Microscopy upgrades to study biological motility and cell signaling at surfaces

所有的生物都会对信号做出反应。在细胞水平，分子信号与活细胞中的膜相关蛋白相互作用。我们的研究团队指导圭尔夫大学的56名HQP，专注于回答有关表面生物运动和信号传导的基本问题，无论是在真核生物或原核生物的活细胞膜中，还是在从纯化蛋白质重建的系统中。我们面临着一个共同的挑战，即获得参与运动和信号传导的分子的高分辨率图像，因为它们在细胞表面与它们的相互作用伙伴进行活动。为了获得这些图像，我们需要最先进的显微镜，提供在表面工作的分子的最佳分辨率。我们需要一个能够进行高分辨率全内反射荧光（TIRF）和Förster共振能量转移（FRET）测量的显微镜系统，该系统带有一个加热台，以微调我们正在寻找的相互作用类型，但这样的系统在圭尔夫并不存在。 使用TIRF显微镜，载玻片表面的一个非常薄（约100 nm）的层被照亮，允许分析集中在表面相关的分子上。我们的团队需要TIRF显微镜来成像运动细胞中的粘着斑动力学，跨膜蛋白对缺氧相关信号的反应，以及哺乳动物细胞中的钙信号。TIRF也将是我们团队回答有关细菌分裂体动态的基本问题的核心，细菌分裂体负责细胞分裂和细菌膜蛋白变化对不同生长条件的响应，并研究表面上重组肌肉蛋白的相互作用。通过FRET测量，可以在活细胞中真实的时间内观察到~10 nm半径内的分子相互作用。在我们的团队中，FRET将用于测量钙信号动力学和未来的第二信使信号和激酶活性使用生物传感器。 在本提案中，我们要求升级现有的新尼康Ti 2-E显微镜，以增加TIRF和FRET显微镜功能。还包括一个抗振表和加热阶段和目标夹克，以提供所需的稳定性和一致性。目前在圭尔夫的FRET显微镜是过时的，没有功能TIRF。所要求的系统将被安置在高级分析中心，该中心是我们整个地区的研究人员都可以使用的设施，有专门的显微镜师来维护系统并为HQP提供培训和指导。 高分辨率显微镜正在成为生命科学研究的标准，从单个分子到活细胞。圭尔夫大学对高分辨率显微镜的需求至关重要。对拟议基础设施的资助将为新的研究途径打开大门，为我们的研究团队，他们的HQP和合作者提供尖端技术，并为当地和区域的下一代科学家提供未来成功的技能。

All living things move in response to signals. At the cellular level, molecular signals interact with membrane-associated proteins in living cells. Our team of researchers mentoring 56 HQP at the University of Guelph are focused on answering fundamental questions about biological motility and signaling at surfaces, be it in living cell membranes in eukaryotes or prokaryotes or in systems reconstituted from purified proteins. We share a common challenge of obtaining high-resolution images of the molecules involved in motility and signaling as they go about their activities at cell surfaces with their interaction partners. To obtain these images, we need state of the art microscopy that provides the best resolution of molecules working at surfaces. We need a microscopy system capable of high-resolution Total Internal Reflection Fluorescence (TIRF) and Förster Resonance Energy Transfer (FRET) measurements with a heated stage to finetune for the types of interactions we are looking for, but such a system does not exist at Guelph. With TIRF microscopy, a very thin (~100 nm) layer at the slide surface is illuminated allowing analysis to be focused on surface-associated molecules. TIRF microscopy is needed by our team to image focal adhesion dynamics in motile cells, the response of transmembrane proteins to signals related to hypoxia, and calcium signaling in mammalian cells. TIRF would also be central for our team to answer fundamental questions about the dynamics of the bacterial divisome responsible for cell division and bacterial membrane protein changes responding to different growth conditions, and to study the interactions of reconstituted muscle proteins on surfaces. With FRET measurements, molecular interactions within a ~10 nm radius can be observed in living cells in real time. Within our team, FRET will be used to measure calcium signal dynamics and future second messenger signaling and kinase activity using biosensors. In this proposal, we request upgrades to an existing new Nikon Ti2-E microscope to add TIRF and FRET microscopy capabilities. Also included is an antivibration table and heated stage and objective jacket to provide needed stability and consistency. Current FRET microscopy at Guelph is outdated and there is no functional TIRF. The requested system will be housed in the Advanced Analysis Centre, a facility accessible to researchers throughout our region with a dedicated microscopist to maintain the system and provide HQP with training and guidance. High-resolution microscopy is becoming the standard for life science research, from single molecules to live cells. The need for high-resolution microscopy at the University of Guelph is critical. Funding of the proposed infrastructure will open the door to new avenues of research, offer cutting-edge technology to our research team, their HQP and collaborators, and provide the next generation of scientists, both locally and regionally, with the skills for future success.