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Membrane shape transition control in cellular membrane trafficking phenomena

细胞膜运输现象中的膜形状转变控制

基本信息

批准号：
10798657
负责人：
Tobias Baumgart
金额：
$ 8.21万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2024-08-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY Dynamic changes in membrane shape are at the heart of cellular membrane trafficking phenomena such as endocytosis, where intracellular transport vehicles form from plasma membrane invaginations. Progress in research aimed to mechanistically understand endocytosis has been challenged in part by the existence of several different endocytic pathways. Clathrin-mediated endocytosis is the best characterized internalization route – it is responsible for the bulk of endocytic trafficking and is comparatively slow. Alternative pathways enable cells to rapidly respond to signals at the plasma membrane. A recently discovered pathway achieves fast responsiveness trough the assembly of transient complexes containing endophilin: a BAR domain protein, and lamellipodin: a multivalent adaptor protein. In this process termed fast endophilin-mediated endocytosis (FEME), dynamic complexes are formed through multivalent interactions that are stabilized through interactions with activated receptors, including members of the receptor tyrosine kinase (RTK), and G-protein coupled receptor (GPCR) classes. Whereas RTKs interact with endophilin only indirectly, involving additional adaptor proteins, several GPCRs show direct endophilin interactions, mediated by the receptors’ third intracellular loop (TIL), which becomes exposed upon receptor engagement by ligand. The combination of GPCR, endophilin, and lamellipodin, therefore represents an ideal system upon which to build an in-depth biophysical description of the mechanisms behind receptor internalization. Our goal is to investigate such mechanisms with the help of model membranes consisting primarily of giant unilamellar vesicles (GUVs) which allow the study of membrane shape transitions under precise control of membrane tension, using techniques that we have developed and refined over the course of this project. A second challenge towards a complete mechanistic understanding of plasma membrane internalization transitions is that membrane shape changes involve interactions in several different layers, all of which must receive due attention. We address this challenge in three aims that each are focused on a single layer, roughly defined by their distance from the membrane. The first aim furthers the understanding of mechanisms that determine the spontaneous bending preference (spontaneous curvature) of the bilayer itself – a prerequisite for rigorous design of the following two aims. A second aim asks how TIL binding modulates endophilin function on the membrane. The third aim considers multivalent interactions occurring distal from the membrane. Specifically, we will test the hypothesis that multivalent interactions involving endophilin’s SH3 domain and lamellipodin’s proline-rich domains, could give rise to critical density fluctuations near a protein-protein (demixing) phase transition, that couples with membrane shape transitions after stabilization through receptor engagement. We believe that findings from this project could translate to the understanding of additional membrane trafficking phenomena involving cooperative interactions under healthy and pathological conditions.

项目摘要膜形状的动态变化是细胞膜运输现象的核心，如胞吞作用，其中细胞内运输媒介物由质膜内陷形成。进展旨在从机理上理解内吞作用的研究受到了部分挑战，几种不同的内吞途径。网格蛋白介导的内吞作用是最具特征的内化途径----它负责大部分内吞转运，并且相对缓慢。替代途径使细胞能够迅速响应质膜上的信号。最近发现的一种途径通过含有内亲和素的瞬时复合物的组装的快速反应性：一种BAR结构域蛋白，和lamellipodin：一种多价衔接蛋白。这个过程称为快速内嗜蛋白介导的内吞作用（FEME），动态复合物通过多价相互作用形成，所述多价相互作用通过与活化受体的相互作用，包括受体酪氨酸激酶（RTK）和G蛋白偶联受体（GPCR）类。而RTKs仅间接地与内啡肽相互作用，衔接蛋白，几个GPCR显示直接的亲内蛋白相互作用，介导的受体的第三个细胞内环（TIL），其在受体与配体接合时暴露。的组合因此，GPCR、内啡肽和片状脂质蛋白代表了一种理想的系统，在此基础上可以建立一个深入的研究。受体内化机制的生物物理学描述。我们的目标是研究这种机制的帮助下，模型膜主要由巨大的单层囊泡（GUV），其允许在精确控制下研究膜形状转变，膜张力，使用我们在这个项目中开发和完善的技术。第二个挑战是对质膜内化的完整机制的理解过渡是膜形状的变化涉及几个不同层的相互作用，所有这些都必须得到应有的重视。我们通过三个目标来应对这一挑战，每个目标都集中在一个层面上，由它们与膜的距离来定义。第一个目标是进一步了解确定双层本身的自发弯曲偏好（自发曲率）-这是严格设计以下两个目标。第二个目标是询问TIL结合如何调节内啡肽功能在膜上。第三个目标考虑发生在膜远端的多价相互作用。具体地说，我们将测试的假设，多价相互作用涉及内亲蛋白的SH 3结构域和 lamellipodin的富含脯氨酸的结构域，可以引起蛋白质-蛋白质附近的临界密度波动，（分层）相变，通过受体稳定后与膜形状转变耦合订婚我们相信，从这个项目的结果可以转化为额外的理解，涉及健康和病理条件下的协同相互作用的膜运输现象。