocal microscope. Z-stacks were complex collected and analyzed; the data presented represents a single optical plane. Whole lenses: Whole E10 lenses were fixed in 3.7% formaldehyde, permeabilized by exposure to 0.25% Triton X-100, immunostained with antibody to γ-catenin (BD Transduction, Previous work from our lab has shown that the formation of stable San Jose, CA) followed by rhodamine-conjugated secondary antibody and then incu- N-cadherin junctions is required for lens cell morphogenetic differ- bated with Alexa488-conjugated phalloidin (Invitrogen-Molecular Probes Eugene, OR) entiation (Ferreira-Cornwell et al., 2000). We now have examined if to detect F-actin. Fluorescence stained samples were imaged at a single optical plane there are specific properties of N-cadherin complexes that allow them (1.13 μm) at the basal aspects of cortical fiber cells by confocal microscopy using a Zeiss – fi LSM510 META confocal microscope. to strengthen and stabilize cell cell junctions as lens ber cells differentiate. For this study lens cells were extracted in a Triton/ Results Octylglucoside buffer, which solubilizes membrane-associated pro- teins including those associated with the actin cytoskeleton and those The lens — a unique embryonic tissue in which to examine the in lipid rafts (Gonen et al., 1979; Hooper, 1999). We investigated the modulation of cadherin junctions in development dynamics of N-cadherin complex formation in relation to lens cell differentiation in vivo by co-immunoprecipitation analysis; immuno- The chick embryo lens is a unique developmental paradigm for precipitating N-cadherin from differentiation-specific fractions of the studying differentiation-state specific changes in cell adhesion junc- embryonic lens isolated by microdissection (EC, EQ, FP and FC) tions. This unusual tissue is completely isolated from its surrounding followed by Western Blotting for β-orγ-catenin (Fig. 1B). These two tissues by its basement membrane capsule and contains no blood catenins bind directly to the cadherin cytoplasmic domain in a vessels or nerves. This property made it possible for us to perform mutually exclusive manner that can define cadherin function. Our biochemical analysis on lens cells without any contamination from study revealed that the principal N-cadherin junction of undiffer- M. Leonard et al. / Developmental Biology 319 (2008) 298–308 301 entiated lens epithelial cells (EC) was the N-cadherin/β-catenin were concentrated at the vertices of the hexagonally-shaped fiber junctional complex. Importantly, the level of association of β-catenin cells (Fig. 2E). In order to confirm this unique localization of γ- with N-cadherin changed very little with lens cell differentiation state catenin in lens fiber cells we performed immunostaining and (Figs. 1B, C). In contrast, there was only a low level of association of confocal imaging of whole E10 chicken embryo lenses (Fig. 2F). γ-catenin with N-cadherin in undifferentiated lens epithelial cells, For these studies the immunostained whole lenses were imaged which increased substantially upon the initiation of differentiation directly at the basal aspects of the fiber cells. This approach made it in the equatorial region (EQ) (Figs. 1B, C). Linkage of γ-catenin to possible to obtain a clear end-on view of γ-catenin localization near N-cadherin was even greater in the actively differentiating cortical fiber the posterior aspects of these cells. This study confirmed the very cell region (FP) and remained a significant component of N-cadherin unique and distinct pattern of localization for γ-catenin to the cell junctions in the nuclear fiber zone (FC) (Figs. 1B, C). The differentiation- vertices (Fig. 2F, closed arrow), which was first detected as the lens specific increase in γ-catenin association with N-cadherin was not cells enter the cortical fiber cell region, a region of the embryonic driven by changes in expression of these proteins (unpublished lens where lens cytoarchitecture is first established (Menko, 2002). observation, M. Leonard). These results show that assembly of the Imaging of these whole lenses co-stained with phalloidin demon- N-cadherin/γ-catenin junction was directly correlated with morpho- strated that the localization pattern for γ-catenin was inverse to that genetic differentiation of lens fiber cells and suggested that these of F-actin. γ-catenin-rich cell vertices were devoid of filamentous junctions are involved in establishing the precise hexagonal packing actin, and actin filaments were concentrated at lateral cell–cell arrangement and elongated structure of lens fiber cells and maintaining interfaces (Fig. 2F, open arrow). Using cross-sectional analysis we the unique cytoarchitecture of the lens. confirmed the restriction of F-actin to lateral borders of lens fiber cells (Fig. 2G, open arrow) and its absence from vertices of these Distinct localization of γ-catenin to cell vertices and β-catenin/F-actin to cells (Fig. 2G, solid arrow). Similar analysis demonstrated that N- lateral cell borders of hexagonally-packed differentiating lens fiber cells cadherin was distributed all along the cell–cell borders of lens fiber cells, both at lateral cell membranes (Fig. 2H, open arrow) co- Much of what is known about classical cadherin junctions is incident with both β-catenin and F-actin, and at the cell vertices focused on the function of cadherin/β-catenin complexes. Far less (Fig. 2H, solid arrow) with γ-catenin. While classical cadherins such attention has been paid to the role of γ-catenin in these cadherin as N-cadherin are typically thought to be associated with the actin junctions. While γ-catenin has been localized to plasma membranes cytoskeleton, our immunolocalization of N-cadherin and γ-catenin of differentiating lens fiber cells both in culture (Ferreira-Cornwell to vertices of this region of differentiating lens fiber cells, where et al., 2000) and in vivo (Franke et al., 1987), there has yet to be a actin is not present, suggests the existence of a novel N-cadherin/γ- comprehensive examination of the localization of this cadherin catenin complex that must interact with other cytoskeletal complex protein during the lens cell differentiation process. Here, elements, such as the vimentin intermediate filament proteins we used confocal imaging to obtain high resolution images of the that are highly expressed in these differentiating cells. lens cell membranes and investigate the possibility that there is a unique organization to the N-cadherin/γ-catenin junction of Identification of a novel N-cadherin junctional complex associated with differentiating lens fiber cells. Immunostaining of lens sections intermediate filament proteins in the lens with antibodies for N-cadherin, β-catenin and γ-catenin confirmed the results of our co-immunoprecipitation studies by showing that Despite the fact that the lens contains no desmosomes, 1) N-cadherin junctions of undifferentiated lens epithelial cells are desmosomal cadherins (Straub et al., 2003) or hemidesmosomes, comprised, principally, of N-cadherin linked to β-catenin; only low there are high levels of intermediate filament proteins (Bloemendal levels of γ-catenin were detected at cell–cell junctions of the central et al., 1981; Ellis et al., 1984; Merdes et al., 1991; Ramaekers et al., lens epithelium, 2) N-cadherin/β-catenin junctions persist through- 1982; Ramaekers et al., 1980). These intermediate filament proteins out lens differentiation, and 3) N-cadherin/γ-catenin junctions are have been localized near the lens plasma membrane, but little is assembled as lens fiber cells begin to differentiate (Figs. 2A–C). known about how they are anchored to the membrane (Blankenship Immunostaining for vimentin demonstrated that these intermediate et al., 2001; Bloemendal et al., 1981; Merdes et al., 1991; Quinlan et filaments are present in all cells of the E10 lens regardless of al., 1996; Sandilands et al., 1995). We used a co-immunoprecipita- differentiation state. As is typical of intermediate filaments vimentin tion approach to examine the possibility that the developing lens filaments extended across the cytoplasm of lens cells. While in contains a novel N-cadherin/γ-catenin junction to which inter- hexagonally packed differentiating lens fiber cells actin filaments mediate filament proteins such as vimentin could be linked. Since rearranged to a classical cortical localization concentrated at cell– our studies above showed that the localization of γ-catenin to the cell interfaces but absent from the cell vertices, the vimentin lens plasma membrane was differentiation-state specific, the filaments maintained their distribution throughout the cells (Weber embryonic lens was microdissected into four differentiation-specific and Menko, 2006 and Figs. 2B–D). It is reasonable to expect that, as regions (see Fig. 1A) prior to extraction in Triton/Octylglucoside occurs in most other cell types, the vimentin filaments in buffer. Co-immunoprecipitation analyses (immunoprecipitation of γ- differentiating fiber cells are linked to cell adhesion junctions, a catenin followed by immunoblotting for vimentin) showed that property that is essential to the role of intermediate filaments in indeed vimentin was recruited to γ-catenin junctions in the providing structural stability to the cell. embryonic lens and that this linkage increased during lens fiber The exact localization of β- and γ-catenins in hexagonally cell differentiation (Fig. 3A). While the association between packed differentiating lens fiber cells was most clearly demon- vimentin and γ-catenin was observed in the equatorial zone (EQ) strated in cross-sections of the embryonic lens along the posterior where differentiation is initiated, much greater linkage of vimentin tips of these cells. These imaging studies provided, for the first time, to γ-catenin occurred in the cortical fiber zone (FP), the principal evidence that β-catenin and γ-catenin-containing junctions have region of lens fiber cell morphogenesis. This association decreased distinct locations on the lens fiber cell membrane. β-catenin in the region of lens fiber cell maturation (FC); the reason for this localized only to the lateral cell interfaces of these differentiating decrease is elucidated in studies presented below. The pattern of fiber cells, where they co-localized with F-actin and both were association of vimentin with γ-catenin was paralleled in co- absent from the cell vertices (Fig. 2E). This localization pattern of F- immunoprecipitation analyses which examined the recruitment of actin in fiber cells has been reported previously (Bassnett et al., vimentin to N-cadherin complexes. These studies showed that the 1999; Weber and Menko, 2006). In contrast, γ-catenin junctions greatest linkage of vimentin to the N-cadherin complex was in the 302 M. Leonard et al. / Developmental Biology 319 (2008) 298–308 cortical fiber zone (Fig. 3B). The specificity of both γ-catenin and N- 3C). These results demonstrate for the first time that a differentia- cadherin association with vimentin was confirmed through co- tion-specific N-cadherin/vimentin junction forms in vivo and immunoprecipitation studies using non-immune mouse IgG (Fig. suggest that linkage of these proteins is mediated by γ-catenin. M. Leonard et al. / Developmental Biology 319 (2008) 298–308 303

Fig. 2. N-cadherin/γ-catenin junctions are assembled as lens fiber cells begin to differentiate and have a distinct localization to vertices of the hexagonally packed lens fiber cells. E10 lenses were immunostained with antibodies against N-cadherin, β-catenin, γ-catenin, vimentin; all were co-stained for F-actin except for N-cadherin. (A) N-cadherin becomes localized at cell–cell borders with the initiation of differentiation in EQ and this organization is maintained in the fiber cell region (FP/FC). β-catenin is present at cell–cell interfaces and is co-incident with F-actin throughout lens cell differentiation (B–D). This is most noticeably observed at the basal aspects of the hexagonally packed lens fiber cells (E). In contrast, γ-catenin does not become highly organized at cell–cell interfaces until the initiation of lens cell differentiation in EQ (C); at all stages of lens cell differentiation γ-catenin is not exactly co-incident with F-actin (B–E). Interestingly, while actin is localized along lateral cell–cell interfaces (F, open arrow) γ-catenin localizes to the vertices of the hexagonally packed lens fiber cells (F, solid arrow) where both β-catenin and actin are not present (E, F), suggesting its linkage to another cytoskeletal component. (Note that occasionally γ-catenin also was detected along the basal aspects of these lens fiber cells with a pattern similar to that of the previously identified lens basement membrane complex (F, arrowhead), a structure shown to contain both integrin and cadherin receptors (Bassnett et al., 1999)). Typical of intermediate filaments, vimentin is present throughout the cytoplasm and its localization is separate and distinct from that of F-actin at all stages of differentiation (B–D). The absence of F-actin from vertices of hexagonally-packed lens fiber cells was confirmed by cross-sectional analysis of whole E10 lenses (G, solid arrow). F-actin localized only to lateral cell–cell interfaces of the fiber cells (G, open arrow) while N-cadherin localized to both lateral cell–cell interfaces as well as the vertices of lens fiber cells (H) where γ-catenin localizes. Analysis of panel B–H was performed by confocal microscopy. Z-stacks were collected and analyzed; the data presented represents a single optical plane.

Desmoplakin is a component of the N-cadherin/γ-catenin junctions of While all these tissues and cell types expressed N-cadherin, γ-catenin, differentiating lens fiber cells and vimentin (Fig. 3E), the only tissue that paralleled the lens with a vimentin linked N-cadherin junction was brain tissue (Fig. 3F). Desmoplakin is the classical linker between γ-catenin and Interestingly, most of the tissues or cell types in which this novel intermediate filaments (Cowin et al., 1986; Franke et al., 1994; Fuchs junction was not detected are known to contain other junctional types and Cleveland, 1998; Gallicano et al., 1998; Getsios et al., 2004; Koch that provide the tensile strength necessary to maintain tissue cyto- and Franke, 1994; Troyanovsky et al., 1994). We now show, by Western architecture. Heart and skin contain desmosomes (Cheng and Koch, Blot analysis, that desmoplakin is expressed at all stages of E10 chick 2004) and in HUVECs, VE-cadherin, is associated with vimentin inter- lens cell differentiation, with an increased expression corresponding mediate filaments (Kowalczyk et al., 1998). Our finding of vimentin- with the initiation of differentiation in the equatorial region (Fig. 3D). linked N-cadherin junctions in brain tissue demonstrates that these More importantly, co-immunoprecipitation analysis (immunopreci- junctions are not unique to the lens and suggests that similar junc- pitation of N-cadherin followed by immunoblotting for desmoplakin) tions will be found in other cell types requiring structural stability in revealed that desmoplakin not only was expressed in the E10 chick the absence of desmosomal junctions. lens but also that it was associated with N-cadherin junctions (Fig. 3D). Desmoplakin association with N-cadherin peaks in the cortical Movement of N-cadherin and γ-catenin to a Highly Insoluble fiber cell region, similar to the pattern of vimentin association with Vimentin-rich cell compartment with differentiation both N-cadherin and γ-catenin. These results demonstrated that desmoplakin is associated with N-cadherin junctions in differentiat- The decrease in vimentin linkage to both N-cadherin and γ-catenin ing lens fiber cells where it likely functions as the linker between N- in the most differentiated fiber cells of the embryonic lens (FC, co- cadherin/γ-catenin junctions and vimentin intermediate filaments. immunoprecipitation analysis of Triton/Octylglucoside cell extracts) led us to investigate whether, as lens fiber cells matured, this unique Association of vimentin intermediate filament proteins with N-cadherin N-cadherin junction became resistant to extraction with the Triton/ is tissue specific but not unique to lens Octylglycoside buffer. This outcome would be consistent with the known resistance of vimentin to extraction by mild detergents. Here, To determine whether N-cadherin linkage to vimentin was unique we examined the detergent solubility properties of N-cadherin, to the lens we performed co-immunoprecipitation analysis in a variety γ-catenin, and β-catenin in each of the differentiation-specific zones of tissue samples, including brain, skeletal muscle, heart, and skin, as of the E10 lens. We were particularly interested in those proteins that well as in cultured human umbilical vein endothelial cells (HUVECs). were insoluble in Triton and Octylglucoside as this protein fraction 304 M. Leonard et al. / Developmental Biology 319 (2008) 298–308

Fig. 3. Identification of a novel vimentin intermediate filament-linked N-cadherin/γ-catenin junction that is tissue specific but not unique to lens. (A) Co-immunoprecipitation analysis of samples extracted with a Triton/Octylglucoside buffer demonstrated that in the cortical fiber zone γ-catenin was highly associated with vimentin. This linkage was diminished in Triton/Octylglucoside extracts of the more differentiated nuclear fiber cells. (B) Co-immunoprecipitation studies reveal the presence of a novel vimentin-linked N-cadherin junction in differentiating lens fiber cells. (C) Co-immunoprecipitation studies using non-immune mouse IgG in FP samples demonstrate that vimentin association with γ-catenin and N-cadherin is not the result of non-specific binding to antibody. (D) Western Blot analysis show that desmoplakin is expressed at all stages of lens cell differentiation, with increased expression corresponding to the initiation of differentiation in EQ, and co-immunoprecipitation studies reveal that N-cadherin is associated with desmoplakin in a pattern similar to that of vimentin. (E) Western Blot analysis and co-immunoprecipitation reveal that N-cadherin, γ-catenin and vimentin are all expressed in a variety of E10 chicken tissues (brain, heart, skeletal muscle and skin) as well as cultured human umbilical vein endothelial cells. (F) Co-immunoprecipitation analysis demonstrate that N-cadherin only associated with vimentin in brain tissue. Results are representative of at least two independent studies. was expected to be enriched in intermediate filament proteins and differentiation in the cortical fiber region (Figs. 4B, C). In the most their associated adhesion complex proteins. Microdissected lens differentiated fiber cells of the embryonic lens (FC), both N-cadherin fractions were extracted sequentially with Triton buffer followed by and γ-catenin were highly associated with the HI fraction, while the Octylglucoside buffer, and proteins in the Triton/Octylglucoside inso- association of β-catenin with the HI fraction changed very little with luble fraction, referred to as the Highly Insoluble (HI) fraction, were differentiation state. There was however a significant increase in solubilized in a 4% SDS buffer. Western Blot analysis was used to N-cadherin/β-catenin association with the Octylglucoside fraction. compare the distribution of N-cadherin, γ-catenin and β-catenin The observation that N-cadherin and γ-catenin were present in the HI, relative to vimentin in the Triton soluble, Triton insoluble/OG soluble vimentin-rich fraction once fiber cell elongation had begun supports and HI fractions of each differentiation-specific zone of the E10 chick the possibility that a differentiation-specific N-cadherin/γ-catenin/ embryo lens. intermediate filament junction exists. The properties of this novel In undifferentiated lens epithelial cells (EC) vimentin was highly junction suggest that it is likely to have a role in the morphogenetic insoluble in Triton and Octylglycoside (Fig. 4A). While N-cadherin, differentiation of lens fiber cells, the maintenance of their elongated β-catenin and γ-catenin were expressed in these undifferentiated lens structure and their acquisition of highly ordered packing. Taken cells, they were primarily detergent soluble (Figs. 4B, C) and did not together these results suggest that γ-catenin, but not β-catenin is fractionate with vimentin. Therefore, in undifferentiated lens epithe- involved in N-cadherin linkage to vimentin. lial cells N-cadherin junctions were not linked to the HI vimentin fraction. Vimentin became increasingly associated with the HI fraction N-cadherin and γ-catenin in the HI fraction are linked to vimentin with increasing lens differentiation state (Fig. 4A). Interestingly, while intermediate filament protein N-cadherin, β-catenin and γ-catenin were primarily Triton soluble in lens epithelial cells, only N-cadherin and γ-catenin moved signifi- The sequential detergent extraction studies suggest that after the cantly to the HI fraction as lens cells began their morphogenetic N-cadherin complexes are stabilized by linking to the intermediate M. Leonard et al. / Developmental Biology 319 (2008) 298–308 305

Fig. 4. Identification of a highly insoluble, vimentin intermediate filament rich fraction, to which N-cadherin and γ-catenin become increasingly associated with differentiation. Microdissected lens fractions (EC, EQ, FP and FC) were extracted sequentially in Triton, Triton/Octylglucoside and SDS (4%) buffers and the compartmentalization of (A) vimentin, (B) N-cadherin, (C) γ-catenin, and (D) β-catenin to these different detergent compartments determined by Western Blot analysis, densitometric data was used to determine the percent of the total protein present in each fraction. The SDS extract is referred to as the Highly Insoluble (HI) compartment. In undifferentiated lens epithelial cells (EC) N-cadherin (B) and γ-catenin (C) were primarily Triton soluble, while vimentin (A) was mostly in the HI fraction. While β-catenin was present in the HI fraction in all regions of lens fiber cell differentiation, its presence increased only slightly in the HI fraction with differentiation (D). In contrast, in regions of lens fiber cell differentiation, both N-cadherin and γ-catenin moved into the HI, vimentin-rich fraction (B, C). Results are representative of at least three independent studies.

filament cytoskeleton, they become insoluble in Triton and Octyl- tion analysis of the RIPA extract of the FP fraction using non-immune glucoside. Since linkage of cell junctions to intermediate filaments mouse IgG confirmed that association of N-cadherin and γ-catenin is known to provide cells with tensile strength, we investigated with vimentin is not due to non-specific antibody–protein interac- whether N-cadherin and γ-catenin in the HI fraction were associated tions (Fig. 5C). It is likely that γ-catenin mediates the interaction with the intermediate filament vimentin by performing standard co- between N-cadherin and vimentin. immunoprecipitation analysis. These co-immunoprecipitation ana- lyses were conducted on the RIPA extract of the HI fraction from each A novel double IP protocol confirms that the association of N-cadherin differentiation-specific zone of the lens obtained by microdissection with vimentin is mediated by γ-catenin as described above. Immunoprecipitation of N-cadherin revealed that vimentin was indeed associated with N-cadherin complexes in the HI Until now it has been impossible to biochemically separate fraction, and that this association increased with differentiation of the N-cadherin/γ-catenin complexes from N-cadherin/β-catenin com- lens fiber cells (Fig. 5A). These studies demonstrate that our inability plexes. Therefore, while our studies presented above demonstrated to detect vimentin-linked N-cadherin junctions in the most differ- that both γ-catenin and vimentin were linked to N-cadherin junctions entiated zone of the embryonic lens (FC) when cells were extracted in and suggested that vimentin was associated with N-cadherin com- Triton/Octylglucoside resulted from the movement of this junction to plexes through γ-, not β-, catenin, this did not prove that N-cadherin, the HI, vimentin-rich cell fraction. Furthermore, the association of γ-catenin and vimentin were associated in the same junctional N-cadherin with vimentin in the HI fraction was strikingly similar to complex. Therefore, it was necessary to develop a new approach in the pattern of association between N-cadherin and γ-catenin (Fig. 5A), order to demonstrate that the linkage of the intermediate filament supporting the likelihood that the formation of this novel N-cadherin/ protein vimentin to N-cadherin junctions as lens fiber cells differ- intermediate filament complex is mediated by γ-catenin. Immuno- entiated in vivo specifically involved the N-cadherin/γ-catenin precipitation of γ-catenin complexes from the HI fraction provided complex. For this purpose, we developed a novel double immuno- further evidence for the existence of an intermediate filament-linked precipitation technique. This protocol made it possible to isolate an N-cadherin/γ-catenin complex. γ-catenin association with vimentin N-cadherin complex based on whether it is linked to β-orγ-catenin in the HI fraction had the same differentiation-specific pattern as and then to identify whether vimentin was associated specifically N-cadherin association with vimentin (Fig. 5B). Co-immunoprecipita- with the N-cadherin/γ-catenin complex. First, N-cadherin antibody 306 M. Leonard et al. / Developmental Biology 319 (2008) 298–308

observation, M. Leonard). Specificity of the linkage of vimentin to N-cadherin/γ-catenin complexes was verified by performing the same studies using immobilized non-immune mouse IgG on the ProFound Co-Immunoprecipitation column (Pierce) with HI-associated proteins from the FP zone (Fig. 6C). These data show that the linkage of N-cadherin to the vimentin intermediate filament cytoskeleton was mediated specifically by N-cadherin/γ-catenin junctions. Our results also demonstrate for the first time that this novel interme- diate filament-linked N-cadherin junction is assembled in vivo, in a differentiation-specific manner, with properties consistent with a role in establishing and maintaining the stability of lens fiber cell–cell interactions required for their differentiation.

Discussion

It is well established that both β- and γ-catenins bind directly to the cadherin cytoplasmic tail and that this interaction occurs in a

Fig. 5. Both N-cadherin and γ-catenin are highly linked to vimentin intermediate filament protein in the Highly Insoluble fraction. The RIPA extract of the HI com- partment of microdissected lens fractions (EC, EQ, FP and FC) was immunoprecipitated for (A) N-cadherin or (B) γ-catenin and Western Blotted for N-cadherin, γ-catenin and vimentin. This co-immunoprecipitation analysis revealed a differentiation-specific inc- rease in the recruitment of γ-catenin and vimentin to N-cadherin complexes in the HI fraction (A). A similar pattern of association of vimentin with γ-catenin indicates that the linkage of vimentin to N-cadherin in this novel junction is mediated by γ-catenin (B). (C) Co-immunoprecipitation analysis using non-immune mouse IgG in the RIPA extract of the HI fraction of FP confirmed the specificity of the observed interactions between N-cadherin and γ-catenin with vimentin. Results are representative of at least three independent studies. was immobilized on a column (ProFound Co-Immunoprecipitation Kit, Pierce) and used to isolate the intact N-cadherin complexes present in the HI fraction of each differentiation-specific zone of the E10 lens. These N-cadherin complexes were isolated by non-reducing elution from the antibody linked to the column and therefore free of any associated antibody. This feature made it possible to perform a second immunoprecipitation for another member of the N-cadherin complex; here we used either antibody to γ-catenin or β-catenin. This approach was completely efficacious in isolating either N-cadherin/ γ-catenin or N-cadherin/β-catenin complexes, on which further analysis could be performed to determine association with individual cytoskeletal proteins, or other proteins of interest in the complex. Control studies were performed in which isolated N-cadherin/ γ-catenin complexes were blotted for β-catenin and N-cadherin/ Fig. 6. Vimentin linkage to N-cadherin is specific to N-cadherin/γ-catenin junctions. A β-catenin complexes were blotted for γ-catenin, proving the efficacy novel double immunoprecipitation approach (described in detail in the methods) that of this protocol to purify only the targeted complexes (Figs. 6A, B). made it possible to separate N-cadherin/β-catenin from N-cadherin/γ-catenin com- Following this confirmation, the linkage of vimentin to isolated plexes in the HI fraction was used to investigate the catenin protein that linked γ β N-cadherin to vimentin. For these studies isolated N-cadherin complexes were immu- N-cadherin/ -catenin and N-cadherin/ -catenin complexes was noprecipitated with antibodies to either γ-catenin (A) or β-catenin (B) and then blotted determined by Western Blot analysis. These studies showed that in for N-cadherin, γ-catenin, β-catenin and vimentin. The results confirm that γ-catenin, the HI fraction vimentin was linked to N-cadherin/γ-catenin junc- not β-catenin, links vimentin to the novel N-cadherin junctions that form in tional complexes (Fig. 6A), but not to N-cadherin/β-catenin complexes differentiating fiber cells of the embryonic lens. (C) Control co-immunoprecipitation analysis using immobilized non-immune mouse IgG followed by immunoprecipitation (Fig. 6B). In contrast, the intermediate filament protein desmin, a for γ-catenin in the FP fraction confirmed the specificity of vimentin association with component of the HI fraction, was not detected in N-cadherin/ the N-cadherin/γ-catenin complex. Results are representative of at least three γ-catenin junctions using the double IP protocol (unpublished independent studies. M. Leonard et al. / Developmental Biology 319 (2008) 298–308 307 mutually exclusive manner (Cowin and Burke, 1996; Hulsken et al., cytoskeletal linkages in the same cells suggests that N-cadherin/ 1994; Nagafuchi et al., 1991; Peifer et al., 1994). While the cadherin/ γ-catenin and N-cadherin/β-catenin junctions may have very differ- β-catenin complex has been studied intensively, particularly in its ent functions in lens development and homeostasis. role in the formation and function of the adherens junction, far less We believe that the novel N-cadherin-intermediate filament- attention has been paid to the role of classical cadherin/γ-catenin linked junction we have identified in lens fiber cells may be critical complexes in cell–cell adhesion. It is not yet understood why classi- to lens morphogenesis and the maintenance of lens cytoarchitecture. cal cadherins link to γ-catenin instead of β-catenin, or whether Formation and maintenance of the highly ordered structural packing the cadherin/γ-catenin cell junctions function differently from the of lens fiber cells is necessary for proper lens function. Therefore, it is cadherin/β-catenin cell junctions. not surprising that differentiating lens fiber cells have very different Although γ- and β-catenins are highly homologous (DeMarais and cell–cell adhesion requirements than their undifferentiated counter- Moon, 1992), γ-catenin is the only junctional protein present in both part, the lens epithelial cell. These N-cadherin/γ-catenin junctions adherens junctions and desmosomes (Troyanovsky et al., 1996; Wahl likely contribute to the strengthening of cell–cell adhesions critical for et al., 1996). Desmosomal junctions are strengthened by their asso- lens fiber cell morphogenesis and maintenance of the unique ciation with intermediate filament cytoskeletal networks; an interac- structure of the differentiating lens fiber cells. We believe that this tion mediated by γ-catenin and desmoplakin (Cowin et al., 1986; novel intermediate filament-linked complex may be functionally Franke et al., 1994; Fuchs and Cleveland, 1998; Gallicano et al., 1998; analogous to desmosomal complexes, providing lens fiber cells with Getsios et al., 2004; Koch and Franke, 1994; Troyanovsky et al., 1994). the tensile strength necessary to maintain the structural integrity of Desmosomes provide cells with the structural strength to maintain the lens. Furthermore, we showed that this junction is not unique to the integrity of a tissue, which is especially important for tissues lens fiber cells, but that cadherin/γ-catenin junctions may foster subjected to mechanical stress, such as the heart and skin (Fuchs and interactions with intermediate filament cytoskeletal networks in Cleveland, 1998; Huber, 2003). γ-catenin has been demonstrated to be other cell types which lack desmosomes. The novel double immuno- critical to this desmosomal function (Bierkamp et al., 1999). While the precipitation protocol used to identify the N-cadherin/γ-catenin/ function of γ-catenin in cell–cell adhesion has not been extensively intermediate filament junction has great potential to enhance our studied outside of its role in desmosomal junctions, it is likely that understanding of protein scaffolds and signaling complexes of the cell. γ-catenin may be equally important in strengthening cell–cell adhesions between cells lacking desmosomes. Acknowledgments Typically, classical cadherins are believed to interact with the actin cytoskeleton, however, Valiron et al. (1996) demonstrated that VE- This work was supported by grants from the NIH: EY10577,EY014258 cadherin co-localizes with desmoplakin and vimentin in endothelial and EY014798 to ASM, and T32E5007283 to ML. We also thank Iris cells. In a related study, transfection of fibroblasts with VE-cadherin Wolff and Ni Zhai for their technical assistance; Janice L. Walker and and desmoplakin results in association between these two proteins Marilyn Woolkalis for their critical reading of the manuscript; and only if the cells are also transfected with γ-catenin (Kowalczyk et al., Terry Hyslop for consultation on statistical analysis of our data. 1998). This study shows that non-desmosomal cadherins have the Grant Information: NIH Grants EY10577, EY014258, EY014798 and potential to associate with intermediate filaments through γ-catenin. T32E5007283. 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