CAREER: Exploring the Functional Diversity of Cytoplasmic Dynein

该奖项全部或部分由《2021年美国救援计划法案》（公法117-2）资助。细胞是所有生物体的基本组成部分。所有活细胞都必须能够迅速重组其内容物以正常运作。为了促进细胞内运动，运动蛋白在真核细胞内运输细胞物质。一些马达沿着被称为微管的轨道运动，微管起着细胞高速公路的作用。几乎所有必须在细胞内移动的东西都是由运动蛋白运输的，从较小的蛋白质到像线粒体这样的细胞器。如果运动蛋白的功能受损，细胞就会死亡或失灵。值得注意的是，只有一种叫做动力蛋白的马达能够长距离地将货物运送到细胞内部。作为唯一的这样的马达，动力蛋白必须能够结合和运输许多不同类型的细胞货物，即使每种货物可能需要以不同的速度移动，在大小上有很大的不同，或者只在特定的时间移动。第二组蛋白质，称为接头，与动力蛋白结合，使其能够运输不同类型的货物。本提案的目标是了解不同的接头蛋白如何使动力蛋白马达在不同时间以不同速度移动不同大小的货物。预期的结果将揭示接头蛋白如何改变动力蛋白的特征以显示不同的运动特性。该研究项目还将培训本科生和研究生在显微镜和生物物理技术方面的知识，并通过舞蹈和戏剧等非传统方式将实验结果传达给广泛而多样化的公众。与密歇根大学自然历史博物馆的合作还将为初高中学生开发动手研究驱动的项目，以了解荧光显微镜和运动蛋白。这个项目的研究将解决一类细胞蛋白，称为适配器，如何使动力蛋白运输不同的货物。初步研究表明，个体的接头蛋白赋予哺乳动物动力蛋白-1马达不同的运动特性。在这一发现的基础上，研究人员将：1)确定为什么不同的适配器对动力蛋白运动有不同的影响；2)确定每个适配器如何调整动力马达组的活动；3)确定每个适配器如何专门调节dynein电机的激活。为了实现这些目标，研究人员将使用单分子荧光成像、蛋白质生物化学、体外复合体重组来在原生样货物上生成马达组，以及微管和溶液中动力蛋白激活的实时荧光显微镜可视化。总之，本提案的结果将提供对衔接蛋白如何增加动力蛋白马达的功能可塑性的深入理解，并促进对整个细胞中运输如何协调的整体理解。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。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).Cells are the building blocks of all organisms. All living cells must be able to reorganize their contents rapidly to function properly. To promote intracellular movements, motor proteins transport cellular material throughout the eukaryotic cell. Some motors move along tracks called microtubules that function as cellular highways. Nearly everything that must move around the cell is transported by motor proteins, from smaller proteins to organelles like mitochondria. If motor proteins’ functions are impaired, cells can die or malfunction. Remarkably, only one type of motor, called dynein, transports cargo towards the interior of the cell for long distances. As the only such motor, dynein must be able to bind and transport many different types of cellular cargo, even though each kind of cargo may need to move at different speeds, be vastly different in size, or move only at specific times. A second set of proteins, called adaptors, bind to dynein and enable it to transport different types of cargos. The goal of this proposal is to understand how different adaptor proteins enable dynein motors to move cargos of different sizes at different speeds at different times. The expected results will reveal how adaptor proteins alter the characteristics of dynein to display different motile properties. This research project will also train both undergraduate and graduate students in microscopy and biophysical techniques, and to communicating experimental findings to a broad and diverse public audience using non-traditional approaches like dance and theater. A collaboration in conjunction with the University of Michigan’s Museum of Natural History will also develop hands-on, research-driven programs for middle and high-school-aged students to learn about fluorescence microscopy and motor proteins.The research of this project will address how a class of cellular proteins, called adaptors, enable dynein to traffic diverse cargoes. Preliminary work has revealed that individual adaptor proteins confer distinct motile properties to the mammalian dynein-1 motor. To build on this finding, the investigators will: 1) Determine why different adaptors have distinct effects on dynein motility; 2) Determine how each adaptor tunes the activity of teams of dynein motors; 3) Determine how each adaptor specifically modulates activation of the dynein motor. To accomplish these goals, the investigators will use single-molecule fluorescence imaging, protein biochemistry, complex in vitro reconstitutions to generate teams of motors on a native-like cargo, and real-time fluorescence microscopic visualization of dynein activation on microtubules and in solution. Together, the results of this proposal will provide an in-depth understanding of how adaptor proteins increase the functional plasticity of the dynein motor, and increase progress towards a holistic understanding of how trafficking is coordinated across the entire cell.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.