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. Author manuscript; available in PMC: 2009 Jul 15.
Published in final edited form as: Dev Biol. 2008 May 8;319(2):298–308. doi: 10.1016/j.ydbio.2008.04.036

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 1, Yim Chan 1, A Sue Menko 1
PMCID: PMC2518943  NIHMSID: NIHMS59869  PMID: 18514185

Abstract

Tissue morphogenesis and maintenance of complex tissue architecture requires a variety of cell-cell junctions. Typically, cells adhere to one another through cadherin junctions, both adherens and desmosomal junctions, strengthened by association with cytoskeletal networks during development. Both β- and γ-catenins are reported to link classical cadherins to the actin cytoskeleton, but only γ-catenin binds to the desmosomal cadherins, which links them to intermediate filaments through its association with desmoplakin. Here we provide the first biochemical evidence that, in vivo, γ-catenin also mediates interactions between classical cadherins and the intermediate filament cytoskeleton, linked through desmoplakin. In the developing lens, which has no desmosomes, we discovered that vimentin became linked to N-cadherin complexes in a differentiation-state specific manner. This newly identified junctional complex was tissue specific but not unique to the lens. To determine whether in this junction N-cadherin was linked to vimentin through γ-catenin or β-catenin we developed an innovative “double” immunoprecipitation technique. This approach 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.

Keywords: γ-catenin, cadherin, intermediate filament, vimentin, lens development, lens fiber cell differentiation

INTRODUCTION

A well-recognized function of cadherin junctions is the establishment and maintenance of the tissue cytoarchitecture (Gumbiner, 1996; Gumbiner, 2005; Halbleib and Nelson, 2006; Takeichi, 1995; Wheelock and Jensen, 1992). In our own studies of the lens we show that proper morphogenesis depends on the ability of lens cells to organize mature N-cadherin cell-cell junctions (Ferreira-Cornwell et al., 2000). To date, the function of classical cadherins like N-cadherin in tissue morphogenesis has been understood to involve their interaction with the actin cytoskeletal network (Cowin and Burke, 1996; Geiger, 1989; Gumbiner, 2005; Pavalko and Otey, 1994). This linkage to the actin cytoskeleton is mediated through α-, β-, and γ-catenins (Cowin and Burke, 1996; Nagafuchi et al., 1991; Peifer et al., 1994), with β-catenin and γ-catenin (plakoglobin) binding directly to the cytoplasmic tail of classical cadherins in a mutually exclusive manner (Cowin and Burke, 1996; Hulsken et al., 1994; Nagafuchi et al., 1991; Peifer et al., 1994). α-catenin then binds to these catenins where it is believed to play a role in the recruitment of actin to 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., 1994). In contrast, in desmosomes, cell-cell junctions that provide cells with tensile strength, the desmosomal cadherins desmocollin and desmoglein link to the intermediate filament cytoskeletal network. This linkage is mediated by γ- but not β-catenin through its association with intermediate filament-linking proteins such as desmoplakin (Cowin et al., 1986; Franke et al., 1994; Fuchs and Cleveland, 1998; Gallicano et al., 1998; Getsios et al., 2004; Koch and Franke, 1994; Troyanovsky et al., 1994). Thus, γ-catenin is a versatile component of cadherin complexes, capable of fostering the interaction of classical cadherins with the actin cytoskeleton in adherens junctions and desmosomal cadherins with the intermediate filament cytoskeleton in desmosomes. While γ-catenin has the potential to link non-desmosomal cadherins to the intermediate filament cytoskeleton, the existence of a cadherin junction with this property has not yet been shown in vivo. Since the lens has no desmosomes to provide tensile strength (Straub et al., 2003), but is 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 structures that could provide tensile strength to tissues without desmosomes.

Evidence that non-desmosomal cadherins might be linked to the intermediate filament cytoskeleton was first shown in studies of VE-cadherin, a Type II classical cadherin (E-cadherin and N-cadherin are Type I classical cadherins), and the principal cadherin in cell-cell contacts of the vascular endothelium (Lampugnani et al., 1992; Navarro et al., 1998; Uccini et al., 1994). While, like the lens, vascular endothelial cells lack desmosomes and desmosomal cadherins (Schmelz and Franke, 1993), in cultured human umbilical vein endothelial cells the vimentin intermediate filament protein co-localizes at the plasma membrane with both VE-cadherin and desmoplakin, an intermediate filament binding protein (Valiron et al., 1996). When VE-cadherin and desmoplakin are co-transfected into cultured fibroblasts they co-localize only if γ-catenin is present (Kowalczyk et al., 1998) and expression of an N-cadherin/estrogen receptor fusion protein by fibroblasts results in its association with endogenous vimentin (Kim et al., 2005); however, there is of yet no biochemical evidence that γ-catenin links classical cadherins to the intermediate filament cytoskeleton in vivo.

The function of cadherins in embryonic development is likely mediated through their interactions with specific cytoskeletal networks, as cadherin junctions provide anchoring sites for cytoskeletal components at the cell membrane (Cowin and Burke, 1996; Pavalko and Otey, 1994). Such cytoskeletal-cell membrane attachments are central to establishing the relationships between cells that are necessary for tissue morphogenesis and for maintaining the structural integrity of tissues following differentiation (Gumbiner, 1996; Halbleib and Nelson, 2006). However, the current repertoire of cell-cell junctions is unlikely to mediate all the types of cell interactions needed to establish and maintain tissue cytoarchitecture. Development of the lens involves dramatic morphogenetic changes that begin after the lens epithelial cells withdraw from the cell cycle and initiate their differentiation. Much of this morphogenetic differentiation occurs in the cortical region of the embryonic lens where the fiber cells become organized with precise hexagonal packing coincident with their tremendous elongation so that they span from the anterior to the posterior aspects of the lens (Bassnett, 2005; Menko, 2002; Piatigorsky, 1981). Stabilization of this elongated morphology and the formation and maintenance of the highly ordered structure of the lens, critical to lens clarity and function, is likely to require the structural and tensile strength of cell-cell junctions, yet the junctions that perform this function are yet to be identified.

N-cadherin is the predominant cadherin in both epithelial and fiber cells of the embryonic chicken lens (Hatta and Takeichi, 1986; Leong et al., 2000), and the formation of N-cadherin cell-cell junctions is required for proper lens morphogenesis (Ferreira-Cornwell et al., 2000). In the developing lens, this cadherin forms adhesion complexes with both β- and γ-catenins (Bagchi et al., 2002; Ferreira-Cornwell et al., 2000; Straub et al., 2003). Conditional knockouts of β-catenin have demonstrated that while β-catenin is required to maintain the lens epithelium, and for the differentiation of epithelial cells into fiber cells, this catenin is not essential for the maintenance of lens fiber cells (Smith et al., 2005). It is therefore likely that an N-cadherin/γ-catenin junction provides the structural and tensile strength necessary to establish and maintain the highly ordered structure of the lens.

Lens cells lack desmosomes, the desmosomal cadherins desmocollin and desmoglein, and the desmosomal catenin plakophilin (Straub et al., 2003), but do express the intermediate filament-linker protein periplakin (Straub et al., 2003) and high levels of intermediate filament proteins including vimentin, desmin, and the lens specific beaded intermediate filament proteins filensin and CP49 (Bloemendal et al., 1981; Ellis et al., 1984; Merdes et al., 1991; Ramaekers et al., 1982; Ramaekers et al., 1980). Although these intermediate filament proteins have been shown to be present at the plasma membrane of lens fiber cells (Blankenship et al., 2001; Bloemendal et al., 1981; Merdes et al., 1991; Quinlan et al., 1996; Sandilands et al., 1995), little is known about how they are anchored to the membrane. Here, we show that the vimentin intermediate filament cytoskeleton is linked to N-cadherin, the principal cell-cell adhesion molecule of differentiating lens fiber cells. Using a novel double immunoprecipitation technique we developed for this study, we separated N-cadherin/γ-catenin junctions from N-cadherin/β-catenin junctions, and demonstrated that the link between N-cadherin and intermediate filaments occurs only in N-cadherin/γ-catenin junctions. In addition, our studies show that these N-cadherin/γ-catenin junctions contain the intermediate filament-linker protein desmoplakin, the classical linker between γ-catenin and intermediate filament proteins. Our studies provide the first evidence of an N-cadherin/γ-catenin/intermediate filament complex in vivo. The existence of this unique intermediate filament-linked cadherin junction is particularly exciting as its properties are likely to provide the structural stability necessary to establish and maintain complex tissue cytoarchitecture.

MATERIALS AND METHODS

Lens microdissection

Lenses were isolated from embryonic day 10 (E10) chicken eggs (Truslow Farms, Chestertown, MD) and microdissected as previously described (Walker and Menko, 1999) to yield four distinct regions of differentiation: the central anterior epithelium (EC), the equatorial epithelium (EQ), cortical fiber cells (FP), and the nuclear fiber zone (FC) (Figure 1A).

Figure 1.

Figure 1

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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-immunopreciptation analysis, and graphically represented in (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.

Protein Extraction, Standard Immunoprecipiatation and Immunoblotting

Tissue samples were extracted in Triton (1% Triton X-100, 100mM NaCl, 1mM MgCl2, 5mM EDTA, 10mM imidazole, pH 7.4), Triton/Octylglucoside (OG) (44.4mM n-Octyl β-D glucopyranoside, 1% Triton X-100, 100mM NaCl, 1mM MgCl2, 5mM EDTA, 10mM imidazole, pH 7.4) or RIPA (5mM EDTA, 150mM NaCl, 1% NP40, 0.1% Sodium deoxycholate, 0.1% SDS, 50mM Tris-HCl, pH 7.4) buffers, as specified in each study. Each extraction buffer contained 1mM sodium vanadate, 0.2mM H2O2, and Protease Inhibitor Cocktail (Sigma, St. Louis, MO). Protein concentrations were quantified using the BCA assay (Pierce, Rockford, IL). For direct immunoblot analysis, 15 μg of protein extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) on precast 4–12% Tris-Glycine gels (Novex, San Diego, CA).

For sequential extraction studies samples were extracted first in Triton, then Triton/OG and lastly in 2X sample buffer ((125 mM Tris-HCl, 4% SDS, 20% glycerol, 2% β-Mercaptoethanol, 0.5% Bromophenol Blue). In these studies, in order to determine the relative distribution of proteins between the different detergent compartments proteins in the Triton soluble fractions were loaded at equal protein concentration (15 μg) and their respective Triton/Octylglucoside and SDS fractions were loaded at volumes equal to the Triton soluble fractions. For analysis of complexes in the highly insoluble fraction samples were extracted first in Triton/OG and then in RIPA.

For each immunoprecipitation study 100 lenses were microdissected into the four differentiation specific fractions, the fractions extracted as specified in the results, and then each cell fraction in its entirety subjected to either standard immunoprecipitation or the new double immunoprecipitation protocol. This approach allowed for similar amounts of N-cadherin to be pulled down from each fraction by immunoprecipitation. For standard immunoprecipitations, samples were incubated at 4° C sequentially with primary antibody and then protein G (Sigma, St. Louis, MO) and the immunoprecipitates subjected to SDS/PAGE as described previously (Walker et al., 2002). Proteins were electrophoretically transferred from the gels onto Immobilon-P membranes (Millipore, Bedford, MA) and detected using standard Western Blot techniques as described previously (Walker et al., 2002). Antibodies included N-cadherin, β-catenin, γ-catenin (BD Transduction, San Jose, CA), actin (Sigma, St. Louis, MO), vimentin (Developmental Studies Hybridoma Bank, Iowa City, IA), and desmoplakin (Abcam Inc., Cambridge, MA). Secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) were detected using ECL reagent from Amersham Biosciences (Piscataway, NJ). Immunoblots were scanned and densitometric analysis was performed using Kodak 1D software (Eastman Kodak Company, Rochester, NY). In co-immunopreciptiation studies the relative, differentiation-specific changes in the association of any specific protein with the immunoprecipitated protein was determined following densitometry analysis. The densitometry data for each protein was first normalized to the EC fraction. This made it possible to control for differences in the antibody and HRP/ECL reactions between the multiple studies included in our statistical analysis. The averages of at least three independent studies are calculated. Then the ratio of the co-immunoprecipitated protein to the immunoprecipitated protein was determined and presented graphically with standard deviations.

Double IP

Antibody directed against N-cadherin (BD Transduction, San Jose, CA) was immobilized using the ProFound Co-Immunoprecipitation Kit from Pierce (Rockford, IL) which allows for the isolation of intact, native protein complexes from a lysate. Antibody immobilization was performed by coupling antibody to aldehyde activated gel beads (AminoLink Plus Coupling Gel) as instructed by the manufacturer. The immobilized antibody was then used to isolate N-cadherin complexes from microdissected, differentiation-specific E10 lens fractions extracted in Triton/Octylglucoside or RIPA buffers as indicated for individual experiments. Lysates were incubated with immobilized antibody at 4° C for 2 hours. Complexes were eluted from the column using a non-reducing elution buffer provided by the manufacturer, and diluted immediately in Triton buffer. Isolated, intact N-cadherin complexes, devoid of N-cadherin antibody were then subjected to a second immunoprecipitation using antibody directed against either γ- or β-catenin, following the standard immunoprecipitation protocol described above.

Immunofluorescence analysis and fluorescence detection of F-actin

Sections: E10 lens cryosections were prepared and immunostained as described previously (Walker and Menko, 1999). Briefly, cryostat sections of methanol (N-cadherin) or formaldehyde (γ-catenin, β-catenin, and vimentin) fixed E10 lenses were prepared and immunostained with antibodies to N-cadherin, γ-catenin, β-catenin (BD Transduction, San Jose, CA) or vimentin (Developmental Studies Hybridoma Bank, Iowa City, IA) followed by rhodamine-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Formaldehyde fixed sections were co-stained for F-actin using Alexa448-conjugated phalloidin (Invitrogen-Molecular Probes Eugene, OR). (N-cadherin immunostaining studies used 8 μ sections examined with a Nikon fluorescent microscope (Nikon Eclipse 80i); or like γ-catenin, β-catenin, vimentin and F-actin, localization was determined using 20μ sections examined by confocal microscopy using a Zeiss LSM510 META confocal microscope. Z-stacks were 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, San Jose, CA) followed by rhodamine-conjugated secondary antibody and then incubated with Alexa488-conjugated phalloidin (Invitrogen-Molecular Probes Eugene, OR) to detect F-actin. Fluorescence stained samples were imaged at a single optical plane (1.13 μm) at the basal aspects of cortical fiber cells by confocal microscopy using a Zeiss LSM510 META confocal microscope.

RESULTS

The lens - a unique embryonic tissue in which to examine the modulation of cadherin junctions in development

The chick embryo lens is a unique developmental paradigm for studying differentiation-state specific changes in cell adhesion junctions. This unusual tissue is completely isolated from its surrounding tissues by its basement membrane capsule and contains no blood vessels or nerves. This property made it possible for us to perform biochemical analysis on lens cells without any contamination from another cell type. Just as importantly, and unique to this developing tissue, multiple stages of differentiation are expressed concurrently at a single stage of development. This characteristic enabled us to isolate significant quantities of the differentiation-specific zones of the embryonic lens (Figure 1A) in order to perform biochemical analysis of cadherin complexes and investigate the dynamic changes in these junctions as lens cells differentiate in vivo. For this study lenses were removed at day 10 of chick embryo development and four distinct regions of differentiation were separated by microdissection (Walker and Menko, 1999). These differentiation-specific zones included the anterior epithelium, which contains undifferentiated lens epithelial cells (Figure 1A, EC); the region at the lens equator where lens epithelial cells initiate their differentiation (Figure 1A, EQ); cortical fiber cells in the peripheral fiber zone, both the principal region of lens fiber cell morphogenetic differentiation and the zone where lens-specific cytoarchitecture is first established (Figure 1A, FP); and the central, nuclear fiber cell zone, the region of lens fiber cell maturation (Figure 1A, FC).

Differentiation-specific assembly of the N-cadherin/γ-catenin junctional complex

Previous work from our lab has shown that the formation of stable N-cadherin junctions is required for lens cell morphogenetic differentiation (Ferreira-Cornwell et al., 2000). We now have examined if there are specific properties of N-cadherin complexes that allow them to strengthen and stabilize cell-cell junctions as lens fiber cells differentiate. For this study lens cells were extracted in a Triton/Octylglucoside buffer, which solubilizes membrane-associated proteins including those associated with the actin cytoskeleton and those in lipid rafts (Gonen et al., 1979; Hooper, 1999). We investigated the dynamics of N-cadherin complex formation in relation to lens cell differentiation in vivo by co-immunoprecipitation analysis; immunoprecipitating N-cadherin from differentiation-specific fractions of the embryonic lens isolated by microdissection (EC, EQ, FP and FC) followed by Western Blotting for β-or γ-catenin (Figure 1B). These two catenins bind directly to the cadherin cytoplasmic domain in a mutually exclusive manner that can define cadherin function. Our study revealed that the principal N-cadherin junction of undifferentiated lens epithelial cells (EC) was the N-cadherin/β-catenin junctional complex. Importantly, the level of association of β-catenin with N-cadherin changed very little with lens cell differentiation state (Figure 1B, C). In contrast, there was only a low level of association of γ-catenin with N-cadherin in undifferentiated lens epithelial cells, which increased substantially upon the initiation of differentiation in the equatorial region (EQ) (Figure 1B, C). Linkage of γ-catenin to N-cadherin was even greater in the actively differentiating cortical fiber cell region (FP) and remained a significant component of N-cadherin junctions in the nuclear fiber zone (FC) (Figure 1B, C). The differentiation-specific increase in γ-catenin association with N-cadherin was not driven by changes in expression of these proteins (unpublished observation, M. Leonard). These results show that assembly of the N-cadherin/γ-catenin junction was directly correlated with morphogenetic differentiation of lens fiber cells and suggested that these junctions are involved in establishing the precise hexagonal packing arrangement and elongated structure of lens fiber cells and maintaining the unique cytoarchitecture of the lens.

Distinct localization of γ-catenin to cell vertices and β-catenin/F-actin to lateral cell borders of hexagonally-packed differentiating lens fiber cells

Much of what is known about classical cadherin junctions is focused on the function of cadherin/β-catenin complexes. Far less attention has been paid to the role of γ-catenin in these cadherin junctions. While γ-catenin has been localized to plasma membranes of differentiating lens fiber cells both in culture (Ferreira-Cornwell et al., 2000) and in vivo (Franke et al., 1987), there has yet to be a comprehensive examination of the localization of this cadherin complex protein during the lens cell differentiation process. Here, we used confocal imaging to obtain high resolution images of the lens cell membranes and investigate the possibility that there is a unique organization to the N-cadherin/γ-catenin junction of differentiating lens fiber cells. Immunostaining of lens sections with antibodies for N-cadherin, β-catenin and γ-catenin confirmed the results of our co-immunoprecipitation studies by showing that 1) N-cadherin junctions of undifferentiated lens epithelial cells are comprised, principally, of N-cadherin linked to β-catenin; only low levels of γ-catenin were detected at cell-cell junctions of the central lens epithelium, 2) N-cadherin/β-catenin junctions persist throughout lens differentiation, and 3) N-cadherin/γ-catenin junctions are assembled as lens fiber cells begin to differentiate (Figures 2A–C). Immunostaining for vimentin demonstrated that these intermediate filaments are present in all cells of the E10 lens regardless of differentiation state. As is typical of intermediate filaments vimentin filaments extended across the cytoplasm of lens cells. While in hexagonally packed differentiating lens fiber cells actin filaments rearranged to a classical cortical localization concentrated at cell-cell interfaces but absent from the cell vertices, the vimentin filaments maintained their distribution throughout the cells (Weber and Menko, 2006 and Figure 2B–D). It is reasonable to expect that, as occurs in most other cell types, the vimentin filaments in differentiating fiber cells are linked to cell adhesion junctions, a property that is essential to the role of intermediate filaments in providing structural stability to the cell.

Figure 2.

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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, C, 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 (BE). 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 B–H was performed by confocal microscopy. Z-stacks were collected and analyzed; the data presented represents a single optical plane.

The exact localization of β- and γ-catenins in hexagonally packed differentiating lens fiber cells was most clearly demonstrated in cross-sections of the embryonic lens along the posterior tips of these cells. These imaging studies provided, for the first time, evidence that β-catenin and γ-catenin-containing junctions have distinct locations on the lens fiber cell membrane. β-catenin localized only to the lateral cell interfaces of these differentiating fiber cells, where they co-localized with F-actin and both were absent from the cell vertices (Figure 2E). This localization pattern of F-actin in fiber cells has been reported previously (Bassnett et al., 1999; Weber and Menko, 2006). In contrast, γ-catenin junctions were concentrated at the vertices of the hexagonally-shaped fiber cells (Figure 2E). In order to confirm this unique localization of γ-catenin in lens fiber cells we performed immunostaining and confocal imaging of whole E10 chicken embryo lenses (Figure 2F). For these studies the immunostained whole lenses were imaged directly at the basal aspects of the fiber cells. This approach made it possible to obtain a clear end-on view of γ-catenin localization near the posterior aspects of these cells. This study confirmed the very unique and distinct pattern of localization for γ-catenin to the cell vertices (Figure 2F, closed arrow), which was first detected as the lens cells enter the cortical fiber cell region, a region of the embryonic lens where lens cytoarchitecture is first established (Menko, 2002). Imaging of these whole lenses co-stained with phalloidin demonstrated that the localization pattern for γ-catenin was inverse to that of F-actin. γ-catenin-rich cell vertices were devoid of filamentous actin, and actin filaments were concentrated at lateral cell-cell interfaces (Figure 2F, open arrow). Using cross-sectional analysis we confirmed the restriction of F-actin to lateral borders of lens fiber cells (Figure 2G, open arrow) and its absence from vertices of these cells (Figure 2G, solid arrow). Similar analysis demonstrated that N-cadherin was distributed all along the cell-cell borders of lens fiber cells, both at lateral cell membranes (Figure 2H, open arrow) co-incident with both β-catenin and F-actin, and at the cell vertices (Figure 2H, solid arrow) with γ-catenin. While classical cadherins such as N-cadherin are typically thought to be associated with the actin cytoskeleton, our immunolocalization of N-cadherin and γ-catenin to vertices of this region of differentiating lens fiber cells, where actin is not present, suggests the existence of a novel N-cadherin/γ-catenin complex that must interact with other cytoskeletal elements, such as the vimentin intermediate filament proteins that are highly expressed in these differentiating cells.

Identification of a novel N-cadherin junctional complex associated with intermediate filament proteins in the lens

Despite the fact that the lens contains no desmosomes, desmosomal cadherins (Straub et al., 2003) or hemidesmosomes, there are high levels of intermediate filament proteins (Bloemendal et al., 1981; Ellis et al., 1984; Merdes et al., 1991; Ramaekers et al., 1982; Ramaekers et al., 1980). These intermediate filament proteins have been localized near the lens plasma membrane, but little is known about how they are anchored to the membrane (Blankenship et al., 2001; Bloemendal et al., 1981; Merdes et al., 1991; Quinlan et al., 1996; Sandilands et al., 1995). We used a co-immunoprecipitation approach to examine the possibility that the developing lens contains a novel N-cadherin/γ-catenin junction to which intermediate filament proteins such as vimentin could be linked. Since our studies above showed that the localization of γ-catenin to the lens plasma membrane was differentiation-state specific, the embryonic lens was microdissected into four differentiation-specific regions (see Figure 1A) prior to extraction in Triton/Octylglucoside buffer. Co-immunoprecipitation analyses (immunoprecipitation of γ-catenin followed by immunoblotting for vimentin) showed that indeed vimentin was recruited to γ-catenin junctions in the embryonic lens and that this linkage increased during lens fiber cell differentiation (Figure 3A). While the association between vimentin and γ-catenin was observed in the equatorial zone (EQ) where differentiation is initiated, much greater linkage of vimentin to γ-catenin occurred in the cortical fiber zone (FP), the principal region of lens fiber cell morphogenesis. This association decreased in the region of lens fiber cell maturation (FC); the reason for this decrease is elucidated in studies presented below. The pattern of association of vimentin with γ-catenin was paralleled in co-immunoprecipitation analyses which examined the recruitment of vimentin to N-cadherin complexes. These studies showed that the greatest linkage of vimentin to the N-cadherin complex was in the cortical fiber zone (Figure 3B). The specificity of both γ-catenin and N-cadherin association with vimentin was confirmed through co-immunoprecipitation studies using non-immune mouse IgG (Figure 3C). These results demonstrate for the first time that a differentiation-specific N-cadherin/vimentin junction forms in vivo and suggest that linkage of these proteins is mediated by γ-catenin.

Figure 3.

Figure 3

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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.

Desmoplakin is a component of the N-cadherin/γ-catenin junctions of differentiating lens fiber cells

Desmoplakin is the classical linker between γ-catenin and intermediate filaments (Cowin et al., 1986; Franke et al., 1994; Fuchs and Cleveland, 1998; Gallicano et al., 1998; Getsios et al., 2004; Koch and Franke, 1994; Troyanovsky et al., 1994). We now show, by Western Blot analysis, that desmoplakin is expressed at all stages of E10 chick lens cell differentiation, with an increased expression corresponding with the initiation of differentiation in the equatorial region (Figure 3D). More importantly, co-immunoprecipitation analysis (immunoprecipitation of N-cadherin followed by immunoblotting for desmoplakin) revealed that desmoplakin not only was expressed in the E10 chick lens but also that it was associated with N-cadherin junctions (Figure 3D). Desmoplakin association with N-cadherin peaks in the cortical fiber cell region, similar to the pattern of vimentin association with both N-cadherin and γ-catenin. These results demonstrated that desmoplakin is associated with N-cadherin junctions in differentiating lens fiber cells where it likely functions as the linker between N-cadherin/γ-catenin junctions and vimentin intermediate filaments.

Association of vimentin intermediate filament proteins with N-cadherin is tissue specific but not unique to lens

To determine whether N-cadherin linkage to vimentin was unique to the lens we performed co-immunoprecipitation analysis in a variety of tissue samples, including brain, skeletal muscle, heart, and skin, as well as in cultured human umbilical vein endothelial cells (HUVECs). While all these tissues and cell types expressed N-cadherin, γ-catenin, and vimentin (Figure 3E), the only tissue that paralleled the lens with a vimentin linked N-cadherin junction was brain tissue (Figure 3F). Interestingly, most of the tissues or cell types in which this novel junction was not detected are known to contain other junctional types that provide the tensile strength necessary to maintain tissue cytoarchitecture. Heart and skin contain desmosomes (Cheng and Koch, 2004) and in HUVECs, VE-cadherin, is associated with vimentin intermediate filaments (Kowalczyk et al., 1998). Our finding of vimentin-linked N-cadherin junctions in brain tissue demonstrates that these junctions are not unique to the lens and suggests that similar junctions will be found in other cell types requiring structural stability in the absence of desmosomal junctions.

Movement of N-cadherin and γ-catenin to a Highly Insoluble Vimentin-rich cell compartment with differentiation

The decrease in vimentin linkage to both N-cadherin and γ-catenin in the most differentiated fiber cells of the embryonic lens (FC, co-immunoprecipitation analysis of Triton/Octylglucoside cell extracts) led us to investigate whether, as lens fiber cells matured, this unique N-cadherin junction became resistant to extraction with the Triton/Octylglycoside buffer. This outcome would be consistent with the known resistance of vimentin to extraction by mild detergents. Here, we examined the detergent solubility properties of N-cadherin, γ-catenin, and β-catenin in each of the differentiation-specific zones of the E10 lens. We were particularly interested in those proteins that were insoluble in Triton and Octylglucoside as this protein fraction was expected to be enriched in intermediate filament proteins and their associated adhesion complex proteins. Microdissected lens fractions were extracted sequentially with Triton buffer followed by Octylglucoside buffer, and proteins in the Triton/Octylglucoside insoluble fraction, referred to as the Highly Insoluble (HI) fraction, were solubilized in a 4% SDS buffer. Western Blot analysis was used to compare the distribution of N-cadherin, γ-catenin and β-catenin relative to vimentin in the Triton soluble, Triton insoluble/OG soluble and HI fractions of each differentiation-specific zone of the E10 chick embryo lens.

In undifferentiated lens epithelial cells (EC) vimentin was highly insoluble in Triton and Octylglycoside (Figure 4A). While N-cadherin, β-catenin and γ-catenin were expressed in these undifferentiated lens cells, they were primarily detergent soluble (Figure 4B, C) and did not fractionate with vimentin. Therefore, in undifferentiated lens epithelial cells N-cadherin junctions were not linked to the HI vimentin fraction. Vimentin became increasingly associated with the HI fraction with increasing lens differentiation state (Figure 4A). Interestingly, while N-cadherin, β-catenin and γ–catenin were primarily Triton soluble in lens epithelial cells, only N-cadherin and γ-catenin moved significantly to the HI fraction as lens cells began their morphogenetic differentiation in the cortical fiber region (Figure 4B, C). In the most differentiated fiber cells of the embryonic lens (FC), both N-cadherin and γ-catenin were highly associated with the HI fraction, while the association of β-catenin with the HI fraction changed very little with differentiation state. There was however a significant increase in N-cadherin/β-catenin association with the Octylglucoside fraction. The observation that N-cadherin and γ-catenin were present in the HI, vimentin-rich fraction once fiber cell elongation had begun supports the possibility that a differentiation-specific N-cadherin/γ-catenin/intermediate filament junction exists. The properties of this novel junction suggest that it is likely to have a role in the morphogenetic differentiation of lens fiber cells, the maintenance of their elongated structure and their acquisition of highly ordered packing. Taken together these results suggest that γ-catenin, but not β-catenin is involved in N-cadherin linkage to vimentin.

Figure 4.

Figure 4

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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.

N-cadherin and γ-catenin in the HI fraction are linked to vimentin intermediate filament protein

The sequential detergent extraction studies suggest that after the N-cadherin complexes are stabilized by linking to the intermediate filament cytoskeleton, they become insoluble in Triton and Octylglucoside. Since linkage of cell junctions to intermediate filaments is known to provide cells with tensile strength, we investigated whether N-cadherin and γ-catenin in the HI fraction were associated with the intermediate filament vimentin by performing standard co-immunoprecipitation analysis. These co-immunoprecipitation analyses were conducted on the RIPA extract of the HI fraction from each differentiation-specific zone of the lens obtained by microdissection as described above. Immunoprecipitation of N-cadherin revealed that vimentin was indeed associated with N-cadherin complexes in the HI fraction, and that this association increased with differentiation of the lens fiber cells (Figure 5A). These studies demonstrate that our inability to detect vimentin-linked N-cadherin junctions in the most differentiated zone of the embryonic lens (FC) when cells were extracted in Triton/Octylglucoside resulted from the movement of this junction to the HI, vimentin-rich cell fraction. Furthermore, the association of N-cadherin with vimentin in the HI fraction was strikingly similar to the pattern of association between N-cadherin and γ-catenin (Figure 5A), supporting the likelihood that the formation of this novel N-cadherin/intermediate filament complex is mediated by γ-catenin. Immunoprecipitation of γ-catenin complexes from the HI fraction provided further evidence for the existence of an intermediate filament-linked N-cadherin/γ-catenin complex. γ-catenin association with vimentin in the HI fraction had the same differentiation-specific pattern as N-cadherin association with vimentin (Figure 5B). Co-immunoprecipitation analysis of the RIPA extract of the FP fraction using non-immune mouse IgG confirmed that association of N-cadherin and γ-catenin with vimentin is not due to non-specific antibody-protein interactions (Figure 5C). It is likely that γ-catenin mediates the interaction between N-cadherin and vimentin.

Figure 5.

Figure 5

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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 compartment 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 increase 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.

A novel double IP protocol confirms that the association of N-cadherin with vimentin is mediated by γ-catenin

Until now it has been impossible to biochemically separate N-cadherin/γ-catenin complexes from N-cadherin/β-catenin complexes. Therefore, while our studies presented above demonstrated that both γ-catenin and vimentin were linked to N-cadherin junctions and suggested that vimentin was associated with N-cadherin complexes through γ-, not β-, catenin, this did not prove that N-cadherin, γ-catenin and vimentin were associated in the same junctional complex. Therefore, it was necessary to develop a new approach in order to demonstrate that the linkage of the intermediate filament protein vimentin to N-cadherin junctions as lens fiber cells differentiated in vivo specifically involved the N-cadherin/γ-catenin complex. For this purpose, we developed a novel double immunoprecipitation technique. This protocol made it possible to isolate an N-cadherin complex based on whether it is linked to β- or γ-catenin and then to identify whether vimentin was associated specifically with the N-cadherin/γ-catenin complex. First, N-cadherin antibody 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/β-catenin complexes were blotted for γ-catenin, proving the efficacy of this protocol to purify only the targeted complexes (Figure 6A, B). Following this confirmation, the linkage of vimentin to isolated N-cadherin/γ-catenin and N-cadherin/β-catenin complexes was determined by Western Blot analysis. These studies showed that in the HI fraction vimentin was linked to N-cadherin/γ-catenin junctional complexes, but not to N-cadherin/β-catenin complexes (Figure 6B). In contrast, the intermediate filament protein desmin, a component of the HI fraction, was not detected in N-cadherin/γ-catenin junctions using the double IP protocol (unpublished 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 (Figure 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 intermediate 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.

Figure 6.

Figure 6

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Vimentin linkage to N-cadherin is specific to N-cadherin/γ-catenin junctions. A novel double immunoprecipitation approach (described in detail in the methods) that made it possible to separate N-cadherin/β-catenin from N-cadherin/γ-catenin complexes in the HI fraction was used to investigate the catenin protein that linked N-cadherin to vimentin. For these studies isolated N-cadherin complexes were immunoprecipitated with antibodies to either γ-catenin (A) or β-catenin (B) and then blotted for N-cadherin, γ-catenin, β-catenin and vimentin. The results confirm that γ-catenin, not β-catenin, links vimentin to the novel N-cadherin junctions that form in differentiating fiber cells of the embryonic lens. (C) Control co-immunoprecipitation analysis using immobilized non-immune mouse IgG followed by γ-catenin in the FP fraction confirmed the specificity of vimentin association with the N-cadherin/γ-catenin complex. Results are representative of at least three independent studies.

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 mutually exclusive manner (Cowin and Burke, 1996; Hulsken et al., 1994; Nagafuchi et al., 1991; Peifer et al., 1994). While the cadherin/β-catenin complex has been studied intensively, particularly in its role in the formation and function of the adherens junction, far less attention has been paid to the role of classical cadherin/γ-catenin complexes in cell-cell adhesion. It is not yet understood why classical cadherins link to γ-catenin instead of β-catenin, or whether the cadherin/γ-catenin cell junctions function differently from the cadherin/β-catenin cell junctions.

Although γ- and β-catenins are highly homologous (DeMarais and Moon, 1992), γ-catenin is the only junctional protein present in both adherens junctions and desmosomes (Troyanovsky et al., 1996; Wahl et al., 1996). Desmosomal junctions are strengthened by their association with intermediate filament cytoskeletal networks; an interaction mediated by γ-catenin and desmoplakin (Cowin et al., 1986; Franke et al., 1994; Fuchs and Cleveland, 1998; Gallicano et al., 1998; Getsios et al., 2004; Koch and Franke, 1994; Troyanovsky et al., 1994). Desmosomes provide cells with the structural strength to maintain the integrity of a tissue, which is especially important for tissues subjected to mechanical stress, such as the heart and skin (Fuchs and Cleveland, 1998; Huber, 2003). γ-catenin has been demonstrated to be critical to this desmosomal function (Bierkamp et al., 1999). While the function of γ-catenin in cell-cell adhesion has not been extensively studied outside of its role in desmosomal junctions, it is likely that γ-catenin may be equally important in strengthening cell-cell adhesions between cells lacking desmosomes.

Typically, classical cadherins are believed to interact with the actin cytoskeleton, however, Valiron, et al (1996) demonstrated that VE-cadherin co-localizes with desmoplakin and vimentin in endothelial cells. In a related study, transfection of fibroblasts with VE-cadherin and desmoplakin results in association between these two proteins only if the cells are also transfected with γ-catenin (Kowalczyk et al., 1998). This study shows that non-desmosomal cadherins have the potential to associate with intermediate filaments through γ-catenin. The possibility that N-cadherin could link to intermediate filaments was suggested by studies in which an N-cadherin/estrogen receptor fusion protein transfected into mouse fibroblast cultures co-immunoprecipitated with endogenous vimentin (Kim et al., 2005). Our data provided the first evidence that an N-cadherin/γ-catenin/desmoplakin/intermediate filament-linked cell-cell junction was formed in vivo, and showed that this junction became organized temporally as lens cells form stable cell-cell adhesions for their differentiation.

In order to identify the cytoskeletal linkage of the N-cadherin/γ-catenin junctional complexes we developed an innovative double immunoprecipitation protocol. This approach made it possible to isolate cadherin/γ-catenin from cadherin/β-catenin complexes (and visa versa) and then determine the linkage of these cadherin/catenin complexes to cytoskeletal proteins. Using this double immunoprecipitation approach we isolated N-cadherin/γ-catenin complexes from the intermediate filament-rich HI fraction of differentiating lens fiber cells and demonstrated for the first time the presence of a novel N-cadherin/γ-catenin junction that is linked to vimentin intermediate filaments in vivo. N-cadherin/β-catenin complexes present in the same cell fraction were not linked to vimentin. The intermediate filament linker proteins that mediated the association of vimentin with the N-cadherin/γ-catenin junction was desmoplakin. Immunolocalization studies in the lens were consistent with extensive literature that demonstrates that N-cadherin/β-catenin junctions are linked to the actin cytoskeleton. These studies showed that in differentiating lens fiber cells β-catenin localized to the lateral cell-cell interfaces, coincident with F-actin. In contrast, γ-catenin, which links to vimentin, localized primarily to the vertices of these hexagonally packed cells, where actin is absent. These results suggest that N-cadherin/γ-catenin and N-cadherin/β-catenin complexes in the developing lens link cell-cell junctions to different components of the cytoskeleton. The presence of N-cadherin junctions with different cytoskeletal linkages in the same cells suggests that N-cadherin/γ-catenin and N-cadherin/β-catenin junctions may have very different functions in lens development and homeostasis.

We believe that the novel N-cadherin-intermediate filament-linked junction we have identified in lens fiber cells may be critical to lens morphogenesis and the maintenance of lens cytoarchitecture. Formation and maintenance of the highly ordered structural packing of lens fiber cells is necessary for proper lens function. Therefore, it is not surprising that differentiating lens fiber cells have very different cell-cell adhesion requirements than their undifferentiated counterpart, the lens epithelial cell. These N-cadherin/γ-catenin junctions likely contribute to the strengthening of cell-cell adhesions critical for lens fiber cell morphogenesis and maintenance of the unique structure of the differentiating lens fiber cells. We believe that this novel intermediate filament-linked complex may be functionally analogous to desmosomal complexes, providing lens fiber cells with the tensile strength necessary to maintain the structural integrity of the lens. Furthermore, we showed that this junction is not unique to lens fiber cells, but that cadherin/γ-catenin junctions may foster interactions with intermediate filament cytoskeletal networks in other cell types which lack desmosomes. The novel double immunoprecipitation protocol used to identify the N-cadherin/γ-catenin/intermediate filament junction has great potential to enhance our understanding of protein scaffolds and signaling complexes of the cell.

Acknowledgments

This work was supported by grants from the NIH: EY10577, EY014258 and EY014798, and T32E5007283 to ASM. We also thank Iris Wolff and Ni Zhai for technical assistance; Janice L. Walker and Marilyn Woolkalis for critical reading of the manuscript; and Terry Hyslop for consultation on statistical analysis of our data.

Grant Information: NIH Grants EY10577, EY014258, EY014798 and T32E5007283

Footnotes

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