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

先进的光学显微镜方法研究调节细胞粘附和迁移的分子机制

• 批准号: RGPIN-2018-06508
• 负责人: Brown, Claire
• 金额: $ 4.66万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=763283
• 关键词: Advanced; light; microscopy; methods; study

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.

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。在细胞迁移过程中，细胞经历了一个循环过程：1)膜突起，2)许多粘附物的形成和稳定，这些粘附物将细胞与其前面的底物连接起来，3)细胞向前移位，4)粘附物的解体和细胞后部的脱落。粘附力必须足够强，以产生细胞向前推进的牵引力，但又不能太强，以至于细胞无法移动。因此，随着细胞的移动，必须对黏附的位置、形成和分解在空间和时间上进行严格的调节。大量的蛋白质参与了黏附的形成和调节，但对其发挥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黏附适配蛋白paxlin及其结合伙伴。我们将测量细胞迁移、蛋白质在整个细胞粘连中的分布和动态，以及已知在调节细胞迁移中发挥作用的关键结合伙伴之间的相互作用。我们将重点研究作为主要调节者的蛋白“开启”和“关闭”的激酶和磷酸酶。这些先进的显微技术将使我们能够确定调控细胞迁移的复杂分子机制的新细节。我们揭示的分子细节将导致对细胞迁移的新的、更精细的理解，我们产生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。然而，在用荧光显微镜对活样本进行成像的过程中，随着时间的推移，可能会产生光毒性副作用。因此，我们已经开发了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协议。这项计划每年将培训两名硕士学生和两名暑期学生，在整个资助过程中总共至少有八名学员。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以及指导其他学生。这将为他们未来在学术、企业或政府职位上的职业生涯做好准备。这项研究的跨学科性质、麦吉尔大学生理学系的培训环境以及先进生物成像设备(ABIF)中最先进的技术的使用，将确保这些受训人员受到独特的培训，并为成为未来职位的领导者做好准备。