Kin5 knockdown in Tetrahymena thermophila using RNAi blocks cargo transport of Gef1.
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.
Awan
Aashir
A
Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
Bell
Aaron J
AJ
Satir
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D015800:Protozoan Proteins