New probes for optical switching of protein interactions and protein fluorescence

描述（由申请人提供）：用于蛋白质相互作用和光开关和蛋白质荧光的新型探针。细胞突出的特征是膜受体、信号蛋白和第二信使（包括PDGF受体、Ca2+、PIP2/PIP3、CapG和肌动蛋白丝的刺端）的浓度、相互作用和活性的时空波动。这些分子事件与肌动蛋白细丝在靠近活化受体的位置的快速聚合相结合。阐明细胞突起机制的努力变得困难，部分原因是缺乏探针来绘制倒刺端特定的相互作用，并且需要专门的技术来快速绘制、操纵和测量活细胞中特定信号分子及其靶蛋白的相互作用和活动。我们描述了一种技术/假设驱动的方法来满足研究目标，该方法引入了新的光学探针，包括光学开关和成像技术，这些探针将用于确定受体介导的信号传导过程中产生的分子信号如何与capg -刺端复合体、肌动蛋白聚合和细胞突起的空间和时间调节相耦合。该提案有四个具体目标：目标1：设计和表征新的无功光开关及其共轭物。目的2：设计、合成和表征可逆调节细胞Ca2+的光开关。目的3：了解细胞Ca2+的变化如何耦合到自由倒刺末端的调节。目的4：建立PIP2/PIP3依赖性刺端脱帽在细胞突出过程中的作用。通过主要的初步成果和最近的出版物证明了拟议研究的可行性。我们相信，本提案中介绍的新型光开关和相关技术的独特特性将刺激新的显微镜技术的发展，并推进细胞和分子生理学复杂和动态过程的机制驱动研究。

DESCRIPTION (provided by applicant): New probes for optical switching of protein interactions and protein fluorescence. Cell protrusion is characterized by spatial and temporal fluctuations in the concentrations, interactions and activities of membrane receptors, signaling proteins and 2nd messengers that include PDGF receptor, Ca2+, PIP2/PIP3, CapG and the barbed end of the actin filament. These molecular events are coupled to the rapid polymerization of actin filaments at sites close to the activated receptor. Efforts to elucidate the mechanism of cell protrusion are made difficult, in part, because of the paucity of probes to map specific interactions at the barbed end, and the need for specialized techniques to rapidly map, manipulate and measure the interactions and activities of specific signaling molecules and their target proteins in live cells. We describe a technology/hypothesis-driven approach to meet the research objective that introduces new optical probes including optical switches and imaging techniques that will be used to establish how molecular signals generated during receptor-mediated signaling are coupled to the spatial and temporal regulation of the CapG-barbed end complex, actin polymerization and cell protrusion. The proposal has four specific aims: Aim 1: To design and characterize new reactive optical switches and their conjugates. Aim 2: To design, synthesize and characterize optical switches to reversibly modulate cell Ca2+. Aim 3: To understand how a change in cell Ca2+ is coupled to the regulation of free barbed ends. Aim 4: To establish the role of PIP2/PIP3- dependent uncapping of barbed-ends during cell protrusion. The feasibility of the proposed studies is demonstrated through key preliminary achievements and recent publications. We believe that the unique properties of the new optical switches and associated technologies introduced in this proposal will spur the development of new microscope techniques and advance mechanism-driven studies of complex and dynamic processes in cell and molecular physiology.