Acquisition of an iLas2 TIRF/FRAP imaging platform

项目摘要。控制细胞运动和膜形态动力学的信号传导途径是紧密的， 在亚细胞水平上受到调节，并以快速动力学发生。因此，最好的方法来调查 参与这些过程的分子是实时观察和控制它们。为了实现这一目标，我们需要 开发分子探针，报告目标分子和显微镜系统的活动，以可视化和 在活细胞中控制这些探针。在父母奖中，我们建议开发光遗传学控制的 调节Dbl家族鸟嘌呤交换因子活性的抑制剂。这些分子是 Rho家族GTP酶的主要激活剂，细胞膜动力学的主要调节剂。在这份补充中， 我们申请资金购买iLas 2 TIRF/FRAP成像平台，以表征GEF-GTP酶信号传导， 通过应用我们的工程抑制剂，在活细胞中实时进行。iLas 2 TIRF/FRAP系统将 通过提供以下能力，极大地增强和扩展了家长奖的科学前提：1）观察 生物学事件，它们主要发生在细胞和盖玻片之间的接触部位，由于 TIRF能力; 2）以准确的亚细胞精度快速释放工程化抑制剂; 3）同时 使用荧光生物传感器观察GT3活性。

Project Abstract. Signaling pathways that control cell movement and membrane morphodynamics are tightly regulated at the subcellular level and occur with rapid kinetics. Therefore, the best way to investigate the molecules involved in these processes is to observe and control them in real-time. To achieve this, we need to develop molecular probes that report the activity of target molecules and microscopy systems to visualize and control these probes in living cells. In the parent award, we propose to develop optogenetically controlled inhibitors that modulate the activity of Guanine Exchange Factors of the Dbl family. These molecules are the main activators of Rho family GTPases, the major regulators of cell membrane dynamics. In this supplement, we request funds to acquire an iLas2 TIRF/FRAP imaging platform to characterize GEF-GTPases signaling in real-time in living cells through the application of our engineered inhibitors. The iLas2 TIRF/FRAP system will greatly enhance and expand the scientific premise of the parent awards by providing the ability to: 1) observe biological events where they primarily occur, at the contact sites between the cell and the coverslip, due to the TIRF capability; 2) rapidly uncage engineered inhibitors with accurate subcellular precision; 3) simultaneously observe GTPase activity using fluorescent biosensors.