Detecting Cadherin Interactions Across Living Cell Junctions

检测活细胞连接处的钙粘蛋白相互作用

基本信息

批准号：
7230085
负责人：
ERIN M SCHUMAN
金额：
$ 16.69万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-05-01 至 2009-04-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：突触连接的突触前和突触后元件通过细胞粘附分子连接在一起：细胞粘附分子是在稳定、信号传导和调节突触强度方面发挥关键作用的蛋白质。钙粘蛋白是一种粘附分子，通过在 1 个膜中形成同型二聚体将 2 个相邻的细胞膜连接在一起，该同型二聚体在连接处（突触间隙）与相对的细胞膜中形成的同型二聚体相互作用。在过去的十年中，人们致力于了解经典钙粘蛋白的结构和分子关联。晶体学和生物物理学研究产生了一些相互矛盾的结果，并且目前尚不清楚钙粘蛋白胞外结构域在二聚化过程中的相互作用。为了更好地了解钙粘蛋白相互作用的动态，我们正在开发基于荧光共振能量转移（FRET）的传感器来监测跨细胞连接的钙粘蛋白关联。 FRET 的独特之处在于它能够提供对 1-10 nm 范围内分子内或分子间距离变化敏感的信号，远低于传统荧光显微镜的固有衍射极限。钙粘蛋白的胞外结构域将被荧光标记； FRET 供体和 FRET 受体将在连接前和连接后细胞中表达，并监测粘附强度。人们早就知道钙粘蛋白的结构完整性取决于局部 Ca2+ 浓度。在缺乏 Ca2+ 的情况下，钙粘蛋白的棒状结构会发生可逆性损失并塌陷。实验数据和模拟预测 Ca2+ 在突触间隙中受到动态调节。我们预测，裂隙 Ca2+ 的改变对钙粘蛋白-钙粘蛋白粘附和信号转导具有重要影响。我们将使用 FRET 动态监测活细胞中钙粘蛋白相互作用以及由细胞外钙和突触活动变化引起的构象变化。由于其在突触间隙的定位以及与细胞质蛋白（如β-连环蛋白）的相互作用，钙粘蛋白占据了关键位置，可能导致与疾病相关的突触功能障碍。例如，在缺乏早老素 1（一种在某些形式的阿尔茨海默氏病中发生突变的蛋白质）的情况下，钙粘蛋白在粘附连接处变得不稳定，并且无法正确定位。这种错误定位可能会导致突触结构和功能的深刻改变。