Kin5 knockdown in Tetrahymena thermophila using RNAi blocks cargo transport of Gef1

• Published in: PloS one Vol. 4; no. 3; p. e4873
• Main Authors: Awan, Aashir, Bell, Aaron J, Satir, Peter
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 17-03-2009; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Analysis; Animals; Anterograde transport; Base Sequence; Biology; Biosynthesis; Cargo transportation; Cell Biology; Cell Biology/Cell Signaling; Cell Biology/Cytoskeleton; Cellular signal transduction; Chlamydomonas; Cilia; DNA Primers; Dynein; Fluorescent Antibody Technique; Gene Knockdown Techniques; Genes; Genomes; Guanine; Immunofluorescence; Kinesin; Lethality; Molecular Sequence Data; Paramecium; Photoreceptors; Promoter Regions, Genetic; Protein Transport; Proteins; Protozoa; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; RNA Interference; RNA-mediated interference; Sequence Homology, Amino Acid; Studies; Tetrahymena; Tetrahymena thermophila - genetics; Tetrahymena thermophila - metabolism; New York; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0004873

A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality.