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

Washington University, Saint Louis MO

Investigators

Abstract

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 IMPACTS This 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.