Both cAMP and cGMP are required for maximal ciliary beat stimulation in a cell-free model of bovine ciliary axonemes

Previously, we have shown that the ATPase-dependent motion of cilia in bovine bronchial epithelial cells (BBEC) can be regulated through the cyclic nucleotides, cAMP via the cAMP-dependent protein kinase (PKA) and cGMP via the cGMP-dependent protein kinase (PKG). Both cyclic nucleotides cause an increase in cilia beat frequency (CBF). We hypothesized that cAMP and cGMP may act directly at the level of the ciliary axoneme in BBEC. To examine this, we employed a novel cell-free system utilizing detergent-extracted axonemes. Axoneme movement was whole-field analyzed digitally with the Sisson-Ammons video analysis system. A suspension of extracted axonemes remains motionless until the addition of 1 mM ATP that establishes a baseline CBF similar to that seen when analyzing intact ciliated BBEC. Adding 10 microM cAMP or 10 microM cGMP increases CBF beyond the established ATP baseline. However, the cyclic nucleotides did not stimulate CBF in the absence of ATP. Therefore, the combination of cAMP and cGMP augments ATP-driven CBF increases at the level of isolated axoneme.     

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.     

Characterization of an A-kinase anchoring protein in human ciliary axonemes

Although protein kinase A (PKA) activation is known to increase ciliary beat frequency in humans the molecular mechanisms involved are unknown. We demonstrate that PKA is associated with ciliary axonemes where it specifically phosphorylates a 23-kDa protein. Because PKA is often localized to subcellular compartments in proximity to its substrate(s) via interactions with A-kinase-anchoring proteins (AKAPs), we investigated whether an AKAP was also associated with ciliary axonemes. This study has identified a novel 28 kDa AKAP (AKAP28)that is highly enriched in airway axonemes. The mRNA for AKAP28 is up-regulated as primary airway cells differentiate and is specifically expressed in tissues containing cilia and/or flagella. Additionally, both Western blot and immunostaining data show that AKAP28 is enriched in airway cilia. These data demonstrate that we have identified the first human axonemal AKAP, a protein that likely plays a role in the signaling necessary for efficient modulation of ciliary beat frequency.     

Control of ciliary orientation through cAMP-dependent phosphorylation of axonemal proteins in paramecium caudatum

Ciliary reorientations in response to cAMP do not take place after a brief digestion with trypsin in ciliated cortical sheets from Triton-glycerol-extracted Paramecium. In this study, we examined the effects of tryptic digestion on the cAMP-dependent phosphorylation of axonemal proteins to clarify the relationship between phosphorylation and ciliary reorientation. As reported for Paramecium tetraurelia, cAMP stimulated phosphorylations of the 29 kDa and 65 kDa axonemal polypeptides also in Paramecium caudatum. After a brief digestion of axonemes by trypsin, none of the cAMP-dependent phosphorylations occurred. On the other hand, the 29 kDa polypeptide still remained to be labeled after a brief digestion of axonemes that had previously been labeled with (32)P in the presence of cAMP, which indicates that this brief digestion breaks down endogenous cAMP-dependent protein kinases but not phosphorylated proteins. This must be the reason that trypsin-treated cilia on the sheets cannot reorient towards the posterior part of the cell. Our results indicate that cAMP regulates not only the beat frequency but also the ciliary orientation via phosphorylation of dynein subunits in Paramecium.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes

The endogenous protein kinases of isolated Paramecium tetraurelia cilia phosphorylated approximately 30 ciliary polypeptides in vitro. Labeling with [gamma-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 mol wt) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000, and 45,000 mol wt); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cyclic AMP, and cyclic GMP, but the labeling pattern was not affected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10(-5) or 10(-4) M), or EGTA. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the molecular weights and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Six behavioral mutants of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant, however, showed a reduction in cyclic GMP-stimulated protein kinase activity.     

Membrane control of ciliary movement in ciliates

Ciliary movement is generated in the axoneme by the unidirectional sliding of the outer doublets of microtubules produced by the adenosine triphosphate (ATP)-energized dynein arms. It is composed of an effective stroke phase and a passive recovery stroke phase. Two parameters are modulated to determine swimming characteristics of the cell (speed and direction): beat frequency; direction of the effective stroke. They are linked to the internal Ca++ level and to the membrane potential. The membrane governs the internal Ca++ level by regulating Ca++ influx and efflux. It contains voltage-sensitive Ca++ channels through which a passive Ca++ influx, driven by the electrochemical gradient, occurs during step depolarization. The rise of the Ca++ level, up to 6.10-7M triggers ciliary reversal and enhances beat frequency. Ca+ is extruded from cilia by active transport. Ca++ also activates a multistep enzymatic process, the first component of which is a membrane calmodulin-dependent guanylate cyclase. cGMP interacts with Ca++ to modulate the parameters of the ciliary beat. The phosphorylation-dephosphorylation cycle of axoneme and membrane proteins seems to play a major role in controlling ciliary movement. Hyperpolarization of the membrane enhances beat frequency by an unknown mechanism. It could be a modification of the ratio of axonemal bound Ca++ and Mg++, or activation by cyclic adenosine monophosphate (cAMP) produced by a membrane adenylate cyclase. The ciliary membrane behaves as a receptor able to detect modifications of external parameters, and as a transductor transmitting the detected signal by a second or third messengers toward the interior of the cilia. These messengers. acting at different levels, modulate the parameters of the mechanism that generates ciliary movement.     

Augmented ciliary reorientation response and cAMP-dependent protein phosphorylation induced by glycerol in triton-extracted Paramecium

In the presence of 30% glycerol, the cilia of a permeabilized cell model from Paramecium exhibit dynamic orientation changes while displaying only a restricted cyclic beating with a very small amplitude. The direction of cilia under these conditions corresponds to the direction of the effective power stroke of cilia beating in the absence of glycerol, i.e., pointing posteriorly in the absence of Ca2+ and anteriorly at > 10(-6) M Ca2+. Ciliary reorientation toward the posterior in response to the removal of Ca2+ is particularly conspicuous; all the cilia become predominantly pointing to the posterior end all through their beating phases. Previous studies suggested that the effect of glycerol is caused through modification of cAMP-dependent protein phosphorylation. To determine whether glycerol in fact affects ciliary reorientation through changes in protein phosphorylation, here we examined protein phosphorylation in the axonemes. Glycerol stimulated cAMP-induced phosphorylation of 29-kDa and 65-kDa proteins. The stimulation of phosphorylation was found to be partly due to the inhibition of endogenous phosphodiesterase (PDE), and partly due to the inhibition of the dephosphorylation of the 29-kDa and 65-kDa phosphoproteins within the axoneme. Thus glycerol appears to cause predominant posterior orientation of cilia by stimulating cAMP-dependent phosphorylation on those proteins. In addition, glycerol appears to inhibit ciliary beating through inhibition of dynein ATPase.     

Reactivation of newt lung cilia: evidence for a possible temperature- and MgATP-induced activation mechanism.

Optimal conditions have been developed for the isolation and reactivation of highly coupled, demembranated ciliary axonemes from newt lungs [Hard, Cypher, and Schabtach, 1988, Cell Motil. Cytoskeleton 10:271-284]. In the present study, the motility of these cilia was further characterized by examining the effects of nucleotides, divalent cations, and temperature on beat frequency. When exposed to a reactivating solution containing Mg2+ and ATP, nearly 100% of the axonemes were motile and beat at frequencies of 0-50 Hz, depending on [MgATP] and temperature. Divalent cations were required for movement, with Mg2+ 2-3 times more effective than Ca2+. There was no absolute requirement for Ca2+ for motility. The beat frequencies obtained with fixed ATP and varying Mg2+ concentrations indicate that MgATP serves as the actual substrate. The effects of MgATP on beat frequency depended on the degree of mechanochemical coupling and temperature. When highly coupled preparations were reactivated at 21 degrees C, double reciprocal plots of beat frequency vs. [MgATP] were biphasic with extrapolated Fmax values of 22 and 44.8 Hz. However, when reactivated at 10 degrees C and 30 degrees C, linear plots were generated with Fmax values of 18.3 and 48.9 Hz, respectively. The beat frequencies of cultured cells and reactivated axonemes also varied biphasically with temperature. Our data suggest that newt lung respiratory cilia possess an intra-axonemal activation mechanism involving a temperature- and MgATP-induced transition between two distinct states whose maximum beat frequencies differ by 200-300%.     

Evidence for Two Extremes of Ciliary Motor Response in a Single Swimming Microorganism

Because arrays of motile cilia drive fluids for a range of processes, the versatile mechano-chemical mechanism coordinating them has been under scrutiny. The protist Paramecium presents opportunities to compare how groups of cilia perform two distinct functions, swimming propulsion and nutrient uptake. We present how the body cilia responsible for propulsion and the oral-groove cilia responsible for nutrient uptake respond to changes in their mechanical environment accomplished by varying the fluid viscosity over a factor of 7. Analysis with a phenomenological model of trajectories of swimmers made neutrally buoyant with magnetic forces combined with high-speed imaging of ciliary beating reveal that the body cilia exert a nearly constant propulsive force primarily by reducing their beat frequency as viscosity increases. By contrast, the oral-groove cilia beat at a nearly constant frequency. The existence of two extremes of motor response in a unicellular organism prompts unique investigations of factors controlling ciliary beating.     

Evidence for Two Extremes of Ciliary Motor Response in a Single Swimming Microorganism

Because arrays of motile cilia drive fluids for a range of processes, the versatile mechano-chemical mechanism coordinating them has been under scrutiny. The protist Paramecium presents opportunities to compare how groups of cilia perform two distinct functions, swimming propulsion and nutrient uptake. We present how the body cilia responsible for propulsion and the oral-groove cilia responsible for nutrient uptake respond to changes in their mechanical environment accomplished by varying the fluid viscosity over a factor of 7. Analysis with a phenomenological model of trajectories of swimmers made neutrally buoyant with magnetic forces combined with high-speed imaging of ciliary beating reveal that the body cilia exert a nearly constant propulsive force primarily by reducing their beat frequency as viscosity increases. By contrast, the oral-groove cilia beat at a nearly constant frequency. The existence of two extremes of motor response in a unicellular organism prompts unique investigations of factors controlling ciliary beating.     

Adaptations of ciliary systems for the propulsion of water and mucus

1. The characteristics of ciliary systems are determined by the dominance of viscous effects over inertial effects. 2. The velocity of water propulsion depends on ciliary length, beat frequency, pattern of beating, the arrangement of the cilia and their co-ordination. Beating cilia influence a layer of water only two or three cilium lengths deep, with maximal velocity near the ciliary tip. 3. Mucus is propelled by the tips of short cilia that penetrate the mucus; these cilia are closely spaced on epithelia, and achieve slow propulsion that is relatively independent of load and does not require strong ciliary co-ordination.     

Outer dynein arm light chain 1 is essential for controlling the ciliary response to cyclic AMP in Paramecium tetraurelia

The individual role of the outer dynein arm light chains in the molecular mechanisms of ciliary movements in response to second messengers, such as Ca(2+) and cyclic nucleotides, is unclear. We examined the role of the gene termed the outer dynein arm light chain 1 (LC1) gene of Paramecium tetraurelia (ODAL1), a homologue of the outer dynein arm LC1 gene of Chlamydomonas reinhardtii, in ciliary movements by RNA interference (RNAi) using a feeding method. The ODAL1-silenced (ODAL1-RNAi) cells swam slowly, and their swimming velocity did not increase in response to membrane-hyperpolarizing stimuli. Ciliary movements on the cortical sheets of ODAL1-RNAi cells revealed that the ciliary beat frequency was significantly lower than that of control cells in the presence of ≥ 1 mM Mg(2+)-ATP. In addition, the ciliary orientation of ODAL1-RNAi cells did not change in response to cyclic AMP (cAMP). A 29-kDa protein phosphorylated in a cAMP-dependent manner in the control cells disappeared in the axoneme of ODAL1-RNAi cells. These results indicate that ODAL1 is essential for controlling the ciliary response by cAMP-dependent phosphorylation.     

Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum

Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.     

Differential regulation of Paramecium ciliary motility by cAMP and cGMP

cAMP and cGMP had distinct effects on the regulation of ciliary motility in Paramecium. Using detergent-permeabilized cells reactivated to swim with MgATP, we observed effects of cyclic nucleotides and interactions with Ca2+ on the swimming speed and direction of reactivated cells. Both cAMP and cGMP increased forward swimming speed two- to threefold with similar half-maximal concentrations near 0.5 microM. The two cyclic nucleotides, however, had different effects in antagonism with the Ca2+ response of backward swimming and on the handedness of the helical swimming paths of reactivated cells. These results suggest that cAMP and cGMP differentially regulate the direction of the ciliary power stroke.     

The Paramecium circadian clock: synchrony of changes in motility, membrane potential, cyclic AMP and cyclic GMP

The behavior of a ciliate protozoan, Paramecium, is known to represent the electrical state of the cell membrane, and regulation of the membrane potential and ciliary motion are known to involve cAMP and cGMP. The present study shows the synchrony of circadian changes in motility, resting membrane potential and cyclic nucleotides in P. multimicronucleatum. Using an automated system for tracking isolated single microorganisms, the isolated Paramecium cells are confirmed to swim fast and straight during the day (and subjective day) and slowly, with frequent turning, at night (and subjective night). The resting membrane potential is more negative during the day than at night. cAMP and cGMP concentrations oscillate in a manner, such that both cAMP and cGMP are higher during the day (or subjective day) than at night (or subjective night). The ratio of cGMP to cAMP during the light and dark cycle (LD) fluctuates, paralleling the fluctuation of the resting membrane potential measured during the LD. These results suggest that the Paramecium will provide an excellent model to explore daily and circadian orchestration of second messengers mediating signals from ambient light/dark cycles and circadian pacemaker to ion channels and cilia, directly involved in daily and circadian cellular outputs of resting membrane potential and motility. Accepted: 23 January 1997     

Isolation and reactivation of highly-coupled newt lung cilia

The paired lungs of the newt, Taricha granulosa, are simple, unbranched sacs, 3.5-5.0 cm in length. The inner epithelium overlying the pulmonary vein is differentiated into a mucociliary tract that extends the entire length of the lung. Populations of single, demembranated ciliary axonemes, 12-13 micron in length, can be isolated by extracting whole lungs or primary cultures of the ciliated epithelium with Triton X-100. The motile capabilities of the isolated axonemes are the highest yet obtained for any ciliary model. When exposed to a suitable reactivating medium containing Mg2+ and ATP, nearly 100% of the axonemes become motile. Uniform reactivation of high quality requires short extraction times, minimization of mechanical damage, and strict adherence to optimal conditions throughout the extraction, storage, and reactivation procedures. Significant deviations from either pH 7.0 or 0.12 M salt can lead to a rapid, irreversible decrease in the beat frequency of reactivated axonemes. Both DTT and EDTA serve to stabilize their motility. The isolated axonemes beat at 29.5 Hz in the presence of 1.75 mM ATP at 21 degrees C, matching the beat frequencies measured for cultured cells at the same temperature. With 5 mM ATP, beat frequencies over 40 Hz are measured. Our results show that neither the plasma membrane, accessory structures, nor hydrodynamic coupling of cilia are required for this activity and imply that the lack of these factors is not responsible for the low motile capabilities of ciliary models isolated previously.     

Regulation of ciliary motility by membrane potential in Paramecium: a role for cyclic AMP

The membrane potential of Paramecium controls the frequency and direction of the ciliary beat, thus determining the cell's swimming behavior. Stimuli that hyperpolarize the membrane potential increase the ciliary beat frequency and therefore increase forward swimming speed. We have observed that 1) drugs that elevate intracellular cyclic AMP increased swimming speed 2-3-fold, 2) hyperpolarizing the membrane potential by manipulation of extracellular cations (e.g., K+) induced both a transient increase in, and a higher sustained level of cyclic AMP compared to the control, and 3) the swimming speed of detergent-permeabilized cells in MgATP was stimulated 2-fold by the addition of cyclic AMP. Our results suggest that the membrane potential can regulate intracellular cAMP in Paramecium and that control of swimming speed by membrane potential may in part be mediated by cAMP.     

Phosphorylation of endogenous proteins of cilia from Paramecium tetraurelia in vitro

Several endogenous substrate proteins of cilia from axenically grown Paramecium tetraurelia were phosphorylated in vitro by inherent protein kinases (PKs). Labeling was stimulated by cAMP and to a lesser extent by cGMP. ATP breakdown was most rapid in cilia and subciliary fractions. Using multiple substrate additions during incubations it was shown that phosphorylation was almost completed within 30 s. Very little dephosphorylation by phosphoprotein phosphatases occurred during 5 min of incubation. Proteins of molecular weight of 103 000 and 46 000 were shown to be particularly associated with axonemal structures of the cilia. No distinct differences in phosphorylation patterns were apparent in ciliary membrane vesicles of low and high buoyant density, which exhibit differential enzyme patterns. cAMP receptor proteins were identified by use of the photoaffinity label 8-azido-[32P]cAMP. Receptor proteins with apparent molecular weights of 43 000, 39 000, 37 000, 31 000 and 30 000 were probably related to the regulatory subunits of cAMP-dependent protein kinases as evidenced by inhibition of incorporation of the photoaffinity label by low concentrations of cAMP. Tagging of a protein of 85 000 molecular weight was specifically inhibited by cGMP, thus in all likelihood it corresponded to a cGMP-dependent protein kinase. Corresponding autophosphorylated protein bands were observed with gamma-[32P]ATP. A functional role for protein phosphorylation in cilia of Paramecium remains to be established.     

Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate and Concanavalin A and fractionated with Triton X-114.

Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from greater than 350 kDa to less than 20 kDa, were resolved. The major surface 50-60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg(2+)-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Reactivation of outer-arm-depleted lung axonemes: evidence for functional differences between inner and outer dynein arms in situ.

Demembranated axonemes isolated from newt lung ciliated cells show a complex beat frequency response to varying [MgATP] and temperature [Hard and Cypher, 1992, Cell Motil. Cytoskeleton 21:187-198]. The present study was undertaken to ascertain whether the beat frequency of outer-arm-depleted newt lung axonemes is controlled in a manner similar to that of intact axonemes. Populations of demembranated ciliary axonemes were isolated by Triton X-100 extraction of lungs from the newt, Taricha granulosa. Aliquots of the demembranated axonemes were further treated with solutions containing high salt (0.375 M KC1) and 1.25 mM MgATP. This treatment resulted in the selective removal of outer dynein arms and a concomitant decrease in beat frequency to a stable level, 33-35% of control values. The effects of pH, salt concentration, nucleotides, and temperature on the beat frequency of reactivated outer-arm-depleted axonemes were ascertained and compared with those of intact axonemes. Some reactivation properties, such as nucleotide specificity, the effect of pH on beat frequency and the threshold [MgATP] required for reactivation (approximately 5 microM) were similar to those observed for intact axonemes. Other properties, such as the relationship between beat frequency and varying [MgATP] or salt concentration, differed both qualitatively and quantitatively from those of control axonemes, as did their response to temperature over the range, 5 degrees-32 degrees C. The nature of the results obtained with temperature and MgATP suggests that inner and outer dynein arms are not functionally equivalent in situ.