Advanced light microscopy methods to study the molecular mechanisms regulating cell adhesion and migration

细胞迁移是许多基本生物过程的核心，如组织组织，神经发育，伤口愈合和免疫反应。在迁移期间，细胞经历以下循环过程：1）膜突出，2）在前部将细胞连接到其基底的许多粘附物的形成和稳定，3）细胞在向前方向上的移位，4）粘附物的分解和在细胞后部的分离。粘附必须足够强，以产生牵引力，使细胞前进，但不能太强，使细胞不能移动。因此，当细胞移动时，粘附的位置、形成和分解在空间和时间上必须有严格的调节。 数百种蛋白质参与粘附形成和调节，但对起作用的分子机制知之甚少。在这个项目中，我们将使用先进的光学显微镜技术来研究粘附适配蛋白桩蛋白及其结合伙伴。我们将测量细胞迁移，蛋白质分布和动态粘附在整个细胞和关键的结合伙伴之间的相互作用，已知在调节细胞迁移中发挥作用。我们将集中在激酶和磷酸酶，作为主调节器的作用，把蛋白质的“开”和“关”。这些先进的显微镜技术将使我们能够确定有关调节细胞迁移的复杂分子机制的新细节。我们发现的分子细节将导致对细胞迁移的新的和更精细的理解，我们生成的定量数据可以应用于细胞迁移的分子模型。 为了观察细胞并测量细胞和蛋白质动力学，我们表达了感兴趣的荧光蛋白的融合体。然而，在用荧光显微镜随时间成像活样品的过程中，可能存在光毒性副作用。因此，我们已经开发出程序来观察具有高空间和时间分辨率但光毒性最小的生命系统。为了保持细胞健康，我们将继续开发，应用和完善荧光照明光工程方案。 该项目每年将培训两名硕士生和两名暑期学生，在资助期间总共至少培训八名学员。这些学员将学习先进的成像技术，细胞生物学，定量图像处理和分析，科学数据准备和演示，并指导其他学生。这将为他们未来在学术，企业或政府职位的职业生涯做好准备。该研究的跨学科性质、麦吉尔大学生理学系的培训环境以及高级生物成像设施（ABIF）中最先进的技术将确保这些学员得到独特的培训并做好准备成为未来职位的领导者。

Cell migration is central to many fundamental biological processes such as tissue organization, neural development, wound healing and the immune response. During migration cells undergo a cyclic process of: 1) membrane protrusion, 2) formation and stabilization of many adhesions that link the cell to its substrate at the front 3) translocation of the cell in the forward direction, 4) disassembly of adhesions and detachment at the cell rear. Adhesions have to be strong enough to create traction for the cell to proceed forward but not so strong that the cell cannot move. Thus, there must be a tight regulation of adhesion location, formation and disassembly in space and time as the cell moves. Hundreds of proteins are involved in adhesion formation and regulation, but little is known about the molecular mechanisms at play. In this project we will used advanced light microscopy techniques to study the adhesion adaptor protein paxillin and its binding partners. We will measure cell migration, protein distributions and dynamics within adhesions across the cell and interactions between key binding partners that are known to play a role in regulating cell migration. We will focus on kinases and phosphatases that act to turn proteins "on" and "off" as master regulators. These advanced microscopy techniques will allow us to determine new details about the complex molecular mechanisms at play in regulating cell migration. The molecular details we uncover will lead to a new and more refined understanding of cell migration and the quantitative data we generate can be applied to molecular models of cell migration. To watch the cells and measure cellular and protein dynamics we express fusions of fluorescent proteins of interest. However, in the process of imaging live samples over time with fluorescence microscopy there can be photo-toxic side effects. So, we have developed procedures to watch living systems with high spatial and temporal resolution but minimal photo-toxicity. To maintain cell health we will will continue to develop, apply and refine fluorescence illumination light engineering protocols. This project will train two master's students and two summer students each year for a total of at least eight trainees over the course of the funding. These trainees will learn advanced imaging techniques, cell biology, quantitative image processing and analysis, scientific data preparation and presentation, and mentoring other students. This will prepare them for their future careers in academic, corporate or government positions. The cross-discipline nature of the research, the training environment at McGill University in the Department of Physiology and access to state-of-the-art technology in the Advanced BioImaging Facility (ABIF) will ensure these trainees well be uniquely trained and prepared to be leaders in their future positions.