IDENTIFICATION OF CILIARY MICROTUBULE CAPPING PROTEINS

University Of Washington, Seattle WA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cilia and eukaryotic flagella move cells, distribute growth factors, and transport fluids over epithelial tissues. Cilia also are sensory organelles, involved with chemo, mechano, and photo- sensation. Defects in ciliary assembly or motility have been grouped in a family of ciliopathies, whose symptoms include primary ciliary dyskinesia, situs inversus, hydrocephaly, polycystic liver and kidney disease, classes of retinitis pigmentosa, Bardet-Biedl syndrome, Alstrom syndrome and Meckel-Gruber syndrome. Many of these ciliopathies are due to failures in the assembly of primary cilia, present in one copy/cell and were discovered when genes discovered to be important for the assembly of Chlamydomonas flagella were compared with patients with what are now known to be ciliopathies. Ciliary assembly requires the synthesis and transport of proteins from the cell body into each cilium by intraflagellar transport (IFT). The regulation of IFT activity, the unloading and assembly of ciliary proteins onto microtubules, and regulation of membrane release all occurs at the ciliary tips, at which novel capping structures link the microtubules to the membrane. These caps are the only known structures that link the inner surface of microtubules to membranes and that are exclusively positioned at microtubule ends. Despite their importance, we have no understanding of their composition or function. This project will identify and sequence cap proteins using mass spectrometry. Potential cap protein genes will be tagged with GFP, expressed in Tetrahymena thermophila, and will be identified by GFP fluorescence at ciliary tips. This pilot project will produce at least one tagged cap protein and will serve as a foundation to isolate and characterize these important tip structures found in all known cilia and flagella.