Sub-cellular propagation of GPCR signaling; functional imaging in space and time

蛋白质平衡和蛋白质运输是控制细胞内稳态和对外界刺激做出反应的能力的中心过程。这些过程发挥核心作用的蛋白质家族之一是G蛋白偶联受体(GPCR)。这些受体形成了参与跨生物膜信息传输的最大蛋白质家族。在经典的表达方式中，GPCR检测细胞表面的信号，并通过质膜传递信息，导致附着在质膜内表面的异源三聚体G蛋白的激活。然后，G蛋白相互作用，调节酶和通道等效应器的活动。在激活之后，受体的磷酸化促进辅助蛋白çarrestin的募集，导致受体内吞和信号停滞。然而，这种传统的观点正在迅速演变，配体促进的信号复合体向脂筏、小窝、内小体、核、高尔基体、线粒体和内质网的转运或直接靶向，已经与这些细胞器中的非规范信号和调节功能联系在一起。然而，大多数细胞器特异性gpr活性的证据都是间接的，在某些情况下，还存在争议。直接监测细胞器特有的蛋白质运输和信号传播仍然是一项艰巨的挑战。我们NSERC支持的研究计划的长期目标是开发一个全面的生物物理平台，允许实时监测活细胞中细胞器特定的运输和活动。该平台将以GPCR为原型进行开发。在目前的应用中，我们建议：1)开发一系列新的基于生物发光共振能量转移(BRET)的生物传感器来检测细胞器特异性的GPCR运输，以及2)使用高分辨率和时间分辨的显微镜来可视化划分的信号活动。为了实现这些特定的目标，我们将设计和验证一系列BRET探头，以将其定位限制在单独的隔室。这些研究将产生关于区隔信号的动力学和作用的新的基础知识。由于它们的便携性，所开发的生物物理和成像工具将用于较长期的项目，以评估其他细胞器特有的生物过程的多样性。

Proteostasis and protein trafficking are central processes controlling the homeostasis of cells and their ability to respond to external stimuli. One of the protein families for which these processes play a central role is the G protein-coupled receptors (GPCR). These receptors form the largest family of proteins involved in the transfer of information across biological membranes. In their classical representation, GPCR detect signals at the cell surface and transmit the information across the plasma membrane, leading to the activation of heterotrimeric G proteins attached to the inner face of the plasma membrane. The G proteins then interact and regulate the activity of effectors such as enzymes and channels. Following activation, phosphorylation of the receptor promotes the recruitment of the accessory protein ßarrestin, leading to receptor endocytosis and signal arrest. This traditional view is however rapidly evolving and ligand-promoted trafficking or direct targeting of signaling complexes to lipid raft, caveolae, endosomes, nucleus, Golgi, mitochondria and endoplasmic reticulum, have been linked to non-canonical signaling as well as regulatory functions in these organelles. Yet, most of the evidence for organelle-specific GPCR activity are indirect and, in some cases, controversial. Directly monitoring organelle-specific protein trafficking and signal propagation remains a daunting challenge. The long-term objective of our NSERC-supported research program is to develop a comprehensive biophysical platform allowing the real-time monitoring of organelle-specific trafficking and activity in living cells. GPCR will be used as prototypical models to develop this platform. In the present application, we propose to: 1) develop a collection of new bioluminescence resonance energy transfer (BRET)-based biosensors detecting organelle-specific GPCR trafficking and, 2) use high-resolution and time-resolved microscopy to visualize the compartmentalized signalling activity. To reach these specific objectives, we will design and validate a collection of BRET probes engineered to restrict their localization to individual compartments. The studies will generate new fundamental knowledge about the dynamics and role of compartmentalized signaling. Because of their portability, the biophysical and imaging tools developed will be used in longer-term projects to asses a diversity of other organelle-specific biological processes.