PI3K Signaling in Single Lymphocytes Analyzed by LMS

描述(申请人提供)：我们的实验室研究淋巴细胞中磷脂酰肌醇3-激酶(PI3K)的信号机制。我们目前由#1RO1-AI50831-01资助，以确定单个PI3K亚型在淋巴细胞激活中的具体作用。这项工作依赖于标准的功能和生化分析来表征缺乏特定PI3K亚型的小鼠的淋巴细胞。然而，传统的测量信号酶活性的生化方法有很大的局限性，特别是在应用于初级淋巴细胞时。单细胞信号事件分析的进展为我们提供了强大的工具来促进我们对PI3K以及其他关键信号系统的理解。为了与R21机制的发展重点保持一致，该应用寻求开发一种新兴技术，激光微管系统(LMS)，在单细胞水平上研究PI3K依赖的信号。我们的第一个目标是开发使用LMS研究淋巴细胞的最佳条件。我们的第二个目标是应用这项技术来解决PI3K调节亚型p85α在激活不同下游信号分子中的作用。

DESCRIPTION (provided by applicant): Our laboratory studies mechanisms of phosphoinositide 3-kinase (PI3K) signaling in lymphocytes. We are currently funded by grant # 1 RO1-AI50831-01 to determine the specific roles of individual PI3K isoforms in lymphocyte activation. This work relies on standard functional and biochemical assays to characterize lymphocytes from mice lacking specific PI3K isoforms. However, traditional biochemical approaches to measuring signaling enzyme activity have significant limitations, especially when applied to primary lymphocytes. Advances in single cell analysis of signaling events offer powerful tools to advance our understanding of PI3K, as well as other critical signaling systems. In keeping with the developmental emphasis of this R21 mechanism, this application seeks to develop an emerging technology, the laser micropipet system (LMS), to study PI3K-dependent signals at the single cell level. Our first objective is to develop optimal conditions for studying lymphocytes using LMS. Our second aim is to apply this technology to address the role of the PI3K regulatory isoform p85alpha in activation of different downstream signaling molecules.