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素通过细胞粘附分子联系在一起：在稳定，信号传导和调节突触强度方面起着至关重要的作用的蛋白质。钙粘蛋白是1种粘附分子，通过在1个膜中形成同型二聚体与在相对细胞膜中形成的同型二聚体相互作用，将2相邻细胞膜连接在一起。在过去的十年中，已经努力理解经典钙粘蛋白的结构和分子关联。晶体学和生物物理研究产生了一定的冲突结果，但尚不清楚，在二聚化过程中钙粘蛋白的相互作用的相互作用图片。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）传感器，以监测跨细胞连接的钙粘蛋白的关联。 FRET在提供对1-10 nm范围内或分子间距离变化的信号的能力上是独一无二的，远低于常规荧光显微镜的固有衍射极限。钙粘着蛋白将在其细胞外域中被荧光标记。将在前后的细胞中表达有点折音和fret-peceptor，并将监测粘附的强度。早就知道，钙粘蛋白的结构完整性取决于局部Ca2+浓度。在没有Ca2+的情况下，钙粘蛋白会经历其类似杆状结构并塌陷的可逆损失。实验数据以及模拟预测CA2+在突触裂隙中受到动态调节。我们预测，Ca2+的变化具有重要的分析，对钙粘蛋白 - 钙粘着蛋白的粘附和信号传导具有重要的影响。我们将使用FRET在活细胞，钙粘蛋白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中动态监测。由于它们在突触裂口处的定位及其与细胞质蛋白的相互作用（如β-蛋白），钙粘蛋白占据了一个关键位置，可以导致与疾病相关的突触功能障碍。例如，在缺乏presenilin 1的情况下，这种蛋白质以某些形式的阿尔茨海默氏病突变，钙粘蛋白在粘附连接处不稳定，无法正确定位。这种错误的定位大概会导致突触结构和功能的深刻改变。