Consequences of desmoglein 2 loss for organization and function of intestinal epithelial junctions

肠道上皮保护机体免受管腔病原体的侵袭，同时能够选择性地吸收营养物质。这些双重功能是由顶端连接复合体支持的，顶端连接复合体位于相邻细胞的管腔质膜上，由周向紧密连接链(TJ)、贴壁连接带(AJs)和多个点状桥粒接触部位组成。虽然TJs和AJs在肠道内稳态中的作用已经被详细研究，但对桥粒的了解要少得多。它们是以桥粒蛋白(DSG)和桥粒粘附素类型为核心的多蛋白复合体，通过细胞内斑块蛋白与细胞质角蛋白中间丝细胞骨架相连。我们最近产生了肠道特异的桥粒蛋白2基因(DSG2)敲除，以检测肠道桥粒的桥粒钙粘附素依赖的生物学功能。这些小鼠没有明显的临床表型。然而，桥粒、AJ和TJ蛋白的水平发生了显著变化。此外，基因敲除小鼠对化学诱导的结肠炎非常敏感，表现为体重减轻、结肠缩短、更强的上皮损伤和肠道促炎细胞因子水平增加。该项目的目标是研究Dsg2缺失对顶端连接复合体的组织和功能的影响。我们认为顶端连接复合体的功能受损是DSG2基因敲除对肠道损伤易感性增加的原因。为了实现我们的目标并验证我们的假设，我们将检查我们的基因敲除小鼠和培养的具有稳定DSG2基因敲除的肠道肿瘤细胞。首先，我们将分析桥粒的组成、超微结构和动力学以及桥粒依赖的黏附。我们将进行免疫沉淀实验，研究Dsg2的消融如何影响剩余桥粒蛋白的相互作用。其次，我们将调查Dsg2缺失对TJs和AJs功能的影响。在此背景下，我们将研究肠道屏障的完整性和吸收和分泌的效率。第三，我们将通过检测上皮细胞沿隐窝-绒毛轴的迁移和分化，通过研究肠上皮细胞的机械弹性、粘附性和对凋亡的敏感性，以及通过研究连接素信号和对微生物感染的保护，来研究Dsg2缺失对整体肠上皮生理的影响。该项目将在肠上皮细胞顶端连接复合体的背景下，加深我们对桥粒钙粘附素对桥粒功能的贡献的理解，长期目标是阐明这些重要的细胞-细胞接触部位在维持和控制肠上皮细胞增殖和分化中的作用。

Intestinal epithelia protect the organism against luminal pathogens while enabling selective uptake of nutrients. These dual functions are supported by the apical junctional complex, which is localized at the adluminal plasma membranes of adjacent cells and consists of circumferential tight junction strands (TJs), the adherens junction belt (AJs) and multiple spot-like desmosomal contact sites. While the function of TJs and AJs for intestinal homeostasis has been investigated in detail, much less is known about desmosomes. They are multiprotein complexes with transmembrane cadherins of the desmoglein (Dsg) and desmocollin type at their core that are associated through intracellular plaque proteins to the cytoplasmic keratin intermediate filament cytoskeleton. We recently generated intestine-specific desmoglein 2 gene (DSG2) knockouts to examine desmosomal cadherin-dependent biological functions of intestinal desmosomes. These mice display no obvious clinical phenotype. Yet, the levels of desmosomal, AJ and TJ proteins are significantly altered. Furthermore, the knockout mice are markedly susceptible to chemically-induced colitis as evidenced by increased weight loss, colon shortening, stronger epithelial damage and increased intestinal levels of proinflammatory cytokines. The goal of the project is to examine the consequences of Dsg2 depletion on the organization and function of the apical junctional complex. We propose that compromised function of the apical junctional complex is responsible for the increased susceptibility of DSG2 knockouts to intestinal injury. To pursue our goal and to investigate our hypothesis we will examine our knockout mice and cultured intestinal tumor cells with a stable DSG2 knockdown. First, we will analyze desmosomal composition, ultrastructure and dynamics and desmosome-dependent adhesion. We will perform immunoprecipitation experiments to study how the ablation of Dsg2 affects the interaction of the remaining desmosomal proteins. Secondly, we will investigate the consequences of Dsg2 loss for the function of TJs and AJs. In this context, we will study intestinal barrier integrity and efficiency of resorption and secretion. Thirdly, we will study the effects of Dsg2 depletion on overall intestinal epithelial physiology by examining migration and differentiation of epithelial cells along the crypt-villus axis, by studying mechanical resilience, adhesive properties and susceptibility to apoptosis of intestinal epithelial cells, and by investigating catenin signaling and protection against microbial infections. The project will improve our understanding of the contribution of desmosomal cadherins to desmosomal functions in the context of the apical junctional complex of intestinal epithelia with the long-term goal to elucidate the role of these prominent cell-cell contact sites for the maintenance and control of intestinal epithelial proliferation and differentiation.