Evidence for a role of 13S axonemal ATPase in modulation of ciliary microtubule sliding

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Abstract
We recently demonstrated that elevated concentrations (≥ 20 μM) of the dynein substrate MgATP2− inhibit the spontaneous ATP-induced sliding disintegration of isolated, Triton-demembranted Tetrahymena cilia. We have used a turbidimetric assay (ΔA350 nm) and electron microscopy to examine the effect of ATP on sliding disintegration when activated by other divalent cations. Mg2+, Ca2+, and Mn2+ are each capable of activating sliding, but only with Mg2+ and Mn2+ is disintegration inhibited by elevated ATP concentrations (≥ 1 mM). The two major ATPase activities obtained by KCI extraction of Tetrahymena axonemes differ in their cation specificities such that Mg2+ and Ca2+ activate the 21S dynein ATPase with equal efficiency, whereas the 13S axonemal ATPase activity is reduced by ∼ 50% when CaATP2− replaces MgATP2− as substrate. With 1 mM MgATP2− as substrate, 10−7 to 10−2M added CaC12 alleviates the ATP-dependent inhibition of disintegration and likewise represses 13S MgATPase activity. In contrast, free Ca2+ has no effect on either the disintegration response or MgATPase activity. In contrast to Triton-treated cilia, glycerinated cilia, which beat in 1 mM MgATP2−, are inhibited from beating by high CaATP2− concentrations. These substrate specificities suggest that concentration-dependent, substrate inhibition of sliding disintegration may be a manifestation of a physiological mechanism that is mediated by the 13S axonemal ATPase and that may function to modulate sliding during bend formation. However, the effects of added CaCl2 probably do not reflect a physiological mechanism for regulating beat parameters, but rather may result from CaATP2− competing for MgATP2− binding sites on the 13S ATPase, thereby blocking expression of the 13S ATPase.