CYSTOLIC-FREE CALCIUM AND CELL MOTILITY

这项工作的目的是了解(1) 运动细胞的细胞内钙调节，(2) 钙离子变化与细胞内其他物质的关系 信令机制，以及(3)这些信令的作用 调节细胞运动的机制。拟议的工作将 检查两种类型的吞噬细胞：人中性粒细胞和 小鼠腹膜巨噬细胞。对于这两种类型的单元格， 是信号产生的广泛的生化特征 细胞骨架的调节机制和机制。 还有大量关于动物活动行为的信息 这些细胞对各种刺激做出反应。尽管强度很大 研究表明，在体内控制运动的机制仍然存在 不清楚，尽管(Ca~(2+))i，Phi，Kinase C和其他 信号已被提出并进行了研究。 光学显微镜技术将被用来测量 细胞内信号的产生和运动反应 单细胞。(Ca~(2+))i将以高时间和 空间分辨率。这些测量将同时进行 通过PHI和细胞形态的测量来提供详细的 信号的产生和运动之间的关联 细胞。测量单细胞中激酶C活性的方法将 被开发出来。三个信号((Ca~(2+))i，Phi， 和激酶C)将被检测。趋化性和吞噬作用 表现出脱敏/复敏现象。这一机制 对于这一点，将在分析其特性的基础上进行研究 这三种细胞内信号及其相互关系。 最后，这些信号在形态和细胞周期中的作用 将检查细胞骨架的变化。趋化性和 吞噬作用是需要在单个细胞上研究的过程。 水平，以了解生化机制是如何协调的 产生特定的细胞行为。所描述的微观方法 在该提案中提供了一种研究细胞内信号的方法 以及单细胞水平上的细胞反应。

The goal of this work is to understand (1) the mechanisms of intracellular calcium regulation in motile cells, (2) the relationship between calcium changes and other intracellular signalling mechanisms, and (3) the role of these signalling mechanisms in regulating cell motility. The proposed work will examine two types of phagocytic cells: the human neutrophil and the mouse peritoneal macrophage. For both types of cells, there is extensive biochemical characterization of the signal generating mechanisms and the mechanisms for regulation of the cytoskeleton. There is also substantial information on the motile behavior of these cells in response to various stimuli. Despite intensive study, the mechanisms for controlling motility in vivo remain unclear, although roles for (Ca2+)i, pHi, kinase C, and other signals have been proposed and investigated. Optical microscopy techniques will be used to measure both the generation of intracellular signals and the motile response in single cells. (Ca2+)i will be measured with high temporal and spatial resolution. These measurements will be made simultaneous with measurements of pHi and cell morphology to provide detailed correlations between the generation of signals and the motion of cells. Methods to measure kinase C activation in single cells will be developed. The interplay between three signals ((Ca2+)i, pHi, and kinase C) will be examined. Both chemotaxis and phagocytosis exhibit desensitization/resensitization phenomena. The mechanism for this will be studied based on analysis of the properties of these three intracellular signals and their inter-relationships. Finally, the role of these signals in the morphological and cytoskeletal changes will be examined. Both chemotaxis and phagocytosis are processes that require study at the single cell level to understand how biochemical mechanisms are orchestrated to produce specific cell behavior. The microscopic methods described in this proposal provide a means to study intracellular signals and cell responses at the single cell level.