Detecting Cadherin Interactions Living Cell Junctions

检测钙粘蛋白与活细胞连接的相互作用

• 批准号: 7076757
• 负责人: ERIN M SCHUMAN
• 金额: $ 20.77万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7076757
• 关键词: bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical equipment development; biosensor device; biotechnology; cadherins; calcium flux; cell adhesion; cell cell interaction; cell line; confocal scanning microscopy; conformation; dimer; fluorescence resonance energy transfer; green fluorescent proteins; intercellular connection; molecular /cellular imaging; neurochemistry; protein protein interaction; synapses; synaptic vesicles; transposon /insertion element

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、发信号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个膜中形成同二聚体将2个相邻的细胞膜连接在一起，这些同二聚体穿过连接处（突触间隙）与相对细胞膜中形成的同二聚体相互作用。在过去的十年里，人们一直致力于了解经典钙粘蛋白的结构和分子缔合。晶体学和生物物理学的研究已经产生了一些相互矛盾的结果，但还不清楚，在二聚化过程中的钙粘蛋白胞外结构域的相互作用的图片。为了更好地了解钙粘蛋白相互作用的动力学，我们正在开发基于荧光共振能量转移（FRET）的传感器来监测跨细胞连接的钙粘蛋白缔合。FRET的独特之处在于它能够提供对1-10 nm范围内的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。钙粘蛋白将在其细胞外结构域中被荧光标记; FRET供体和FRET受体将在连接前和连接后细胞中表达，并将监测粘附强度。钙粘蛋白的结构完整性依赖于局部的钙离子浓度。在没有Ca 2+的情况下，钙粘蛋白经历其棒状结构的可逆损失和塌陷。实验数据以及模拟预测，Ca 2+是动态调节的突触间隙。我们预测，在裂缝Ca 2+的改变有重要的分歧，钙粘蛋白-钙粘蛋白粘附和信号。我们将使用FRET动态监测，在活细胞中，钙粘蛋白的相互作用和构象变化引起的细胞外钙和突触活动的变化。由于它们定位于突触间隙以及它们与细胞质蛋白（如β-连环蛋白）的相互作用，钙粘蛋白占据了可以促成与疾病相关的突触功能障碍的关键位置。例如，在缺乏早老素1（在某些形式的阿尔茨海默病中突变的蛋白质）的情况下，钙粘蛋白在粘附连接处变得不稳定，并且不能正确定位。这种错误定位可能导致突触结构和功能的深刻改变。