Inner arm dynein 1 is essential for Ca++‐dependent ciliary reversals in Tetrahymena thermophila

Abstract

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.

Targeted gene disruption of dynein heavy chain 7 ofTetrahymena thermophilaresults in altered ciliary waveform and reduced swim speed

“…It has been previously shown in the free swimming, freshwater ciliate Paramecium that depolarizing stimuli can decrease both beat frequency and forward swimming velocity, whereas hyperpolarization may result in increased beat frequency and faster forward swimming (Sugino and Machemer, 1988). This observation was also made with Tetrahymena in recent behavioral studies (Hennessey et al, 2002). 8 mM KCl is a depolarizing stimulus that is below the threshold necessary for ciliary reversal.…”

“…A Tetrahymena mutant containing a disrupted gene corresponding to inner arm dynein heavy chain 6 (DYH6) displayed slow swimming and abnormal waveform, but retained normal beat frequencies (Angus et al, 2001;Hennessey et al, 2002). This mutant, called KO6, was also defective in its ability to produce ciliary reversals and consequent backward swimming in response to depolarizing stimuli (Hennessey et al, 2002).…”

“…A Tetrahymena mutant containing a disrupted gene corresponding to inner arm dynein heavy chain 6 (DYH6) displayed slow swimming and abnormal waveform, but retained normal beat frequencies (Angus et al, 2001;Hennessey et al, 2002). This mutant, called KO6, was also defective in its ability to produce ciliary reversals and consequent backward swimming in response to depolarizing stimuli (Hennessey et al, 2002). Similar studies in which gene disruptions were introduced into the inner arm heavy chain genes DYH8, DYH9 and DYH12 of Tetrahymena suggested that DYH8 may actually play a role in determining ciliary beat frequency, whereas DYH9 and DYH12 may play roles in generating waveform (Liu et al, 2004).…”

Keeping an eye on I1: I1 dynein as a model for flagellar dynein assembly and regulation

“…These phenotypic data demonstrate that I1 dynein is a critical component of a complex system, involving multiple axonemal dyneins, which controls axonemal bending. Extensive in vitro functional and pharmacological data also indicate I1 dynein is required for control of axonemal sliding [Habermacher and Sale, 1997;King and Dutcher, 1997;Hendrickson et al, 2004], and that the mechanism of regulation involves reversible phosphorylation of the I1 subunit IC138 in a process that also may be controlled by calcium [Hennessey et al, 2002;Smith, 2002a;Dymek and Smith, 2006].…”

“…For example, not only is I1-dynein activity regulated by phosphorylation in Chlamydomonas, recent data suggests that I1 activity plays a role in regulating motility in Tetrahymena thermophila [Hennessey et al, 2002;Deckman and Pennock, 2004].…”