Transport of cell adhesion molecules: How does Rab21 organise integrin trafficking on a mechanistic level?

许多疾病，如癌症进展、炎症和退行性疾病都是细胞粘附和迁移失控的结果。跨膜细胞表面受体整合素家族对肿瘤和免疫细胞的迁移和侵袭能力具有决定性影响。此外，多种病原体利用整合素侵入宿主细胞进行感染，扩展了整合素与传染病的相关性。越来越多的证据表明，整合素的激活/失活以及胞内/胞外循环在调节整合素的功能中起着重要作用。小的ras家族gtp结合蛋白Rab21与α -整合素的细胞质尾部结合，是整合素内吞运输的主要调节因子。然而，在分子或结构水平上，由于缺乏已知的rab21相互作用蛋白，其作用方式仍然知之甚少。拟议研究的目的是深入了解Rab21如何在结构机制层面上组织整合素贩运。为了回答这个问题，我们将实施三个项目。A)参与整合素运输的Rab21效应蛋白的鉴定。新的Rab21下游效应蛋白将使用基于定量质谱的蛋白质相互作用筛选进行鉴定，并测试其对整合素运输的影响。B) rab21整合素贩运网络的结构分析。鉴定后，Rab21下游效应物以及α -整合素的细胞质尾部将与Rab21结合结晶，以获得Rab21调控的整合素运输网络的结构快照。x射线结构将提供对运输复合体功能的见解，并回答Rab21如何管理运输货物（整合素）和并行结合效应蛋白的问题。c) Rab21效应蛋白的功能分析。我将研究已鉴定的效应蛋白的生化和细胞生物学功能，并解决这些效应蛋白如何影响整合素内吞动力学的问题。不同的显微镜技术（共聚焦显微镜，活细胞成像，TIRF）将被采用。在这项拟议的研究中，我将采用多学科方法，从生化筛选开始，结合各种最新技术和尖端的细胞和结构生物学工具，以最大限度地提高我们对整合素如何从质膜运输到内体以及这种运输如何影响不同疾病过程的机制理解。

Numerous diseases such as cancer progression, inflammation and degenerative disorders are results of deregulated cell adhesion and migration. The integrin family of transmembrane cell surface receptors have a decisive impact on the migratory and invasive capacities of cancer and immune cells. Furthermore, a variety of pathogens use integrins to invade host cells for infection, extending the relevance of integrins to infectious diseases. It is becoming increasingly evident that not only activation/deactivation, but also the endo-/exocytic cycle of integrins plays an important role in mediating integrin function. The small Ras-family GTP-binding protein Rab21, which binds to the cytoplasmic tail of alpha-integrins, is a major regulator in the endocytic trafficking of integrins. However, on a molecular or structural level the mode of action remains poorly understood mainly due to the lack of known Rab21-interacting proteins.The aim of the proposed research is to gain insights how Rab21 organises integrin trafficking on a structural-mechanistic level. To answer this question, three projects will be implemented.A) Identification of Rab21 effector proteins involved in integrin trafficking.Novel Rab21 downstream effector proteins will be identified using a quantitative mass spectrometry-based protein-protein interaction screen and tested regarding their influence on integrin trafficking.B) Structural analysis of the Rab21-integrin trafficking network. After their identification, Rab21 downstream effectors as well as the cytoplasmic tail of alpha-integrins will be crystallised in complexes with Rab21 to obtain a structural snapshot of the Rab21-regulated integrin trafficking network. The X-ray structures will provide insights into the function of the trafficking complex and answer the question how Rab21 manages to transport cargo (integrin) and bind effector proteins in parallel.C) Functional analysis of the Rab21 effector proteins. I will investigate the biochemical and cell biological function of the identified effector proteins and address the question how these effector proteins effect the dynamics of integrin endocytosis. Different microscopy techniques (confocal microscopy, live cell imaging, TIRF) will be employed.In this proposed research, I will employ a multidisciplinary approach starting with a biochemical screen and combining various state of the art and cutting edge cell and structural biological tools to maximise our mechanistic understanding of the question how integrins are trafficking from the plasma membrane to endosomes and how this trafficking influences different disease processes.