Regulation of intermicrovillar adhesion and microvillar dynamics in epithelial cells by a JAM family adhesion molecule

上皮片在人体生理中起着重要的作用，它在房间隔之间形成机械和生理屏障。机械屏障在受到机械力时保持器官的完整性，生理屏障允许控制地摄取可溶性因子。这种细胞间屏障功能受细胞黏附分子的调节，黏附分子可以感知和抵抗力，并调节细胞旁通透性的选择性。有趣的是，相似的细胞黏附分子定位于不同上皮细胞的顶端区域，分别调节微绒毛之间的相互作用或耳蜗前庭系统中吸入性上皮细胞富含F-肌动蛋白的突起与感觉上皮细胞之间的相互作用。人们对这些黏附分子的作用知之甚少。与细胞间连接处的黏附分子类似，微绒毛间连接处的黏附分子在空间上被限制在特定的(亚微绒毛)位置，并参与机械传感。我们的工作重点是连接黏附分子(JAMs)。我们最近发现，JAM家族中的一个新成员，称为JAM-M，定位于肠上皮细胞的微绒毛，并由小鼠的肠绒细胞特异性表达。我们发现JAM-M在微绒毛的中部到基底部有丰富的表达，而在远端无表达。利用酵母双杂交方法和生化实验，我们发现JAM-M与存在于微绒毛上的两个含有PDZ结构域的支架蛋白，即NHERF1和NHERF2以PDZ结构域依赖的方式相互作用。最后，使用允许异位微绒毛形成的诱导系统，我们观察到JAM-M以PDZ结构域依赖的方式招募到微绒毛。因此，我们的发现表明存在一种新的微绒毛间黏附复合体，该复合体定位于微绒毛的基底部。本研究的目的是了解这种黏附复合体在微绒毛形成中的作用。我们将对该复合体进行详细的生化表征，并分析该复合体在调节Ezrin的定位和活性方面的作用，Ezrin是微绒毛形成所需的质膜-细胞骨架交联蛋白。我们将分析JAM-M对NHERF1和NHERF2动态的影响以及它对微绒毛动态的影响。我们还将分析JAM-M作为黏附分子的生化和生物物理特性。最后，我们将建立两个动物模型，在其中我们将研究JAM-M在肠道发育过程中的作用，以及它在绒毛细胞形成和功能中的作用。通过我们的研究，我们期望对肠道上皮细胞微绒毛形成和调控的分子机制有更深入的了解。

Epithelial sheets play important roles in human physiology by forming mechanical and physiological barriers between compartments. The mechanical barriers maintain the integrity of the organs when subjected to mechanical forces, the physiological barriers allow a controlled uptake of soluble factors. This intercellular barrier function is regulated by cell adhesion molecules, which sense and resist forces and which mediate selectivity in paracellular permeability. Interestingly, similar cell adhesion molecules are localized at the apical domain of various epithelial cells and mediate the interaction between microvilli or between stereocilia, F-actin-rich protrusions of absoptive epithelial cells and sensory epithelial cells in the cochlear/vestibular system, respectively. The role of these adhesion molecules is much less understood. Similar to adhesion molecules at intercellular junctions, the adhesion molecules at intermicrovillar junctions are spatially restricted to specific (submicrovillar) locations and are involved in mechanosensing. The focus of our work are Junctional Adhesion Molecules (JAMs). We have recently found that a novel member of the JAM family of adhesion molecules, called JAM-M, is localized at microvilli of intestinal epithelial cells and is specifically expressed by intestinal tuft cells in mice. We found that JAM-M is enriched at the middle to basal region of microvilli and is absent form the distal tip region. Using a yeast-two hybrid approach and biochemical experiments, we found that JAM-M interacts with two PDZ domain-containing scaffolding proteins present at microvilli, i.e. NHERF1 and NHERF2 in PDZ domain-dependent manner. Finally, using an inducible system allowing ectopic microvilli formation, we observed that JAM-M is recruited to microvilli in a PDZ-domain-dependent manner. Our findings thus point to the existence of a novel intermicrovillar adhesion complex that is localized at the basal region of microvilli. The aim of this study is to understand the role of this adhesion complex in microvillus formation. We will biochemically characterize the complex in detail, and we will analyze the role of this complex in regulating the localization and activity of ezrin, a plasma membrane - cytoskeleton cross-linking protein required for microvilli formation. We will analyze the influence of JAM-M on the dynamics of NHERF1 and NHERF2 as well as its influence on the dynamics of microvilli. We will also analyze the biochemical and biophysical porperties of JAM-M as adhesion molecule. Finally, we will generate two animal models in which we will study the role of JAM-M during intestinal development and its role in the formation and function of tuft cells. From our studies we expect a deeper understanding of the molecular mechanisms underlying the formation and regulation of microvilli in intestinal epithelial cells.