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并对环境做出反应的蛋白质。蛋白质通过一种称为胞吞作用的过程被输送到表面，当细胞在内吞作用中将其表面的部分内化时，蛋白质被移除。平衡这两个事件可以让细胞在任何给定的时间控制哪些蛋白质在表面，并使添加新材料或移除可能有害的受损蛋白质成为可能。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人们知之甚少的内吞作用途径，称为网状蛋白非依赖性内吞作用(CIE)，它存在于许多细胞类型中。通过这项研究，该项目将在多个教育层次提供前沿研究和培训机会，包括本科生和研究生以及高中生。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的新的令人兴奋的发现。这个研究项目使用发芽酵母，这是一种简单但极其强大的遗传模型生物，它提供了对许多基本细胞功能的洞察，这些功能在进化中是保守的，包括胞吐和内吞。虽然酵母最初被认为只使用笼状蛋白介导的内吞作用，但在该途径被阻断的突变菌株中的研究揭示了存在一种新的不依赖于笼状蛋白的途径。第二条途径需要包括Rho1在内的大量蛋白质，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经历了激活和失活的循环，其活性可以被限制在细胞内的特定位置。Rho1活性和位置之间的关系，以及它们与其在内吞作用中的作用之间的关系，目前还知之甚少。因此，该项目将实现以下几个目标：(1)确定调节局部Rho1激活和失活的蛋白质调节胞吞作用的能力；(2)研究调节胞吐和胞吞作用的蛋白质复合体之间的关系；(3)识别在高渗透压条件下被胞苷不依赖的胞吞作用所内化的货物；(4)研究渗透胁迫反应途径在胞吞作用中的作用；以及(5)确定在缺乏胞苷介导的胞吞作用和已知的胞吞作用途径的酵母突变株中促进货物内化的基因，以确定是否存在其他途径。总体而言，这项研究的结果将为控制网状蛋白非依赖性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鲜为人知的过程。使用简单的模式生物，如酵母，可以快速识别这些途径的关键组成部分，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。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.