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

纤毛/鞭毛（细胞表面的毛发状突起）在多种生物体中感知细胞外信息的重要性越来越受到重视。 为了了解纤毛/鞭毛在细胞信号转导过程中的作用，鞭毛膜信号蛋白的转运机制及其鞭毛内转运（IFT）机制将在鞭毛内转运周期的三个部分进行研究：（i）鞭毛进入，（ii）鞭毛内运动，（iii）鞭毛尖端重塑在模式生物衣原体。 对于运输周期的每个部分，该项目将使用定量单分子荧光成像方法来测试竞争模型：（i）在鞭毛进入区域，将确定在细胞质中合成的鞭毛膜信号蛋白是否通过扩散进入鞭毛，或者它们是否通过IFT机器进入鞭毛。 (ii)在鞭毛内，该项目的目的是阐明IFT载体和定量的选定鞭毛膜信号蛋白的贩运动力学。 (iii)在鞭毛尖端，将解决IFT机械组件是否从微管和彼此分离并重新组装以进行逆行旅行，或者是否在没有IFT运输机械分离的情况下发生重新组装。更广泛的影响本项目是具有单分子成像方法和衣原体（藻类）IFT遗传学专业知识的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合并将有利于鞭毛和单分子成像领域。 它将允许应用先进的成像工具来解决细胞信号转导的基本方面的问题，同时为学生提供令人兴奋的机会，进行物理学和生物学的跨学科培训。 特别是，该项目将致力于支持对女研究生和女本科生的培训，包括从主要是本科生的区域学院招收的学生。拟议调查的成功将加强成像科学途径成员之间的合作，成像科学途径是一个生物成像项目，包括华盛顿大学文理学院、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际推广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.