Full Project 4: Time-resolved flow cytometric study of cell signaling

具体目标（摘要）我们将全面运作的杰西卡P.休斯顿博士（NMSU）和罗杰布伦特博士（FHCRC）的实验室之间的持续试点合作。我们提出的工作建立在两个实验室的互补优势之上：休斯顿实验室的仪器工程和信号分析，布伦特实验室的蛋白质工程和遗传学。特别是，这项工作建立在“频域”方法来确定荧光 显微镜的寿命，以及“频域”方法的更先进发展 流式细胞术，休斯顿博士正在成为领导者。 导致不适当的细胞增殖的细胞信号传导功能障碍导致大多数癌症。 虽然对细胞信号传导有很多了解，但关键问题，包括细胞信号传导的原因和后果， 细胞信号和反应的变化，仍然没有答案。在这里，我们将使用荧光寿命方法 为了扩展单细胞测定的能力以量化酵母中细胞信号传导中的关键事件（“开发 平台"）和哺乳动物细胞中。这些测量依赖于绿色荧光蛋白的使用 (GFP)与构成不同细胞信号通路的蛋白质融合的衍生物， 例如将信号复合物募集到细胞膜上。 在未来三年内，我们将发展我们的方法，以解决这些问题，并允许更广泛的 应用程序。特别是，我们将继续改进我们的仪器，用它来开发更好的GFP 衍生物和信号报告，并使用改进的方法来了解细胞间的原因， 酵母信号系统的变异。我们还将使用这些方法开发高信号报告基因， 允许使用流式细胞术定量克隆哺乳动物细胞系中的细胞信号传导。成功的工作将 值得未来的资助。它还应该增加休斯顿实验室的知名度，帮助招募新的学生， 研究人员从代表性不足的少数民族，在这两个网站，到一个活跃的国际领域， 科研

Specific Aims (Abstract) We will make fully operational a continuing pilot collaboration between the labs of Dr. Jessica P. Houston (NMSU) and Dr. Roger Brent (FHCRC). Our proposed work builds on complementary strengths in the two labs: instrumentation engineering and signal analysis in the Houston lab, protein engineering and genetics in the Brent lab. In particular, the work builds on "frequency domain" methods to determine fluorescence lifetimes in microscopy, and on signiflcantly more advanced developments in "frequency domain" methods for flow cytometry, for which Dr. Houston is emerging as a leader. Dysfunctions in cell signaling leading to inappropriate cell proliferation contribute to most cancers. Although much is known about cell signaling, key quesfions, including the causes and consequences of cellto- cell variation in signaling and response, remain unanswered. Here, we will use fluorescent lifefime methods to extend the power of single cell assays to quantify key events in cell signaling in yeast (the "development platform") and in mammalian cells. These measurements depend on the use of Green Fluorescent Protein (GFP) derivatives fused to the proteins that make up the different cell signaling pathways to quantify events such as recruitment of a signaling complex to the cell membrane. During the next three years, we will develop our methods, to address these quesfions and to allow wider applicafions. In particular, we will confinue to improve our instrumentafion, use it to develop better GFP derivatives and signaling reporters, and use the improved methods to understand the causes of cell-to-cell variation in a yeast signaling system. We will also use these methods to develop high-signal reporters that allow quanfificafion of cell signaling in clonal, mammalian cell lines using flow cytometry. Successful work will merit future funding. It should also increase the visibility ofthe Houston lab and help recruit new students and researchers from underrepresented ethnic minorities, at both sites, into an active area of international scientific research.