Detecting Cadherin Interactions Across Living Cell Junctions

描述(申请人提供)：突触连接的突触前和突触后元素由细胞黏附分子连接在一起：细胞黏附分子是在稳定、信号和调节突触强度方面发挥关键作用的蛋白质。钙粘附素是一种黏附分子，通过在一个细胞膜上形成同源二聚体将相邻的两个细胞膜连接在一起，这些同源二聚体跨越连接(突触裂隙)与相对细胞膜中形成的同源二聚体相互作用。在过去的十年里，人们致力于了解经典钙粘附素的结构和分子关联。结晶学和生物物理学的研究已经产生了一些相互矛盾的结果，并且还不清楚，在二聚化过程中钙粘蛋白胞外结构域的相互作用。为了更好地了解钙粘附素相互作用的动力学，我们正在开发基于荧光共振能量转移(FRET)的传感器，以监测细胞连接之间的钙粘附素关联。FRET的独特之处在于它能够提供对分子内或分子间距离在1-10 nm范围内变化敏感的信号，远远低于传统荧光显微镜固有的衍射极限。钙粘附素将在其胞外区域被荧光标记；FRET供体和FRET受体将在连接前和连接后的细胞中表达，黏附强度将被监测。很早以前就知道，钙粘附素的结构完整性取决于局部的钙离子浓度。在没有钙离子的情况下，钙粘附素会经历可逆性的棒状结构丧失和崩溃。实验数据和模拟都预测，突触间隙中的钙离子是动态调节的。我们预测，裂隙钙离子的改变对钙粘附素-钙粘附素的黏附和信号转导有重要的影响。我们将使用FRET来动态监测活细胞中钙粘附素的相互作用以及由细胞外钙和突触活性变化引起的构象变化。由于钙粘附素定位于突触间隙，并与细胞质蛋白如β-连环蛋白相互作用，因此在疾病相关的突触功能障碍中起着关键作用。例如，在缺乏早老素1的情况下，钙粘附素在附着的连接处变得不稳定，无法正确定位。这种错误的定位可能导致突触结构和功能的深刻变化。

DESCRIPTION (provided by applicant): The pre-and post-synaptic elements of synaptic junctions are linked together by cell adhesion molecules: proteins that play crucial roles in stabilizing, signaling and adjusting the strength of the synapse. The cadherins, 1 type of adhesion molecule, link together 2 adjacent cell membranes by forming homodimers in 1 membrane that interact across the junction (synaptic cleft) with homodimers formed in the opposing cell membrane. In the last decade, much effort has been devoted to understanding the structure and molecular associations of the classic cadherins. Crystallographic and biophysical studies have yielded somewhat conflicting results and, as yet an unclear, picture of the interactions of the cadherin extracellular domain during dimerization. To better understand the dynamics of cadherin interactions we are developing Fluorescence Resonance Energy Transfer (FRET) based sensors to monitor cadherin associations across cellular junctions. FRET is unique in its ability to provide signals that are sensitive to changes in intra-or intermolecular distances in the 1-10 nm range, well below the inherent diffraction limit of conventional fluorescence microscopy. Cadherins will be fluorescently tagged in their extracellular domains; FRET-donor and FRET-acceptors will be expressed in pre-and postjunctional cells and the strength of adhesion will be monitored. It has long been known that and the structural integrity of cadherins is dependent on the local Ca2+ concentration. In the absence of Ca2+, cadherins undergo a reversible loss of their rod-like structure and collapse. Experimental data as well as simulations predict that Ca2+ is dynamically regulated in the synaptic cleft. We predict that alterations in cleft Ca2+ have important ramifications for cadherin-cadherin adhesion and signaling. We will use FRET to monitor dynamically, in living cells, cadherin interactions and conformational changes induced by changes in extracellular calcium and synaptic activity. By virtue of their localization at synaptic cleft and their interactions with cytoplasmic proteins, like beta-catenin, the cadherins occupy a pivotal position that can contribute to the synaptic dysfunction associated with disease. For example, in the absence of presenilin 1, a protein mutated in some forms of Alzheimer's disease, cadherins become destabilized at adherent junctions and fail to localize properly. This mislocalization presumably leads to profound alterations in synaptic structure and function.