Polpeptides of the Flagellar Tip Complex

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DESCRIPTION (provided by applicant): Intraflagellar transport (IFT) is a process that is fundamental to the proper function of many cell types in the body, among them cells of the kidney where it is required for the assembly and maintenance of the primary cilia of the cells lining the nephron. Defects in the components of these primary cilia and defects in the machinery of IFT have been shown to cause polycystic kidney disease. The long term goals of this new project are to identify and characterize proteins of the flagellar tip complex and learn how they are involved in controlling particle movement during IFT. Why direct these studies to the flagellar tip? The microtubules (MTs) of the flagellar axoneme assemble and continuously turn over at the flagellar tip. The supply to and removal from the flagella of IFT components requires two motors: the MT-based motor protein kinesin-ll moves cargo from the base to the tip, and cytoplasmic dynein 1b moves cargo from the tip back to the base. Important activities relevant to the proper functioning of IFT occur at the flagellar tip, and these include MT assembly and disassembly, motor protein regulation, and cargo loading and unloading. Because an abrupt change in the direction of particle movement occurs only at the flagellar tip, the motor proteins must be regulated at the tip. Kinesin must be down regulated or turned off, and cytoplasmic dynein must be upregulated or turned on. The mechanisms used to control motor protein regulation and cargo unloading at the tip are unknown, but it is likely that a complex of proteins restricted to the flagellar tip, herein referred to as the flagellar tip complex (FTC), plays a definitive role in these processes. To test this hypothesis, three aims are proposed here: Having identified a polypeptide, CrEB1, that is localized at the flagellar tip. I will use rEB1 as a hook to fish out and identify other FTC proteins that are specific to the flagellar tip. The second aim will employ different approaches to identify structural and enzymatic components of the FTC that do not depend on an interaction with CrEBl. The third aim will characterize the FTC proteins identified via the first two aims and determine their function in IFT. Understanding more about the process of IFT is very important, as recent studies have shown that IFT defects cause polycystic kidney disease specifically, and defects in primary cilia are associated with a range of human disorders in addition to cystic diseases of the kidney, including retinal degeneration, obesity, hypertension, and diabetes, etc.