New probes for optical switching of protein interactions and protein fluorescence

描述(申请人提供)：用于蛋白质相互作用和蛋白质荧光的光学开关的新型探针。细胞突起以膜受体、信号蛋白和第二信使的浓度、相互作用和活性的时空波动为特征，第二信使包括PDGF受体、钙离子、PIP2/PIP3、CAPG和肌动蛋白细丝的带刺末端。这些分子事件与肌动蛋白细丝在激活受体附近的快速聚合有关。解释细胞突起机制的努力变得困难，部分原因是缺乏探针来描绘带刺末端的特定相互作用，以及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目标蛋白的相互作用和活性。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，这些技术将用于确定在受体介导的信号传递过程中产生的分子信号是如何与CAPG带刺末端复合体、肌动蛋白聚合和细胞突起的空间和时间调节相耦合的。该提案有四个具体目标：目标1：设计和表征新的反应式光开关及其共轭。目的2：设计、合成和表征可逆性调节细胞内钙离子的光开关。目的3：了解细胞内钙离子的变化是如何与自由倒钩末端的调节相耦合的。目的：探讨PIP2/PIP3依赖的去掉带刺末端在细胞突起过程中的作用。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，这项提议中引入的新光学开关和相关技术的独特性质将推动新显微镜技术的发展，并推动对细胞和分子生理学中复杂和动态过程的机制驱动的研究。

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.