Department of Cell Biology and Physiology, College of Life Sciences, Brigham Young University, Provo, UT 84602, USA.
Department of Microbiology and Molecular Biology, College of Life Sciences, Brigham Young University, Provo, UT 84602, USA.
Int J Mol Sci. 2023 Jun 6;24(12):9784. doi: 10.3390/ijms24129784.
Cell-adhesion molecules (CAMs) are responsible for cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Claudins (CLDNs), occludin (OCLN), and junctional adhesion molecules (JAMs) are CAMs' components of the tight junction (TJ), the single protein structure tasked with safeguarding the paracellular space. The TJ is responsible for controlling paracellular permeability according to size and charge. Currently, there are no therapeutic solutions to modulate the TJ. Here, we describe the expression of CLDN proteins in the outer membrane of and report its consequences. When the expression is induced, the unicellular behavior of is replaced with multicellular aggregations that can be quantified using Flow Cytometry (FC). Our method, called iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols), allows high-throughput screening (HTS) of small-molecules for interactions with CAMs. Here, we focused on using iCLASP to identify paracellular modulators for CLDN2. Furthermore, we validated those compounds in the mammalian cell line A549 as a proof-of-concept for the iCLASP method.
细胞黏附分子 (CAMs) 负责细胞-细胞、细胞-细胞外基质和细胞-病原体的相互作用。紧密连接 (TJ) 的组成部分包括 Claudins (CLDNs)、occludin (OCLN) 和 junctional adhesion molecules (JAMs)，TJ 是负责根据大小和电荷控制细胞旁通透性的单一蛋白质结构。目前，尚无调节 TJ 的治疗方法。在这里，我们描述了 CLDN 蛋白在外膜中的表达，并报告了其后果。当表达被诱导时， 的单细胞行为被多细胞聚集所取代，后者可以使用流式细胞术 (FC) 进行定量。我们的方法称为 iCLASP（通过 FC 方案检查细胞黏附分子聚集），允许对小分子与 CAMs 的相互作用进行高通量筛选 (HTS)。在这里，我们专注于使用 iCLASP 来鉴定 CLDN2 的细胞旁调节剂。此外，我们还在哺乳动物细胞系 A549 中验证了这些化合物，作为 iCLASP 方法的概念验证。
