IDENTIFICATION OF MAMMALIAN INTRAFLAGELLAR TRANSPORT COMPLEXES FOR PRIMARY CILI

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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. Primary cilia play an important, but as yet, undefined role in a wide variety of developmental and pathological processes. Central to these functions is the ability to polarize a cell and produce the microtubule-based projection. In the green algae, Chlamydomonas Reinhardtii, a process termed intraflagellar transport is responsible for the establishing and maintaining flagella. Some of the mammalian equivalents have now been shown to perform a similar function in vivo and produce pathologies such as polydactyly, cystic kidneys and degenerative blindness seen in a variety of human diseases (e.g. Bardet-Beidl syndrome, Polycystic Kidney disease and Nephronophthsis). As yet there does not exist a well-defined proteomic catalogue of the intraflagellar transport machinery in mammalian cells. Moreover, this transport machinery will also be linked to cargos destined for the cilium, such as receptors, signaling proteins and structural factors, and the motors responsible for the transport process. To this end, we have generated a murine kidney epithelial cell line, one that produces cilia at high propensity, stably expressing a tandem affinity fusion to an IFT component. This cell line will be used to generate substantial material from which IFT complexes will be purified and analyzed by mass spectrometry. In parallel with the proteomic analysis, we are also carrying out microscopic analyses to visualize the process of Intraflagellar Transport and identify the dynamics of transport and their relation to establishing the primary cilium and its maintenance.