Imaging Actin Dynamics at the Ventral Surface of Live Cells

活细胞腹面肌动蛋白动力学成像

• 批准号: 7618490
• 负责人: Ben Ovryn
• 金额: $ 27.93万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7618490
• 关键词: Actins; Adhesions; Biosensor; Cell surface; Cells; Computer software; Event; Fluorescence; Fluorescence Microscopy; Focal Adhesions; GTP-Binding Proteins; Image; Interference Microscopy; Lasers; Lateral; Life; Light Microscope; Lighting; Microscopy; Optics; Outcomes Research; Physiologic pulse; Reporting; Surface; fluorescence imaging; imaging modality; optical imaging; polymerization; two-dimensional

DESCRIPTION (provided by applicant): The mechanisms responsible for pathfinding are largely unexplored. Pathfinding is defined in two-dimensional culture as the formation of ventral protrusions that are required for focal contact formation. The reason that the relationships between ventral protrusions, lateral protrusions and adhesion formation have not been adequately studied to date is in part due to the inability of current light microscopes to image actin polymerization dynamics at the ventral surface in live cells during stimulated protrusion. In this application we describe how recent advances in optical imaging may be combined with existing biosensors in order to elucidate the choreographed sequence of spatial and temporal events that are involved in ventral protrusions and their relationship to focal contact formation. We will: 1. Extend imaging methods for observation of ventral cell surface dynamics to live cells. a) Develop hardware and software to enable total internal reflection fluorescence (TIRF) imaging using excitation from a pulsed laser. b) Develop hardware and software to enable fluorescence lifetime imaging microscopy (FLIM) using TIRF illumination. c) Develop hardware and software in order to optimize interference microscopy (IRM). 2. Visualize ventral protrusions in live cells and determine the actin compartments on the ventral surface that contribute to ventral protrusion activity and the formation of local contacts.

描述（由申请人提供）：在很大程度上尚未探索导致探路的机制。途径在二维培养物中定义为腹接触形成所需的腹侧突起的形成。迄今为止，尚未对腹侧突起，侧向突起和粘附形成之间的关系进行充分研究，部分原因是当前光显微镜无法在刺激的突起期间在活细胞中腹侧细胞的肌动蛋白聚合动力学图像肌动蛋白聚合动力学。在此应用中，我们描述了如何将光学成像的最新进展与现有生物传感器结合在一起，以阐明与腹侧突起有关的空间和时间事件的编排序列及其与焦点接触形态的关系。我们将：1。扩展成像方法，以观察腹细胞表面动力学到活细胞。 a）开发硬件和软件，以使用脉冲激光器的激发来实现总内反射荧光（TIRF）成像。 b）开发硬件和软件，以使用TIRF照明来实现荧光寿命成像显微镜（FLIM）。 c）开发硬件和软件以优化干扰显微镜（IRM）。 2。可视化活细胞中的腹侧突起，并确定有助于腹侧突出活性和局部接触形成的腹表面上的肌动蛋白室。