Development Project 4

我们将使Jessica P. Houston博士（NMSU）和Roger布伦特博士（FHCRC）的实验室之间的持续试点合作全面运作。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”方法的基础上，以确定显微镜中的荧光寿命，并在“频域”方法的流式细胞术中取得了更先进的发展，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。尽管人们对细胞信号传导了解很多，但关键问题，包括细胞间信号传导和反应变异的原因和后果，仍然没有答案。在这里，我们将使用荧光寿命方法来扩展单细胞测定的能力，以量化酵母（“开发平台”）和哺乳动物细胞中细胞信号传导的关键事件。这些测量依赖于使用与构成不同细胞信号传导途径的蛋白质融合的绿色荧光蛋白（GFP）衍生物来量化事件，例如信号传导复合物向细胞膜的募集。 在未来三年内，我们将开发我们的方法，以解决这些问题，并允许更广泛的应用。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酵母信号系统中细胞间变异的原因。我们还将使用这些方法来开发高信号报告，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。成功的工作将值得今后提供资金。它还应该提高休斯顿实验室的知名度，并帮助招募来自代表性不足的少数民族的新学生和研究人员进入国际科学研究的活跃领域。

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 significantly 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 questions, including the causes and consequences of cell-to-cell variation in signaling and response, remain unanswered. Here, we will use fluorescent lifetime 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 questions and to allow wider applications. In particular, we will continue to improve our instrumentation, 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 quantification of cell signaling in clonal, mammalian cell lines using flow cytometry. Successful work will merit future funding. It should also increase the visibility of the Houston lab and help recruit new students and researchers from underrepresented ethnic minorities into an active area of international scientific research.