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

JAM家族粘附分子对上皮细胞微绒毛间粘附和微绒毛动力学的调节

• 批准号: 425854143
• 负责人: Professor Dr. Klaus Thomas Ebnet
• 金额: --
• 依托单位: Institut für Medizinische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/425854143?language=en
• 关键词: Regulation; intermicrovillar; adhesion; microvillar; dynamics

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.

上皮细胞通过在细胞间形成机械和生理屏障在人体生理中发挥重要作用。当受到机械力时，机械屏障维持器官的完整性，生理屏障允许可溶性因子的受控摄取。这种细胞间屏障功能是由细胞粘附分子调节的，细胞粘附分子感知和抵抗力，并介导细胞旁通透性的选择性。有趣的是，类似的细胞粘附分子定位于各种上皮细胞的顶端域，分别介导耳蜗/前庭系统中微绒毛或立体纤毛、吸收性上皮细胞富f -肌动蛋白突起和感觉上皮细胞之间的相互作用。人们对这些粘附分子的作用知之甚少。与细胞间连接处的粘附分子类似，微绒毛间连接处的粘附分子在空间上被限制在特定的（微绒毛下）位置，并参与机械传感。我们的工作重点是连接粘附分子（Junctional Adhesion Molecules, JAMs）。我们最近发现了JAM粘附分子家族的一个新成员JAM- m，它定位于肠上皮细胞的微绒毛，并在小鼠肠簇细胞中特异性表达。我们发现JAM-M在微绒毛的中至基底区富集，而在远端端区不存在。通过酵母- 2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PDZ结构域的支架蛋白NHERF1和NHERF2以PDZ结构域依赖的方式相互作用。最后，使用诱导系统允许异位微绒毛形成，我们观察到JAM-M以pdz结构域依赖的方式被募集到微绒毛中。因此，我们的研究结果指出存在一种新的微绒毛间粘连复合物，它位于微绒毛的基底区。本研究的目的是了解这种粘附复合物在微绒毛形成中的作用。我们将详细描述该复合物的生化特征，并分析该复合物在调节ezrin（微绒毛形成所需的质膜-细胞骨架交联蛋白）的定位和活性中的作用。我们将分析JAM-M对NHERF1和NHERF2动力学的影响，以及对微绒毛动力学的影响。我们还将分析JAM-M作为粘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，研究JAM-M在肠道发育过程中的作用及其在簇细胞形成和功能中的作用。通过我们的研究，我们期望对肠上皮细胞中微绒毛形成和调控的分子机制有更深入的了解。