FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.
Bottom Line: A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility.Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong.Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo.
Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA 30602.Show MeSH
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Mentions: The cryo–electron tomography data place FAP206 in the RS2 front prong, a location close to the surface of the A-tubule of the outer doublet microtubules, opening the possibility that FAP206 might directly interact with the microtubule and thus contribute to RS2 docking. To test whether FAP206 has microtubule-binding activity in vivo, we briefly (3 h) overexpressed GFP-FAP206 fusion protein under the cadmium-inducible promoter in vegetatively growing cells that also expressed the native FAP206. The induced GFP-FAP206 localized to cilia in two distinct patterns. In a subset of cilia that tended to be relatively short (and therefore were likely in the process of assembly during the period of transgene induction), overproduced GFP-FAP206 was distributed along the entire cilium length (Figure 6, white arrowheads), indicating that the tagged protein can be incorporated into the axoneme during cilia assembly in place of the native protein. The second pattern was seen in a subset of cilia that were mostly full length, where GFP-FAP206 was not detectable along most of the cilium length but strongly accumulated at the distal tip (Figure 6, yellow insets). Likely, these full-length cilia were already assembled at the time of induction, and GFP-FAP206 could not be incorporated into the axonemes because its docking sites were occupied by the native protein, which turns over slowly. Strikingly, in the cell body, overexpressed GFP-FAP206 also strongly colocalized with the network of cytoplasmic microtubules (Figure 6, white insets). Thus, when overproduced, GFP-FAP206 colocalizes with nonciliary microtubules, indicating that FAP206 might have a microtubule-binding activity. To this end, we tested whether GFP-FAP206 purified from overproducing Tetrahymena cells can bind to microtubules in vitro, using a sedimentation assay (Goode and Feinstein, 1994). Unfortunately, GFP-FAP206 (and not GFP alone) sedimented on its own, indicating that it undergoes oligomerization or aggregation (unpublished data).
Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA 30602.