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

细胞表面是一个重要的结构，它定义了细胞内外的边界，并含有帮助细胞与环境相互作用和反应的蛋白质。蛋白质通过胞吐作用（exocytosis）传递到细胞表面，当细胞内化其表面的片段时，蛋白质通过胞吞作用（endocytosis）被移除。平衡这两个事件使细胞能够控制在任何给定时间表面上的蛋白质，并使添加新物质或去除可能有害的受损蛋白质成为可能。本项目结合细胞生物学、分子生物学和遗传学方法，研究存在于许多细胞类型中的胞吞作用途径，称为网格蛋白独立胞吞作用（CIE）。通过这项研究，该项目将为包括本科生和研究生学员以及高中生在内的许多教育水平提供前沿研究和培训机会。一个主要目标是让年轻科学家有机会看到他们在课堂上学到的东西如何应用于膜生物学领域令人兴奋的新发现。该研究项目使用出芽酵母，这是一种简单但功能强大的遗传模式生物，它提供了许多通过进化保守的基本细胞功能的见解，包括胞吐和内吞作用。虽然酵母最初被认为仅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进行的研究揭示了一种新的网格蛋白独立途径的存在。第二种途径需要包括Rho1在内的许多蛋白质，Rho1调节肌动蛋白细胞骨架的聚合并协调酵母细胞壁的修复。Rho1经历激活和失活的循环，其活性可以限制在细胞内的特定部位。Rho1活性和位置之间的关系，以及这些与它在内吞作用中的作用之间的关系，人们知之甚少。因此，本项目将实现几个目标：(1)确定调节局部Rho1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复合物之间的关系；(3)确定在高渗透压条件下通过不依赖网格蛋白的内吞作用内化的货物，已知这些货物促进了这一途径；(4)研究渗透应激反应途径在不依赖网格蛋白的胞吞作用中的作用；(5)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，鉴定促进货物内化的基因，以确定是否存在其他途径。总的来说，这项研究的结果将为控制网格蛋白独立内吞作用的分子机制提供新的见解，这是所有真核细胞中一个重要但知之甚少的过程。使用酵母等简单的模式生物可以快速识别这些途径的关键成分，这反过来又可以更好地了解包括人类在内的其他生物中的相关途径。该奖项反映了美国国家科学基金会的法定使命，并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。

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.