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82135357.Pdf View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Developmental Biology 319 (2008) 298–308 Contents lists available at ScienceDirect Developmental Biology journal homepage: www.elsevier.com/developmentalbiology 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
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