Effects of targeted gene disruption of inner arm dynein heavy chain 7 on axonemal function in Tetrahymena thermophila
Wood, Christopher R
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The ciliated protozoan, Tetrahymena thermophila , swims by the coordinated beating of hundreds of cilia that cover its body. The forward swimming speed is governed by both the frequency and waveform of ciliary beating. It has been proposed that the outer arm dyneins of the ciliary axoneme control beat frequency changes, while the many different inner arm dyneins contribute to the waveform. To test the role of one of these inner arm dynein heavy chains, Dyh7p, a knockout mutant was generated by targeted biolistic transformation of the vegetative macronucleus. Dyh7p is thought to be part of the two-headed I1 dynein complex. Disruption of DYH7, the gene which encodes Dyh7p, was confirmed by PCR examination of both genomic and cDNA templates. Digital video analysis of free swimming cells was used to quantitate swimming behaviors. Both intact and detergent extracted, reactivated cell model preparations of these mutants, which we call DYH7neo3, displayed swim speeds nearly half that of wild-type cells. Although the DYH7neo3 mutants were slower than wild type, they were still able to speed up and slow down in response to mild hyperpolarizing and depolarizing stimuli, suggesting that their defect is in their basal, not stimulated, swim speed. The DYH7neo3 mutants were also able to show avoiding reactions (repeated bouts of backward and forward swimming) in response to strong depolarizing ionic stimuli, suggesting that their ciliary reversal machinery is not impaired. High-speed video microscopy of intact, free-swimming DYH7neo3 mutants revealed an irregular pattern of ciliary beat and waveform. The mutant cilia appeared to be engaging in less coordinated, whip-like movements in which the typical shape, periodicity, and coordination seen in wild type cilia were absent or disturbed. We propose that the axonemal inner arm dynein heavy chain 7 proteins contribute to the formation of normal ciliary waveform which in turn governs the forward swimming velocity of these cells. Supported by NSF grant MCB-0445362 to T.M.H.