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CAREER: Regulation of clathrin-independent endocytosis by modulators of Rho GTPase function

职业：Rho GTPase 功能调节剂调节网格蛋白独立的内吞作用

基本信息

批准号：
1942395
负责人：
Derek Prosser
金额：
$ 127.76万
依托单位：
Virginia Commonwealth University
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1942395&HistoricalAwards=false
关键词：
CAREER Regulation clathrin independent endocytosis

项目摘要

The cell surface is an important structure that defines the boundary between the inside and outside of the cell and contains proteins that help the cell to interact with and respond to its environment. Proteins are delivered to the surface using a process called exocytosis and are removed when a cell internalizes pieces of its surface in a process known as endocytosis. Balancing these two events allows the cell to control which proteins are on the surface at any given time and makes it possible to add new material or remove damaged proteins that may be harmful. This project combines cell biology, molecular biology and genetic approaches to study poorly understood pathways for endocytosis, termed clathrin-independent endocytosis (CIE), that are present in many cell types. Through this research, this project will provide cutting-edge research and training opportunities at many levels of education, including undergraduate and graduate trainees as well as high-school students. A major goal is to give young scientists the opportunity to see how the things they learn in the classroom can be applied to make new and exciting discoveries in the field of membrane biology.This research project uses budding yeast, a simple yet extremely powerful genetic model organism that has provided insight into many basic cellular functions that are conserved through evolution, including exocytosis and endocytosis. Although yeast was originally thought to use only clathrin-mediated endocytosis, studies in a mutant strain where this pathway was blocked revealed the existence of a new clathrin-independent pathway. This second pathway requires numerous proteins including Rho1, which regulates polymerization of the actin cytoskeleton and coordinates repair of the yeast cell wall. Rho1 undergoes cycles of activation and inactivation, and its activity can be restricted to specific sites within a cell. The relationship between Rho1 activity and location, and how these relate to its role in endocytosis, are poorly understood. Thus, this project will achieve several goals: (1) define the ability of proteins that regulate localized Rho1 activation and inactivation to regulate clathrin-independent endocytosis; (2) examine relationships between protein complexes that regulate exocytosis and endocytosis; (3) identify cargos that are internalized by clathrin-independent endocytosis under high osmolarity conditions that are known to facilitate this pathway; (4) examine the role of the osmotic stress response pathway in clathrin-independent endocytosis; and (5) identify genes that promote cargo internalization in mutant yeast strains lacking both clathrin-mediated endocytosis and the known clathrin-independent pathway to determine if additional pathways exist. Overall, the results of this research will provide new insights into the molecular machinery that controls clathrin-independent endocytosis, an important but poorly understood process in all eukaryotic cells. Using a simple model organism such as yeast makes it possible to rapidly identify the key components of these pathways, which in turn will make it possible to better understand related pathways in other organisms, including humans.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.

细胞表面是一个重要的结构，它定义了细胞内外之间的边界，并含有帮助细胞与环境相互作用和响应的蛋白质。蛋白质通过一种称为胞吐作用的过程被传递到表面，当细胞在一种称为胞吞作用的过程中内化其表面的碎片时被去除。平衡这两个事件使细胞能够控制在任何给定时间在表面上的蛋白质，并可以添加新的材料或去除可能有害的受损蛋白质。该项目结合了细胞生物学，分子生物学和遗传学方法来研究在许多细胞类型中存在的内吞作用（称为网格蛋白独立内吞作用（CIE））的知之甚少的途径。通过这项研究，该项目将在许多教育层次提供尖端的研究和培训机会，包括本科生和研究生学员以及高中生。本研究的主要目的是让年轻科学家有机会了解他们在课堂上学到的知识如何应用于膜生物学领域，从而获得令人兴奋的新发现。本研究项目使用了芽殖酵母，这是一种简单但非常强大的遗传模式生物，它提供了对许多在进化过程中保守的基本细胞功能的深入了解，包括胞吐作用和胞吞作用。虽然酵母最初被认为只使用网格蛋白介导的内吞作用，但在该途径被阻断的突变株中的研究揭示了一种新的网格蛋白非依赖性途径的存在。第二种途径需要许多蛋白质，包括Rho 1，它调节肌动蛋白细胞骨架的聚合并协调酵母细胞壁的修复。Rho 1经历激活和失活的循环，其活性可以限制在细胞内的特定位点。Rho 1活性和位置之间的关系，以及这些如何与其在胞吞作用中的作用，知之甚少。因此，本项目将实现以下几个目标：（1）确定调节局部Rho 1激活和失活的蛋白质调节网格蛋白非依赖性内吞作用的能力;（2）研究调节胞吐和内吞作用的蛋白质复合物之间的关系;（3）鉴定在已知促进该途径的高渗透压条件下由网格蛋白非依赖性内吞作用内化的货物;（4）检查渗透应激反应途径在网格蛋白非依赖性内吞作用中的作用;和（5）鉴定在缺乏网格蛋白介导的内吞作用和已知的网格蛋白非依赖性途径的突变酵母菌株中促进货物内化的基因，以确定是否存在另外的途径。总的来说，这项研究的结果将提供新的见解的分子机制，控制网格蛋白独立的内吞作用，一个重要的，但在所有真核细胞了解甚少的过程。利用酵母等简单的模式生物，可以快速确定这些途径的关键成分，进而可以更好地了解包括人类在内的其他生物的相关途径。该奖项反映了NSF的法定使命，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。