Genetic dissection of scaffold protein function in Drosophila

果蝇支架蛋白功能的遗传解析

• 批准号: 46651-2006
• 负责人: Jacobs, Roger
• 金额: $ 2.19万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2006
• 资助国家: 加拿大
• 起止时间: 2006-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=337378
• 关键词: Genetic; dissection; scaffold; protein; function

The organisation of tissues, and effective communication between cells requires that cells become polarised - that is, the cell surface on one side is molecularly different from the other. There is a complicated hierarchy of events that target and then retain specialised signaling or adhesion molecules to certain domains of the cell surface. We seek to understand how this polarisation is maintained, and how it regulates signaling between cells.   Our research program examines two scaffolding proteins, Veli(LIN7) and "Protein Associated with Lin7"(PALS2), that tether cell surface molecules in specialised domains, and link them to the cell's internal skeleton. We are using the genetic model organism, the fruitfly, to manipulate the function of these two proteins, and determine how they perform their function in localising cell surface proteins. We apply genetic approaches to modify protein structure or expression in subsets of cells, and then monitor the consequences in cell morphology and function.   Our previous work has established that Veli is required presynaptically to regulate effective transmitter release. Our working hypothesis is that Veli acts in a scaffold that localises voltage dependent channels at the synapse. We will use a genetic approach to test candidate presynaptic  proteins for a requirement of Veli for efficient targeting and neuromuscular transmission.  We will apply a yeast-2-hybrid approach to uncover which transmembrane proteins bind to Veli at the synapse.   PALS2 works in sheets of cells, in a way that may suppress the growth of cells in tumours. We are extending our analysis of the Veli scaffold to follicle cells, where PALS2 is expressed. Our working hypothesis is that Veli and PALS2 work together in epithelia to localize an  unidentified signaling complex. We will use genetic mosaics, RNAi knockdown and targeted gene expression to characterize the function of Veli and PALS2 in this model epithelium.

组织的组织和细胞之间的有效交流需要细胞变得极化——也就是说，一边的细胞表面与另一边的细胞表面在分子上不同。有一个复杂的层次结构的事件，目标，然后保留专门的信号或粘附分子到细胞表面的某些区域。我们试图了解这种极化是如何维持的，以及它是如何调节细胞之间的信号传导的。我们的研究项目检查了两种支架蛋白，Veli（LIN7）和“与LIN7相关的蛋白”（PALS2），它们将细胞表面分子系在特定区域，并将它们连接到细胞的内部骨架。我们正在使用遗传模式生物果蝇来操纵这两种蛋白质的功能，并确定它们如何在定位细胞表面蛋白质方面发挥作用。我们应用遗传方法来修饰细胞亚群中的蛋白质结构或表达，然后监测细胞形态和功能的后果。我们之前的工作已经确定，Veli需要突触前调节有效的递质释放。我们的工作假设是，Veli在一个支架中起作用，该支架定位了突触上的电压依赖通道。我们将使用遗传方法测试候选突触前蛋白，以满足Veli有效靶向和神经肌肉传递的要求。我们将采用酵母-2杂交方法来发现哪些跨膜蛋白在突触上与Veli结合。PALS2在细胞片中起作用，在某种程度上可能抑制肿瘤细胞的生长。我们正在将Veli支架的分析扩展到PALS2表达的卵泡细胞。我们的工作假设是，Veli和PALS2在上皮中共同作用，定位一种未知的信号复合物。我们将使用遗传镶嵌、RNAi敲除和靶向基因表达来表征Veli和PALS2在该模型上皮中的功能。