Exploiting Notch trafficking to probe mechanisms of endosomal sorting and compartmentation.

在真核细胞中，内溶酶体途径是一种囊泡介导的转运系统，在调节膜信号受体的活性方面起着重要作用。它通过去除细胞表面与配体接触的受体，将受体从细胞质中分离出来，引导它们进入内体腔内囊泡，然后将它们递送给溶酶体降解，从而下调信号传导。然而，内吞途径也可以通过将必要的成分聚集在内体周界膜上，进入适当的信号胜任环境或“信号体”，对信号传导产生积极影响。因此，信号成分在不同内体位置的内吞通量和驻留时间可以以不同的方式塑造信号活动的动态。内体由不同的亚结构域组成，以独特的膜和蛋白质组成为标志。然而，对于内体的亚区隔化及其不同结构域之间的运输通量如何与它们在信号调节中的不同生物学作用相关联，或者内体结构的错误调节如何与缺失的信号活动相关联，我们仍然只有有限的理解。本提案的总体目标将是利用我们对Notch（一种至关重要的信号受体）的内溶酶体调控的新理解，作为解决我们知识中这些重要空白的模型。我们最近的工作表明，Notch可以通过两种不同的途径传递到核内体，这些途径的不同尚不清楚，这些核内体结构域以gpi锚定蛋白的富集或不富集为标志，这些蛋白似乎是较大细胞器结构的亚室。Notch可以在这些区室之间的内体区域以及内体周缘膜和腔内囊泡之间进行分类，这使得Notch调控成为探索内溶酶体途径功能区室的理想模型。现在的挑战是更好地理解内溶酶体途径进入其功能调节单元的区隔化，了解这些单元如何在内体成熟的背景下运作，并定义不同内体环境之间的运输如何通过Notch调节和核心内体分选功能的相互作用与Notch信号的调节相关联。果蝇非常适合作为这项研究的起点，它快速而廉价，使我们能够利用非常有效的遗传策略。果蝇研究在建立现代生物学基础方面发挥了核心作用，为细胞生物学、蛋白质运输、细胞信号传导和发育方面的许多重要进展做出了贡献，并揭示了许多对人类健康和疾病至关重要的保守途径。重要的是，果蝇提供了一个独特的实验平台来解剖体内内体调节和Notch信号之间的相互作用。这是很重要的，因为在细胞培养研究之外，很少有内溶酶体区隔的详细功能特征。许多突变可以破坏不同的成分，从而在完整的生物体中识别Notch上的后果。高效基因编辑技术的最新进步意味着新的突变可以通过设计进入果蝇的生殖系，以创建位点定向突变和插入标签，以促进体内的生物成像研究。考虑到人类和苍蝇共享基础生物学的大多数方面，这种策略快速、成本效益高，并且可能具有高度可翻译性。

In eukaryotic cells the endo-lysosomal pathway is a vesicle mediated transport system that plays an important role in modulating the activity of membrane signalling receptors. It down regulates signalling by removing receptors from access to ligands at the cell surface, sequesters them from the cytoplasm by directing them into intraluminal vesicles of the endosomal lumen and then delivers them to the lysosome for degradation. However the endocytic pathway can also have a positive affect on signalling by bringing necessary components together on the endosomal perimeter membrane into an appropriate signal-competent environment or "signalosome". Hence endocytic flux and residency time of signalling components in different endosomal locations can shape the dynamics of signalling activity in different ways. Endosomes are comprised of different subdomains marked by a distinctive membrane and protein composition. However we still have only a limited understanding of how the sub-compartmentalisation of the endosomes, and the trafficking flux between their different domains, is linked to their different biological roles in signalling regulation, or how misregulation of endosomal architecture is linked to abberent signalling activity. The overarching aim of this proposal will be to take advantage of our new understanding of the endo-lysosomal regulation of Notch, a crucially important signalling receptor, as a model to address these important gaps in our knowledge. Our recent work has revealed that Notch can be delivered to endosomes by two distinct routes to different as yet poorly understood, endosomal domains which are marked by enrichment or not for GPI-anchored proteins, which appear to be subcompartments of the larger organelle stuctures. Notch can be sorted in the endosomal domain between these compartments and also between the endosomal perimeter membrane and the intraluminal vesicles, making Notch regulation an ideal model with which to probe the functional compartments of the endolysosomal pathway. The challenge now is to better understand the compartmentalisation of the endolysosomal pathway into its functional regulatory units, understand how these units operate in the context of endosomal maturation and define how trafficking between different endosomal environments is linked to regulation of Notch signalling through the interplay of Notch regulatory and core endosomal sorting functions. Drosophila is uniquely suited as a starting point for this research, being fast and inexpensive, and enabling us to capitalise on very efficient genetic strategies. Drosophila research has played a central role in establishing the foundations of modern biology, contributing to numerous important advances in cell biology, protein trafficking, cell signalling and development, and uncovering many of the conserved pathways that are fundamental to human health and disease. Importantly Drosophila provides a unique experimental platform to dissect the interaction between endosomal regulation and Notch signalling in vivo. This is important because there have been little detailed functional characterisation of endolysosomal compartmentation outside of cell culture studies. Numerous mutations are available that disrupt different components allowing the consequences on Notch to be discerned in the intact organism. The recent improvements in efficient gene editing techniques now mean that new mutations can be engineered into the Drosophila germline by design, to create site directed mutations and insert tags to facilitate bioimaging studies in vivo. This strategy is fast, cost-effective and likely to be highly translatable given that most aspects of fundamental biology are shared between humans and the fly.