Establishing a common function for ferlin proteins in membrane fusion using novel genetic code expansion and single molecule techniques.

膜运输被认为是真核细胞的一个重要而独特的特征，细胞内运输和分泌事件在稳态和信号传导中起着至关重要的作用。虽然我们对膜转运的了解大多来自于对酵母的研究，但在多细胞生物中，有一些重要的蛋白质参与了膜转运，而在酵母中却没有发现。这些蛋白质中的一个家族是ferlins，这是一个进化上古老的运输蛋白质家族，与越来越多样化的生理活动有关，包括生育、声音编码、肌肉发育和受损细胞膜的修复。这种生理作用的多样性使得为这个家族建立一个共同的潜在分子功能具有挑战性。这个项目将填补我们对Ferlin家族蛋白质知识的空白，从而增加我们对生育、听力和肌肉发育的理解。这项工作还将为本科生提供以膜生物学为重点的实验室研究培训机会和实验课，并为研究生提供最先进的跨学科研究培训。该项目验证了一种观点，即ferlins具有钙敏感支架的共同功能，用于调节胞吐和内吞作用。由于蛋白质的大尺寸，它们的跨膜结构域和ferlins典型的大量内源性半胱氨酸的挑战，使得许多传统的重组蛋白分析无法用于测试这种假设的功能。为了克服这些挑战，一种新的单分子蛋白质-蛋白质相互作用试验已经开发出来，可以探测大的多价膜蛋白的接触。此外，遗传密码扩展技术将被用于将环境敏感的荧光非天然氨基酸纳入ferlins。这些技术将被用来确定费林活动背后的功能。拟议的研究将定义一组潜在的分子水平功能，这些功能将ferlins联合起来，尽管它们的生理作用不同。这些信息将使ferlins整合到膜运输和细胞信号的更大图景中。新型单分子荧光技术的发展将为研究大型多结构域膜蛋白提供一种新的方法，从而在ferlins的研究之外具有应用价值。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Membrane trafficking is regarded as a crucial and distinctive characteristic of eukaryotic cells, with both intracellular transport and secretion events playing an essential role in homeostasis and signaling. While much of what is known about membrane trafficking comes from studies in yeast, there are important proteins involved in membrane trafficking in multicellular organisms not seen in yeast. One family of these proteins are the ferlins, an evolutionarily ancient family of trafficking proteins linked to an increasingly diverse list of physiological activities, including fertility, the encoding of sound, muscle development, and repair of damaged cell membranes. This diversity of physiological roles has made establishing a common underlying molecular function for this family challenging. This project will address this gap in our knowledge of the Ferlin family proteins, and in so doing add to our understanding of fertility, hearing, and muscle development. The proposed work will also develop undergraduate laboratory research training opportunities and lab classes focused on membrane biology and provide graduate students with state-of-the-art training in interdisciplinary research.The project tests the idea that ferlins share a common function as calcium sensitive scaffolds for regulated exocytosis and endocytosis. Challenges due to the large size of the proteins, their transmembrane domains, and the high number of endogenous cysteines typical of ferlins have precluded many of the traditional recombinant protein assays typically used to test this hypothetical function. To overcome these challenges a novel single molecule protein-protein interaction assay that allows for probing the contacts of large multivalent membrane proteins has been developed. In addition, genetic code expansion techniques will be exploited to incorporate environmentally sensitive fluorescent unnatural amino acids into the ferlins. These technologies will be exploited to determine the functions that underlie ferlin activity. The proposed studies will define a set of underlying molecular-level functions that unite the ferlins, despite their disparate physiological roles. This information will allow integrating ferlins into the larger picture of membrane trafficking and cell signaling. The development of a novel single molecule fluorescence technique will provide a method for the study of large multi-domain membrane proteins and thus have applications beyond the study of ferlins.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.