RESUMO
Desmosome diseases are caused by dysfunction of desmosomes, which anchor intermediate filaments (IFs) at sites of cell-cell adhesion. For many decades, the focus of attention has been on the role of actin filament-associated adherens junctions in development and disease, especially cancer. However, interference with the function of desmosomes, their molecular constituents or their attachments to IFs has now emerged as a major contributor to a variety of diseases affecting different tissues and organs including skin, heart and the digestive tract. The first Alpine desmosome disease meeting (ADDM) held in Grainau, Germany, in October 2022 brought together international researchers from the basic sciences with clinical experts from diverse fields to share and discuss their ideas and concepts on desmosome function and dysfunction in the different cell types involved in desmosome diseases. Besides the prototypic desmosomal diseases pemphigus and arrhythmogenic cardiomyopathy, the role of desmosome dysfunction in inflammatory bowel diseases and eosinophilic esophagitis was discussed.
Assuntos
Desmossomos , Doença , Humanos , Adesão Celular , Desmossomos/fisiologia , PênfigoRESUMO
Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Desmossomos/genética , Endocitose/genética , Animais , Adesão Celular/genética , Linhagem Celular , Desmogleínas/genética , Desmogleínas/metabolismo , Epiderme/metabolismo , Humanos , Junções Intercelulares/genética , Queratinócitos/metabolismo , CamundongosRESUMO
RATIONALE: Endothelial barrier function depends on the proper localization and function of the adherens junction protein VE (vascular endothelial)-cadherin. Previous studies have suggested a functional relationship between integrin-mediated adhesion complexes and VE-cadherin yet the underlying molecular links are unclear. Binding of the cytoskeletal adaptor protein talin to the ß-integrin cytoplasmic domain is a key final step in regulating the affinity of integrins for extracellular ligands (activation) but the role of integrin activation in VE-cadherin mediated endothelial barrier function is unknown. OBJECTIVE: To test the requirement of talin-dependent activation of ß1 integrin in VE-cadherin organization and endothelial cell (EC) barrier function. METHODS AND RESULTS: EC-specific deletion of talin in adult mice resulted in impaired stability of intestinal microvascular blood vessels, hemorrhage, and death. Talin-deficient endothelium showed altered VE-cadherin organization at EC junctions in vivo. shRNA (short hairpin RNA)-mediated knockdown of talin1 expression in cultured ECs led to increased radial actin stress fibers, increased adherens junction width and increased endothelial monolayer permeability measured by electrical cell-substrate impedance sensing. Restoring ß1-integrin activation in talin-deficient cells with a ß1-integrin activating antibody normalized both VE-cadherin organization and EC barrier function. In addition, VE-cadherin organization was normalized by reexpression of talin or integrin activating talin head domain but not a talin head domain mutant that is selectively deficient in activating integrins. CONCLUSIONS: Talin-dependent activation of EC ß1-integrin stabilizes VE-cadherin at endothelial junctions and promotes endothelial barrier function.
Assuntos
Antígenos CD/fisiologia , Caderinas/fisiologia , Células Endoteliais/fisiologia , Integrina beta1/fisiologia , Talina/fisiologia , Animais , Antígenos CD/análise , Caderinas/análise , Feminino , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Junções Intercelulares/metabolismo , Masculino , CamundongosRESUMO
Cadherins are the primary adhesion molecules in adherens junctions and desmosomes and play essential roles in embryonic development. Although significant progress has been made in understanding cadherin structure and function, we lack a clear vision of how cells confer plasticity upon adhesive junctions to allow for cellular rearrangements during development, wound healing and metastasis. Endocytic membrane trafficking has emerged as a fundamental mechanism by which cells confer a dynamic state to adhesive junctions. Recent studies indicate that the juxtamembrane domain of classical cadherins contains multiple endocytic motifs, or "switches," that can be used by cellular membrane trafficking machinery to regulate adhesion. The cadherin-binding protein p120-catenin (p120) appears to be the master regulator of access to these switches, thereby controlling cadherin endocytosis and turnover. This review focuses on p120 and other cadherin-binding proteins, ubiquitin ligases, and growth factors as key modulators of cadherin membrane trafficking.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Desenvolvimento Embrionário/fisiologia , Endocitose/fisiologia , Junções Aderentes/fisiologia , Animais , Membrana Celular/fisiologia , Humanos , Transporte ProteicoRESUMO
Desmosomes are macromolecular junctions responsible for providing strong cell-cell adhesion. Because of their size and molecular complexity, the precise ultrastructural organization of desmosomes is challenging to study. Here, we used direct stochastic optical reconstruction microscopy (dSTORM) to resolve individual plaque pairs for inner and outer dense plaque proteins. Analysis methods based on desmosomal mirror symmetry were developed to measure plaque-to-plaque distances and create an integrated map. We quantified the organization of desmoglein 3, plakoglobin and desmoplakin (N-terminal, rod and C-terminal domains) in primary human keratinocytes. Longer desmosome lengths correlated with increasing plaque-to-plaque distance, suggesting that desmoplakin is arranged with its long axis at an angle within the plaque. We next examined whether plaque organization changed in different adhesive states. Plaque-to-plaque distance for the desmoplakin rod and C-terminal domains decreased in PKP-1-mediated hyperadhesive desmosomes, suggesting that protein reorganization correlates with function. Finally, in human epidermis we found a difference in plaque-to-plaque distance for the desmoplakin C-terminal domain, but not the desmoplakin rod domain or plakoglobin, between basal and suprabasal cells. Our data reveal the molecular organization of desmosomes in cultured keratinocytes and skin as defined by dSTORM.
Assuntos
Desmossomos/metabolismo , Microscopia/métodos , Fenômenos Ópticos , Adesão Celular , Humanos , Masculino , Placofilinas/metabolismo , Pele/metabolismo , Processos EstocásticosRESUMO
Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with ß- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions.
Assuntos
Anquirinas/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Endocitose , Multimerização Proteica , Junções Aderentes/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Caderinas/química , Caderinas/genética , Cateninas/metabolismo , Adesão Celular , Linhagem Celular , Sequência Conservada , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imunoprecipitação , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Transporte Proteico , Triptofano/genética , delta CateninaRESUMO
Desmosomes are prominent adhesive junctions present between many epithelial cells as well as cardiomyocytes. The mechanisms controlling desmosome assembly and remodeling in epithelial and cardiac tissue are poorly understood. We recently identified protein palmitoylation as a mechanism regulating desmosome dynamics. In this study, we have focused on the palmitoylation of the desmosomal cadherin desmoglein-2 (Dsg2) and characterized the role that palmitoylation of Dsg2 plays in its localization and stability in cultured cells. We identified two cysteine residues in the juxtamembrane (intracellular anchor) domain of Dsg2 that, when mutated, eliminate its palmitoylation. These cysteine residues are conserved in all four desmoglein family members. Although mutant Dsg2 localizes to endogenous desmosomes, there is a significant delay in its incorporation into junctions, and the mutant is also present in a cytoplasmic pool. Triton X-100 solubility assays demonstrate that mutant Dsg2 is more soluble than wild-type protein. Interestingly, trafficking of the mutant Dsg2 to the cell surface was delayed, and a pool of the non-palmitoylated Dsg2 co-localized with lysosomal markers. Taken together, these data suggest that palmitoylation of Dsg2 regulates protein transport to the plasma membrane. Modulation of the palmitoylation status of desmosomal cadherins can affect desmosome dynamics.
Assuntos
Membrana Celular/metabolismo , Desmogleína 2/metabolismo , Desmossomos/metabolismo , Lipoilação/fisiologia , Substituição de Aminoácidos , Linhagem Celular Tumoral , Membrana Celular/genética , Desmogleína 2/genética , Desmossomos/genética , Humanos , Mutação de Sentido Incorreto , Transporte Proteico/fisiologiaRESUMO
Cadherin-11 (Cad11) cell adhesion molecule plays a role in prostate cancer cell migration. Because disassembly of adhesion complexes through endocytosis of adhesion proteins has been shown to play a role in cell migration, we examined whether Cad11 endocytosis plays a role in Cad11-mediated migration. The mechanism by which Cad11 is internalized is unknown. Using a GST pulldown assay, we found that clathrin binds to the Cad11 cytoplasmic domain but not to that of E-cadherin. Using deletion analysis, we identified a unique sequence motif, VFEEE, in the Cad11 membrane proximal region (amino acid residues 11-15) that binds to clathrin. Endocytosis assays using K(+)-depletion buffer showed that Cad11 internalization is clathrin dependent. Proximity ligation assays showed that Cad11 colocalizes with clathrin, and immunofluorescence assays showed that Cad11 localizes in vesicles that stain for the early endosomal marker Rab5. Deletion of the VFEEE sequence from the Cad11 cytoplasmic domain (Cad11-cla-Δ5) leads to inhibition of Cad11 internalization and reduces Cad11-mediated cell migration in C4-2B and PC3-mm2 prostate cancer cells. These observations suggest that clathrin-mediated internalization of Cad11 regulates surface trafficking of Cad11 and that dynamic turnover of Cad11 regulates the migratory function of Cad11 in prostate cancer cells.
Assuntos
Caderinas/metabolismo , Movimento Celular , Clatrina/metabolismo , Endocitose , Proteínas de Neoplasias/imunologia , Neoplasias da Próstata/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Clatrina/genética , Células HEK293 , Humanos , Masculino , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação ProteicaRESUMO
Desmosomes are cell-cell junctions that mediate adhesion and couple the intermediate filament cytoskeleton to sites of cell-cell contact. This architectural arrangement integrates adhesion and cytoskeletal elements of adjacent cells. The importance of this robust adhesion system is evident in numerous human diseases, both inherited and acquired, which occur when desmosome function is compromised. This review focuses on autoimmune and infectious diseases that impair desmosome function. In addition, we discuss emerging evidence that desmosomal genes are often misregulated in cancer. The emphasis of our discussion is placed on the way in which human diseases can inform our understanding of basic desmosome biology and in turn, the means by which fundamental advances in the cell biology of desmosomes might lead to new treatments for acquired diseases of the desmosome.
Assuntos
Desmossomos/metabolismo , Dermatopatias/metabolismo , Dermatopatias/patologia , Animais , Humanos , Modelos BiológicosRESUMO
Cell junctions integrate extracellular signals with intracellular responses to polarize tissues, pattern organs, and maintain tissue architecture by promoting cell-cell adhesion and communication. In this review, we explore the mechanisms whereby the adhesive junctions, adherens junctions and desmosomes, co-assemble and then segregate into unique plasma membrane domains. In addition, we highlight emerging evidence that these junctions are spatially and functionally integrated with the endoplasmic reticulum to mediate stress sensing and calcium homeostasis. We conclude with a discussion of the role of the endoplasmic reticulum in the mechanical stress response and how disruption of these connections may cause disease.
RESUMO
The desmosome is a cell-cell adhesive junction that provides integrity and mechanical resistance to tissues through its attachment to the intermediate filament cytoskeleton. Defects in desmosomes cause diseases impacting the heart, epidermis, and other epithelia. In this review, we provide a historical perspective on the discovery of the desmosome and how the evolution of cellular imaging technologies revealed insights into desmosome structure and function. We also discuss recent findings using contemporary imaging approaches that have informed the molecular order, three-dimensional architecture, and associations of desmosomes with organelles such as the endoplasmic reticulum. Finally, we provide an updated model of desmosome molecular organization and speculate upon novel functions of this cell junction as a signaling center for sensing mechanical and other forms of cell stress.
Assuntos
Desmossomos , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Humanos , Animais , Adesão Celular , Transdução de SinaisRESUMO
Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to regulate cellular functions. Desmosomes are mechanically resilient adhesive junctions that associate with lipid raft membrane domains, yet the mechanisms directing raft association of the desmosomal proteins, particularly the transmembrane desmosomal cadherins, are poorly understood. We identified the desmoglein-1 (DSG1) transmembrane domain (TMD) as a key determinant of desmoglein lipid raft association and designed a panel of DSG1 TMD variants to assess the contribution of TMD physicochemical properties (length, bulkiness, and palmitoylation) to DSG1 lipid raft association. Sucrose gradient fractionations revealed that TMD length and bulkiness, but not palmitoylation, govern DSG1 lipid raft association. Further, DSG1 raft association determines plakoglobin recruitment to raft domains. Super-resolution imaging and functional assays uncovered a strong relationship between the efficiency of DSG1 TMD lipid raft association and the formation of morphologically and functionally robust desmosomes. Lipid raft association regulated both desmosome assembly dynamics and DSG1 cell surface stability, indicating that DSG1 lipid raft association is required for both desmosome formation and maintenance. These studies identify the biophysical properties of desmoglein transmembrane domains as key determinants of lipid raft association and desmosome adhesive function.
RESUMO
Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.
Assuntos
Caderinas , Proteólise , Ubiquitina-Proteína Ligases , Humanos , Junções Aderentes/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Caderinas/metabolismo , Adesão Celular , Células HEK293 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Adherens junctions are important mediators of intercellular adhesion, but they are not static structures. They are regularly formed, broken, and rearranged in a variety of situations, requiring changes in the amount of cadherins, the main adhesion molecule in adherens junctions, present at the cell surface. Thus, endocytosis, degradation, and recycling of cadherins are crucial for dynamic regulation of adherens junctions and control of intercellular adhesion. In this chapter, we review the involvement of cadherin endocytosis in development and disease. We discuss the various endocytic pathways available to cadherins, the adaptors involved, and the sorting of internalized cadherin for recycling or lysosomal degradation. In addition, we review the regulatory pathways controlling cadherin endocytosis and degradation, including regulation of cadherin endocytosis by catenins, cadherin ubiquitination, and growth factor receptor signaling pathways. Lastly, we discuss the proteolytic cleavage of cadherins at the plasma membrane.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Adesão Celular/fisiologia , Endocitose , Proteólise , Transdução de Sinais , Animais , Humanos , Transporte ProteicoRESUMO
Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.
RESUMO
The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.
Assuntos
Citoesqueleto , Desmossomos , Desmossomos/química , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Citoesqueleto/metabolismo , Queratinas/metabolismo , Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Retículo Endoplasmático/metabolismoRESUMO
In common with other p120-catenin subfamily members, Xenopus ARVCF (xARVCF) binds cadherin cytoplasmic domains to enhance cadherin metabolic stability or, when dissociated, modulates Rho-family GTPases. We report here that xARVCF binds and is stabilized by Xenopus KazrinA (xKazrinA), a widely expressed conserved protein that bears little homology to established protein families, and which is known to influence keratinocyte proliferation and differentiation and cytoskeletal activity. Although we found that xKazrinA binds directly to xARVCF, we did not resolve xKazrinA within a larger ternary complex with cadherin, nor did it co-precipitate with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin, suggesting a potential means by which xKazrinA localizes to cell-cell borders. This was supported by the resolution of a ternary biochemical complex of xARVCF-xKazrinA-xß2-spectrin and, in vivo, by the finding that ectodermal shedding followed depletion of xKazrin in Xenopus embryos, a phenotype partially rescued with exogenous xARVCF. Cell shedding appeared to be the consequence of RhoA activation, and thereby altered actin organization and cadherin function. Indeed, we also revealed that xKazrinA binds p190B RhoGAP, which was likewise capable of rescuing Kazrin depletion. Finally, xKazrinA was found to associate with δ-catenins and p0071-catenins but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin subfamily. Taken together, our study supports the essential role of Kazrin in development, and reveals the biochemical and functional association of KazrinA with ARVCF-catenin, spectrin and p190B RhoGAP.
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Espectrina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Caderinas/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Linhagem Celular , Células Epiteliais/química , Células Epiteliais/enzimologia , Proteínas Ativadoras de GTPase/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Espectrina/genética , Técnicas do Sistema de Duplo-Híbrido , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Pemphigus vulgaris is an autoimmune disease caused by IgG antibodies against desmoglein 3 (Dsg3). Previously, we isolated a pathogenic mAb against Dsg3, AK23 IgG, which induces a pemphigus vulgaris-like phenotype characterized by blister formation. In the present study, we generated a transgenic mouse expressing AK23 IgM to examine B-cell tolerance and the pathogenic role of IgM. Autoreactive transgenic B cells were found in the spleen and lymph nodes, whereas anti-Dsg3 AK23 IgM was detected in the cardiovascular circulation. The transgenic mice did not develop an obvious pemphigus vulgaris phenotype, however, even though an excess of AK23 IgM was passively transferred to neonatal mice. Similarly, when hybridoma cells producing AK23 IgM were inoculated into adult mice, no blistering was observed. Immunoelectron microscopy revealed IgM binding at the edges of desmosomes or interdesmosomal cell membranes, but not in the desmosome core, where AK23 IgG binding has been frequently detected. Furthermore, in an in vitro dissociation assay using cultured keratinocytes, AK23 IgG and AK23 IgM F(ab')(2) fragments, but not AK23 IgM, induced fragmentation of epidermal sheets. Together, these observations indicate that antibodies must gain access to Dsg3 integrated within desmosomes to induce the loss of keratinocyte cell-cell adhesion. These findings provide an important framework for improved understanding of B-cell tolerance and the pathophysiology of blister formation in pemphigus.
Assuntos
Desmogleína 3/química , Imunoglobulina G/química , Imunoglobulina M/química , Pênfigo/imunologia , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Vesícula/imunologia , Adesão Celular , Citometria de Fluxo/métodos , Hibridomas/metabolismo , Imunoglobulina M/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Microscopia Imunoeletrônica/métodos , Modelos Genéticos , Dados de Sequência MolecularRESUMO
RATIONALE: p120-catenin (p120) is an armadillo family protein that binds to the cytoplasmic domain of classical cadherins and prevents cadherin endocytosis. The role of p120 in vascular development is unknown. OBJECTIVE: The purpose of this study is to examine the role of p120 in mammalian vascular development by generating a conditionally mutant mouse lacking endothelial p120 and determining the effects of the knockout on vasculogenesis, angiogenic remodeling, and the regulation of endothelial cadherin levels. METHODS AND RESULTS: A conditional Cre/loxP gene deletion strategy was used to ablate p120 expression, using the Tie2 promoter to drive endothelial Cre recombinase expression. Mice lacking endothelial p120 died embryonically beginning at embryonic day 11.5. Major blood vessels appeared normal at embryonic day 9.5. However, both embryonic and extraembryonic vasculature of mutant animals were disorganized and displayed decreased microvascular density by embryonic day 11.5. Importantly, both vascular endothelial cadherin and N-cadherin levels were significantly reduced in vessels lacking p120. This decrease in cadherin expression was accompanied by reduced pericyte recruitment and hemorrhaging. Furthermore, p120-null cultured endothelial cells exhibited proliferation defects that could be rescued by exogenous expression of vascular endothelial cadherin. CONCLUSIONS: These findings reveal a fundamental role for p120 in regulating endothelial cadherin levels during vascular development, as well as microvascular patterning, vessel integrity, and endothelial cell proliferation. Loss of endothelial p120 results in lethality attributable to decreased microvascular density and hemorrhages.
Assuntos
Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Cateninas/metabolismo , Células Endoteliais/metabolismo , Animais , Antígenos CD/metabolismo , Vasos Sanguíneos/patologia , Padronização Corporal , Antígenos CD8 , Caderinas/metabolismo , Cateninas/deficiência , Cateninas/genética , Proliferação de Células , Células Cultivadas , Perda do Embrião , Células Endoteliais/patologia , Idade Gestacional , Hemorragia/embriologia , Hemorragia/genética , Hemorragia/metabolismo , Imunoglobulinas , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/embriologia , Microvasos/metabolismo , Pericitos/metabolismo , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/genética , Receptor TIE-2 , delta CateninaRESUMO
Desmosomes are macromolecular cell-cell junctions critical for maintaining adhesion and resisting mechanical stress in epithelial tissue. Desmosome assembly and the relationship between maturity and molecular architecture are not well understood. To address this, we employed a calcium switch assay to synchronize assembly followed by quantification of desmosome nanoscale organization using direct Stochastic Optical Reconstruction Microscopy (dSTORM). We found that the organization of the desmoplakin rod/C-terminal junction changed over the course of maturation, as indicated by a decrease in the plaque-to-plaque distance, while the plaque length increased. In contrast, the desmoplakin N-terminal domain and plakoglobin organization (plaque-to-plaque distance) were constant throughout maturation. This structural rearrangement of desmoplakin was concurrent with desmosome maturation measured by E-cadherin exclusion and increased adhesive strength. Using two-color dSTORM, we showed that while the number of individual E-cadherin containing junctions went down with the increasing time in high Ca2+, they maintained a wider desmoplakin rod/C-terminal plaque-to-plaque distance. This indicates that the maturation state of individual desmosomes can be identified by their architectural organization. We confirmed these architectural changes in another model of desmosome assembly, cell migration. Desmosomes in migrating cells, closest to the scratch where they are assembling, were shorter, E-cadherin enriched, and had wider desmoplakin rod/C-terminal plaque-to-plaque distances compared to desmosomes away from the wound edge. Key results were demonstrated in three cell lines representing simple, transitional, and stratified epithelia. Together, these data suggest that there is a set of architectural programs for desmosome maturation, and we hypothesize that desmoplakin architecture may be a contributing mechanism to regulating adhesive strength.