Flagellar Membrane Signaling Protein Transport Mechanisms: A Single-Molecule Study

人们越来越认识到纤毛/鞭毛(细胞表面的毛状突起)在感知多种生物的细胞外信息方面的重要性。为了了解纤毛/鞭毛在细胞信号转导过程中的作用，在模式生物衣藻中，我们将从鞭毛进入、鞭毛内运动和鞭毛顶端重塑三个部分来研究鞭毛膜信号蛋白的转运机制及其鞭毛内转运机制。对于运输周期的每个部分，本项目将使用定量的单分子荧光成像方法来测试竞争模型：(I)在鞭毛进入区，将确定在细胞质中合成的鞭毛膜信号蛋白是通过扩散进入鞭毛，还是通过IFT机械进入鞭毛。(Ii)在鞭毛内部，该项目旨在阐明IFT载体，并对选定的鞭毛膜信号蛋白的运输动力学进行量化。(Iii)在鞭毛顶端，将确定IFT机械部件是否从微管分离并在逆行过程中重新组装，或者是否在不分离IFT运输机械的情况下进行重新组装。BROADER IMPACTS他的项目是拥有单分子成像方法专业知识的实验室和衣藻(藻类)IFT遗传学实验室之间的多学科联合努力。遗传学和成像生物物理学的这种结合将使鞭毛和单分子成像领域都受益。它将允许将先进的成像工具应用于解决细胞信号转导的基本方面的问题，同时为学生提供在物理和生物方面进行跨学科培训的令人兴奋的机会。特别是，该项目将旨在支持对女研究生和本科生的培训，包括从区域本科院校招收的学生。拟议中的调查的成功将加强成像科学路径成员之间的合作，这是一个生物成像项目，包括华盛顿大学艺术与科学学院、工程学院和医学院的教师。此外，对于更广泛的当地社区，该项目针对圣路易斯地区高中生的实践外展活动旨在吸引年轻人选择科学作为职业。

There is a growing recognition of the importance of cilia/flagella (hair-like projections on cell surfaces) in sensing extracellular information in multiple organisms. In order to understand the role cilia/flagella play in cellular signal transduction processes, the transport mechanisms of flagellar membrane signaling proteins and their intraflagellar transport (IFT) machinery will be examined at three parts of the intraflagellar transport cycle: (i) flagellar entry, (ii) intraflagellar motion, and (iii) flagellar tip remodeling in the model organism Chlamydomonas. For each part of the transport cycle, this project will use quantitative single-molecule fluorescence imaging methods to test competing models: (i) In the flagellar entry region, it will be determined whether flagellar membrane signaling proteins synthesized in the cytoplasm enter the flagella by diffusion or if they are carried into the flagella by IFT machinery. (ii) Inside the flagella, the project aims to elucidate the IFT carrier and quantitate the trafficking kinetics for selected flagellar membrane signaling proteins. (iii) At the flagellar tip, it will be resolved whether the IFT machinery components dissociate from microtubules and each other and reassemble for the retrograde trip, or whether the reassembly occurs without dissociation of the IFT transport machinery.BROADER IMPACTSThis project is a multidisciplinary joint effort between laboratories with expertise in single-molecule imaging method, and Chlamydomonas (algal) IFT genetics. This merger of genetics and imaging biophysics will benefit both the flagellar and single molecule imaging fields. It will allow for the application of advanced imaging tools to questions addressing fundamental aspects of cellular signal transduction, while offering students exciting opportunities to carry out interdisciplinary training in physics and biology. In particular, the project will aim to support the training of female graduate and undergraduate students, including students recruited from regional primarily undergraduate colleges. The success of the proposed investigation will strengthen collaborations among members of the Imaging Sciences Pathway, a biological imaging program that includes faculty from the Schools of Arts & Sciences, Engineering, and Medicine at Washington University. In addition, for the broader local community, the project's hands-on outreach activities to high school students in the St. Louis region are designed to entice young minds to choose science as a career.