Development Project 4

我们将使杰西卡·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全面运作。我们提议的工作建立在两个实验室优势互补的基础上：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，这项工作的基础是在显微镜下确定荧光寿命的“频域”方法，以及在流式细胞术的“频域”方法方面取得的更先进的进展，休斯顿博士正在成为这方面的领导者。 导致细胞不适当增殖的细胞信号功能障碍导致了大多数癌症的发生。尽管对细胞信号转导有很多了解，但关键问题，包括细胞间信号转导和反应差异的原因和后果，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分析的能力，以量化酵母(“发育平台”)和哺乳动物细胞中细胞信号中的关键事件。这些测量依赖于绿色荧光蛋白(GFP)衍生品的使用，这些衍生品融合到组成不同细胞信号通路的蛋白质上，以量化诸如信号复合体向细胞膜募集等事件。 在接下来的三年里，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，利用它来开发更好的GFP衍生物和信号报告程序，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程序，允许使用流式细胞术对克隆的哺乳动物细胞系中的细胞信号进行量化。成功的工作将值得未来的资助。它还应该提高休斯顿实验室的知名度，并帮助招收来自未被充分代表的少数民族的新学生和研究人员，进入一个活跃的国际科学研究领域。

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.