Imaging tools to visualize and manipulate signaling in minute spaces

 描述(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并不局限于后退，而是在迁徙前沿被激活。在大多数FRET传感器中，Rho GTP酶直接与效应器片段和FRET对相连。在激活时，传感器采用闭合/绑定构象并改变FRET。然而，从简单的反应扩散考虑，这种类型的传感器存在一个内在的问题。在封闭/结合构象中，它暂时失去了与调节器或效应器相互作用的能力，并在此期间可以从最初的激活部位扩散出去。因此，这种类型的传感器在跟踪信号时会失去保真度，特别是在小空间尺度上。这一问题在我们对粘连和树突棘中RAC和Rho激活的初步研究中得到了明确的证明。在这里，我们建议通过改进现有FRET传感器的动态范围和关闭动力学来解决这个问题。此外，还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的环境基金、GAP和Rho效应器复合体。最后，一个潜在的可推广的光遗传策略将被探索与Rho GTP酶相关的信号靶标。这些互补的成像工具将提供独特的力量来解析微小亚细胞结构内信号的时空动力学。

 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.