Functional analysis of the cytoplasmic dynein-2 complex in Tetrahymena thermophila

Dyneins are large molecular motor complexes containing one or more heavy chains that function as motors and several accessory subunits that bind cargoes and regulate heavy chain function. These dynein complexes perform various cellular functions within the eukaryotic cell. Axonemal outer and inner arm complexes and cytoplasmic dyneins-1 and -2 are the major types of dyneins that exist in a ciliated organism. The cytoplasmic dynein-2 complex is composed of the dynein-2 heavy chain (DYH2) and the dynein-2 light intermediate chain (D2LIC). Cytoplasmic dynein-2 heavy chain is the retrograde motor for intraflagellar transport in Chlamydomonas, C. elegans, Leishmania and mouse (Pazour et al., 1999; Porter et al., 1999; Wicks et al., 2000; May et al., 2005). Mutations of the DYH2 or D2LIC genes in these organisms result in short, stumpy flagella or cilia. In our earlier study, we knocked down the macronuclear copies of the dynein-2 heavy chain gene (DYH2) in Tetrahymena thermophila. Surprisingly, the DYH2 knockdown cells continued to form motile cilia but were misshapened and missized (Lee et al., 1999). Since our previous Tetrahymena DYH2 knockdown results were not consistent with the generally accepted model for dynein-2 in ciliogenesis, I have re-investigated the function of the Tetrahymena dynein-2 complex by a germline knockout strategy. I created knockout heterokaryon cell lines in which the DYH2 or the D2LIC gene is disrupted in the micronucleus. When two heterokaryons were mated, the somatic macronuclei of the resulting progeny were completely devoid of the targeted gene. Cells with knockout of the DYH2 gene (KO-DYH2) and the D2LIC gene (KO-D2LIC) have motile cilia but the cilia were fewer and shorter compared to wild type controls. Because of ciliary defects, the KO-DYH2 and KO-D2LIC cells swam more slowly than wild type control cells. Both KO-DYH2 and KO-D2LIC cells exhibited secondary defects including slow growth. The mutant cultures were always less dense compared to the wild type cultures. Despite the ciliary and swimming defects, both KO-DYH2 and KO-D2LIC cultures could be kept alive indefinitely. In conclusion, the cytoplasmic dynein-2 complex is important but not essential for ciliogenesis in Tetrahymena