Studies on factors affecting axoneme assembly and stability in Tetrahymena thermophila
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Intraflagellar transport (IFT) involves the transport of ciliary precursors on oligomeric protein complexes (A, B) attached to microtubule motors (kinesin-2 and cytoplasmic dynein-1b) for ciliary assembly and maintenance. The assembly of the three axonemal segments (proximal, middle, distal) and the regulation of the different components of the IFT pathway are poorly understood. Moreover, the form in which tubulin is transported into cilia is not known. This work explores the mechanism of IFT, with respect to its regulation and assembly of distinct axonemal segments. I studied the mechanism of suppression in a suppressor of IFT52, a complex B protein. Partial suppressors (IFT52Δsm) are conditional and assemble cilia under: either lower temperature or hypoxia. I showed a novel intragenic mechanism of suppression that occurs in two steps. First by native RNAi-mediated genome rearrangements for deleting micronucleus-specific sequences during conjugation of two heterokaryons carrying micronuclear copies of neo2-disrupted IFT52. Next the flanking sequences controlling neo expression are processed as multiple artificial introns, thereby restoring the translational frame. Chlamydomonas mutants of IFT46 (a complex B protein) are partially suppressed under hypoxia, suggesting a conserved link between oxygen and IFT-regulation. The detergent-soluble fraction of cilia from IFT52Δsm cells was analyzed by twodimensional gels, which showed an increase in the concentration of a single protein spot, identified as tubulin folding cofactor B (TCB). TCB is one of the five cofactors in the tubulin folding pathway that generates αβ-tubulin dimers from monomers. The presence of TCB in the cilium indicated a potential ciliary role of the components of tubulin folding factors in ciliogenesis, such as the transport of tubulin. We show that a knockout of TCB1 in Tetrahymena produces a lethal phenotype associated with a general loss of microtubules. Folding α-tubulin by Tcb1p is its primary role. I explored the functions of DYF-1 (an IFT-associated protein), in axoneme assembly. In C. elegans, Osm-3 kinesin-2 assembles the distal-most segment of the axoneme (with outer singlets). DYF-1 is required for Osm-3 function and was proposed as a motor activator or adaptor. Tetrahymena cells lacking DYF-1 have non-functional cilia with a variety of axonemal defects. Zebrafish dyf-1 mutants assemble non-functional cilia lacking polyglutamylation, an important post-translational tubulin modification. Tetrahymena cells lacking Dyf1p have hyperglutamylated short axonemes. Thus, DYF-1 functions in axoneme assembly and/or stability but the phenotypes from loss of DYF-1 are organism-specific.