Genetic dissection of scaffold protein function in Drosophila

组织的组织和细胞之间的有效交流需要细胞极化--也就是说，细胞一侧的表面在分子上与另一侧不同。有一个复杂的事件层次结构，以细胞表面的某些区域为靶点，然后保留特定的信号或黏附分子。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间的信号传递的。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捆绑在专门的区域中，并将它们与细胞内部骨架联系起来。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改变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结果。我们之前的工作已经确定，Veli在突触前被要求调节有效的递质释放。我们的工作假设是，Veli在突触定位电压依赖通道的支架上发挥作用。我们将使用遗传方法来测试候选突触前蛋白是否需要Veli来有效地靶向和神经肌肉传递。我们将应用酵母-2杂交方法来揭示哪些跨膜蛋白在突触与Veli结合。*PALS2在细胞片中工作，以一种可能抑制肿瘤细胞生长的方式。我们正在将我们对Veli支架的分析扩展到表达PALS2的毛囊细胞。我们的工作假设是Veli和PALS2在上皮细胞中共同作用，定位一个未知的信号复合体。我们将使用遗传嵌合体、RNAi敲除和靶向基因表达来表征Veli和PALS2在该模型上皮细胞中的功能。

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.