Protein Droplets as Catalysts of Coated Vesicle Assembly

蛋白质液滴作为包被囊泡组装的催化剂

基本信息

  • 批准号:
    9925051
  • 负责人:
  • 金额:
    $ 6.53万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2019
  • 资助国家:
    美国
  • 起止时间:
    2019-09-30 至 2022-09-29
  • 项目状态:
    已结题

项目摘要

PROJECT SUMMARY By internalizing cell surface receptors, clathrin-mediated endocytosis enables cellular responses to external cues, regulates nutrient availability, and controls cell signaling. Clathrin-coated pits (CCPs) form at the plasma membrane through the coordinated assembly of dozens of adaptor and coat proteins into a mesh-like network that surrounds a budding vesicle. Within seconds of initiation, nascent CCPs will either mature productively into vesicles or abortively disassemble. What differentiates productive CCPs from their abortive counterparts? It has been suggested that productive CCPs pass through a critical checkpoint, but the checkpoint criteria remain poorly understood. Toward explaining the mechanism that drives CCP progression, preliminary work shows that key CCP initiator proteins, Fcho1 and Eps15, assemble together at membrane surfaces into liquid droplets. The formation of protein droplets via phase separation has recently been shown to provide spatiotemporal control over the catalysis of several pathways including membrane receptor signaling and actin filament nucleation. In a similar way, assembly of a protein droplet at the CCP could function to locally catalyze endocytosis. Fcho1 and Eps15, which are among the earliest factors to arrive at CCPs, recruit other clathrin adaptors and are collectively essential for timely CCP initiation and maturation. Importantly, these initiator proteins bear two hallmarks of phase separating proteins: multivalent interaction motifs and intrinsically disordered regions. Recently I have discovered that Fcho1 and Eps15 assemble into protein liquid droplets at membrane surfaces. This exciting result has the potential to explain both the stochastic assembly of nascent CCPs at discrete endocytic sites and the robust recruitment of a protein network to these sites. Specifically, phase separation of initiator proteins could provide a plausible mechanistic explanation for CCP initiation and maturation. Therefore, the goal of the proposed work is to understand the mechanism by which assembly of the Fcho1/Eps15 initiator proteins contributes to robust endocytosis. Work in Aim 1 will evaluate the biochemical properties of the Fcho1/Eps15 network, testing the working hypothesis that Fcho1 and Eps15 enhance recruitment of each other to the membrane through specific multivalent interactions, and thereby enhance recruitment of downstream binding partners. Work in Aim 2 will evaluate the biophysical properties of the Fcho1/Eps15 network, testing the working hypothesis that the thermodynamic and kinetic properties of Fcho1/Eps15 droplets are consistent with a phase separated system. Work in Aim 3 will evaluate the properties of the Fcho1/Eps15 network in live cells, testing the working hypothesis that the liquid-like behavior of Fcho1/Eps15 is essential for effective catalysis of CCP maturation in cells. The outcome of this research will be a characterization of the presently unknown thermodynamics and kinetics of the CCP initiator complex. More broadly, this work has the potential to introduce a new biophysical paradigm for understanding the roles of protein networks during vesicle formation events throughout the cell.
通过内化细胞表面受体,网格蛋白介导的内吞作用使细胞 对外界信号的反应,调节营养物质的可用性,并控制细胞信号。网格蛋白包被凹坑(CCP) 在质膜上形成,通过几十个衔接子和外壳蛋白的协调组装成一个 围绕着出芽小泡的网状网络。在启动后的几秒钟内,新生的CCP要么成熟, 生产性地进入囊泡或不成功地分解。生产性CCP与流产性CCP的区别是什么? 同行?有人建议,生产CCP通过一个关键的检查点,但检查点 人们对标准仍知之甚少。为了解释推动中共进步的机制,初步 研究表明,关键的CCP起始蛋白Fcho 1和Eps 15在膜表面组装在一起, 液滴通过相分离形成蛋白质液滴最近已经显示出提供了 对包括膜受体信号传导和肌动蛋白在内的几种途径的催化的时空控制 灯丝成核以类似的方式,蛋白质液滴在CCP的组装可以起到局部催化的作用。 内吞作用Fcho 1和Eps 15是最早到达CCP的因子之一,它们招募了其他网格蛋白 它们是适应子,并且对于及时的CCP启动和成熟是共同必需的。重要的是,这些引发剂 蛋白质具有相分离蛋白质的两个特征:多价相互作用基序和内在的 无序区域最近,我发现Fcho 1和Eps 15组装成蛋白质液滴, 膜表面这一令人兴奋的结果有可能解释新生细胞的随机组装, 在离散的内吞位点的CCP和这些位点的蛋白质网络的稳健募集。具体地说, 引发蛋白的相分离可以为CCP引发提供合理的机制解释, 成熟因此,所提出的工作的目标是了解组装的机制, Fcho 1/Eps 15起始蛋白有助于稳健的内吞作用。目标1中的工作将评估生物化学 Fcho 1/Eps 15网络的特性,测试Fcho 1和Eps 15增强的工作假设 通过特定的多价相互作用将彼此募集到膜上,从而增强 下游结合伴侣的募集。目标2中的工作将评估 Fcho 1/Eps 15网络,测试工作假设,即热力学和动力学性质的 Fcho 1/Eps 15液滴与相分离系统一致。目标3中的工作将评估属性 的Fcho 1/Eps 15网络在活细胞中,测试的工作假设,液体样的行为, Fcho 1/Eps 15是有效催化细胞中CCP成熟所必需的。这项研究的结果将是 CCP引发剂络合物的目前未知的热力学和动力学的表征。更 广泛地说,这项工作有可能为理解蛋白质的作用引入一种新的生物物理范式。 在整个细胞中囊泡形成过程中的网络。

项目成果

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Kasey Jill Day其他文献

Kasey Jill Day的其他文献

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{{ truncateString('Kasey Jill Day', 18)}}的其他基金

Protein Droplets as Catalysts of Coated Vesicle Assembly
蛋白质液滴作为包被囊泡组装的催化剂
  • 批准号:
    10244884
  • 财政年份:
    2019
  • 资助金额:
    $ 6.53万
  • 项目类别:
Protein Droplets as Catalysts of Coated Vesicle Assembly
蛋白质液滴作为包被囊泡组装的催化剂
  • 批准号:
    10396786
  • 财政年份:
    2019
  • 资助金额:
    $ 6.53万
  • 项目类别:
Protein Droplets as Catalysts of Coated Vesicle Assembly
蛋白质液滴作为包被囊泡组装的催化剂
  • 批准号:
    9760358
  • 财政年份:
    2019
  • 资助金额:
    $ 6.53万
  • 项目类别:
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