IDENTIFICATION OF EARLY BASAL BODY ASSEMBLY PROTEINS FROM THE ORAL APPARATUS OF

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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. Centrosomes promote the fidelity of chromosome transmission and aberrations in their number and structure is evident in the majority of tumors. Integral to centrosome duplication and organization, centrioles are a core structure around which centrosomes organize. Basal bodies, for the formation of cilia and flagella, are analogous to centrioles with a conserved molecular composition and structural organization. Initial events in the assembly of nascent centrioles and basal bodies are highlighted by the formation of an amorphous disk and cartwheel followed by the microtubule rich blades to form a barrel structure. Centriole study is constrained by the limited number of known components. To identify the constituents that generate these early steps in assembly, we will utilize two procedures for isolating these structural proteins from the basal body rich oral apparatus of Tetrahymena. Initially, we will use increasing salt concentrations to extract and isolate the basal body proteins in a stepwise fashion. Solubilized structures lost from the oral apparatus framework will be confirmed by electron microscopy. Fractions containing cartwheel proteins will then be subjected to MuDPIT. In the second procedure, basal body enriched, biochemical preparations will be isolated from Tetrahymena and placed into cartwheel reassembly conditions. These utilize a preparation that enables cartwheel reassembly, in vitro (Gavin, 1984). Structures resembling early steps in basal body and centriole assembly were reassembled and subjected to mass spectrometry (MudPIT) to identify basal body components necessary for cartwheel assembly. In addition to predicting amorphous disk and cartwheel proteins, these studies will identify a number of known and potentially novel basal body and centriole components.