The flagellar membrane contains a unique set of signaling membrane proteins which the plasma membranes lack. While these proteins play a vital role in the life processes of the cell, it is currently unknown exactly how these proteins, which are synthesized in the cytoplasm, enter the flagella across diffusion barriers at the flagellar base. Further, once these proteins have entered the flagellum they depend heavily on the intraflagellar transport (IFT) machinery of the flagellum to complete necessary cellular signaling pathways. In order to better understand these essential signaling pathways, we employ single-molecule fluorescence imaging methods to investigate the intraflagellar trafficking dynamics of Pkd2-GFP, an important flagellar signaling protein responsible for polycystic kidney disease. Specifically, we use total internal reflection fluorescence microscopy (TIRF microscopy) combined with numerous techniques developed in our group, including single image molecula r analysis (SIMA), to explore the dynamics of the membrane signaling proteins at the flagellar entry region and the flagellar tip. Using these techniques, we aim to uncover the physics of the signaling pathway machinery and address any possible failures thereof.
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