Imaging tools to visualize and manipulate signaling in minute spaces

 描述（由申请人提供）：基于荧光共振能量转移（FRET）的生物传感器是研究Rho GTP酶时空调控的有力工具。早期版本表明，主动RhoA不限于回缩后部，并且在迁移前部的前缘被激活。在大多数这些FRET传感器中，Rho GTdR直接与效应片段和FRET对连接。在激活时，传感器采用闭合/结合构象并改变FRET。然而，从简单的反应扩散考虑，这种类型的传感器存在固有的问题。在闭合/结合构象中，它暂时失去与调节剂或效应物相互作用的能力，并且在此期间可以从初始激活位点扩散。因此，这种类型的传感器在跟踪信号时失去保真度，特别是在小的空间尺度上。在我们对粘连和树突棘中Rac和Rho激活的初步研究中，清楚地证明了这一问题。在这里，我们建议通过改善现有FRET传感器的动态范围和关闭动力学来解决这个问题。此外，将开发一种全新的策略-单分子探针-以超分辨率精确捕获活性GEF，GAP和Rho效应物复合物。最后，将探索与Rho GTP酶相关的信号转导靶标的潜在可推广的光遗传学策略。这些互补的成像工具将提供一个独特的力量，以解决微小的亚细胞结构内的信号的时空动态。

 DESCRIPTION (provided by applicant): Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for studying the spatial and temporal regulation of Rho GTPases. Early versions demonstrated that active RhoA was not restricted to the retracting rear and was activated at the leading edge of the migration front. In most of these FRET sensors, a Rho GTPase is directly tethered with an effector fragment and a FRET pair. Upon activation, the sensor adopts a closed/bound conformation and alters FRET. From a simple reaction-diffusion consideration, however, there is an intrinsic problem with this type of sensor. In the closed/bound conformation it temporarily loses the ability to interact with regulators or effectors and during this time can diffuse away from the initial site of activation. This type of sensor thereby loses fidelity in tracking signals, especially on small spatial scales. This issue was clearly demonstrated in our preliminary studies of Rac and Rho activation in adhesions and dendritic spines. Here we propose to address this issue by improving the dynamic range and off kinetics of the existing FRET sensors. In addition, a completely novel strategy - single molecule probes - will be developed to precisely capture active GEF, GAP and Rho-effector complexes in super resolution. Lastly, a potentially generalizable optogenetic strategy will be explored on signaling targets associated with Rho GTPases. These complementary imaging tools will provide a unique strength to resolve the spatiotemporal dynamics of signaling within minute subcellular structures.