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

具体目标(摘要)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，这项工作建立在确定荧光的“频域”方法上 在显微镜下的寿命，以及在“频域”方法方面的更先进的发展 流式细胞术，休斯顿博士正在成为这一领域的领导者。 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。 尽管人们对细胞信号转导知道很多，但关键问题，包括细胞死亡的原因和后果-- 细胞在信号和反应中的变异，仍然没有答案。在这里，我们将使用荧光救生方法 为了扩展单细胞分析的能力，以量化酵母细胞信号中的关键事件(“发展 平台“)和哺乳动物细胞。这些测量依赖于绿色荧光蛋白的使用 (GFP)衍生物与组成不同细胞信号通路的蛋白质融合以量化事件 例如将信号复合体募集到细胞膜上。 在接下来的三年里，我们将发展我们的方法，以解决这些问题，并允许更广泛的 申请。特别是，我们将局限于改进我们的仪器，利用它来开发更好的GFP 导数和信号报告，并使用改进的方法了解细胞到细胞的原因 酵母信号系统的变异。我们还将利用这些方法来发展高信号记者， 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半定量检测。成功的工作将 值得未来的资金支持。它还应该提高休斯顿实验室的知名度，并帮助招收新学生和 在这两个地点，来自代表性不足的少数民族的研究人员进入了一个活跃的国际领域 科学研究。

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.