Temporal and spatial interaction patterns of bZIP proteins in living C. elegans

蛋白质之间的相互作用在调节和执行所有细胞过程中起着关键作用。尽管对全基因组相互作用网络的研究可以提供线索，以预测每种蛋白质如何通过蛋白质-蛋白质相互作用在细胞中发挥作用，但要证明已确定的相互作用是否具有生物学意义以及这些相互作用在体内是如何在时间和空间上调节的，仍然是一个挑战。该项目的主要目标是使用一种新的双分子荧光互补(BIFC)方法来研究线虫体内蛋白质-蛋白质相互作用在整个发育过程中是如何在时间和空间上进行调节的。该检测的原理是将黄色荧光蛋白(YFP)的一半融合到两种蛋白中的每一种，并在活着的蠕虫中共同表达。这两种融合蛋白之间的任何相互作用都会使两部分非荧光片段接近，并重新构建完整的YFP，从而允许直接观察它们在整个发育过程中的相互作用。具体地说，第一个目标是使用哺乳动物和果蝇Fos和Jun转录因子的两个同源物Ce-Fos-1和Ce-Jun-1作为模型系统，建立一种在活体蠕虫中显示它们的时间和空间相互作用的方法。第二个目标是寻找Ce-FOS-1的其他相互作用伙伴，并研究Ce-FOS-1与其他bZIP蛋白(碱性区域亮氨酸拉链家族转录因子)的时空相互作用模式。第三个目标是使用RNAi和其他遗传方法来确定这些相互作用的功能丧失表型，并将这些表型与时间和空间相互作用模式相关联。最终，将分析所有bZIP蛋白的时间和空间相互作用模式。经过验证的系统将允许研究蠕虫和其他生物体中其他蛋白质家族的时间和空间相互作用。该项目将涉及本科生和研究生的培训，建立的系统将允许生物学学生直接可视化bZIP蛋白质如何相互作用调节特定的发育事件。

Protein-protein interactions play a pivotal role in regulating and executing all cellular processes. Although studies of a genome-wide interaction network can provide clues to predict how each protein might function in cells through protein-protein interactions, it remains a challenge to prove whether the identified interactions are biologically relevant and how the interactions are temporally and spatially regulated in vivo. The major objective of this project is to use a novel bimolecular fluorescence complementation (BiFC) approach to investigate how protein-protein interactions are temporally and spatially regulated throughout development in the living nematode worm, C. elegans. The principle of the assay is to fuse one half of a yellow fluorescent protein (YFP) to each of two proteins and co-express them in living worms. Any interaction between the two fusion proteins brings two halves of non-fluorescent fragments in proximity and reconstitutes an intact YFP, allowing direct visualization of their interactions throughout development. Specifically, the first objective is to use Ce-FOS-1 and Ce-JUN-1, two homologs of mammalian and Drosophila Fos and Jun transcription factors which function as a heterodimer, as a model system to establish an assay for the visualization of their temporal and spatial interactions in living worms. The second objective is to identify other interaction partners of Ce-FOS-1 and examine the temporal and spatial interaction patterns of Ce-FOS-1 with other bZIP proteins (basic region leucine zipper family transcription factors). The third objective is to use RNAi and other genetic approaches to determine the loss-of-function phenotypes of these interactions and correlate the phenotypes to the temporal and spatial interaction patterns. Ultimately, the temporal and spatial interaction patterns of all bZIP proteins will be analyzed. The proven system will allow the study of temporal and spatial interactions of other families of proteins in worms and in other organisms as well. This project will involve the training of undergraduate and graduate students, and the established system will allow biology students to directly visualize how interactions of bZIP proteins regulate specific developmental events.