Mechanochemical coupling in eukaryotic flagella

Quantitative analyses of ATP hydrolysis coupled to movement of eukaryotic flagella is important for understanding the relationship between ATP hydrolysis and movement. The difference in ATPase activity between intact motile axonemes (that is the cytoskeletal core of flagella) and homogenized or immotile axonemes has been assumed to be coupled to movement. However, recent findings on rates of steps in the dynein ATPase cycle and the effect of interaction with microtubules on those steps call for reassessment of movement-coupled ATPase. From these studies, it is clear that dynein ATPase activity is not as tightly coupled to interaction with microtubules as myosin ATPase activity is coupled to interaction with actin. The method by which axonemal movement is inhibited will critically affect the interpretation of difference in ATPase activity. If the homogenization or similar methods uncouple dynein, the difference in ATPase activity is not a useful measurement. Greater understanding of the relationship between dynein kinetics and axonemal movement may be obtained by use of conditions and substrates with known effects at specific steps in the dynein mechanochemical cycle and quantitating their effects on movement.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Dual regulation of mammalian myosin VI motor function

Myosin VI is expressed in a variety of cell types and is thought to play a role in membrane trafficking and endocytosis, yet its motor function and regulation are not understood. The present study clarified mammalian myosin VI motor function and regulation at a molecular level. Myosin VI ATPase activity was highly activated by actin with K(actin) of 9 microm. A predominant amount of myosin VI bound to actin in the presence of ATP unlike conventional myosins. K(ATP) was much higher than those of other known myosins, suggesting that myosin VI has a weak affinity or slow binding for ATP. On the other hand, ADP markedly inhibited the actin-activated ATPase activity, suggesting a high affinity for ADP. These results suggested that myosin VI is predominantly in a strong actin binding state during the ATPase cycle. p21-activated kinase 3 phosphorylated myosin VI, and the site was identified as Thr(406). The phosphorylation of myosin VI significantly facilitated the actin-translocating activity of myosin VI. On the other hand, Ca(2+) diminished the actin-translocating activity of myosin VI although the actin-activated ATPase activity was not affected by Ca(2+). Calmodulin was not dissociated from the heavy chain at high Ca(2+), suggesting that a conformational change of calmodulin upon Ca(2+) binding, but not its physical dissociation, determines the inhibition of the motility activity. The present results revealed the dual regulation of myosin VI by phosphorylation and Ca(2+) binding to calmodulin light chain.     

Properties of an antiserum against native dynein 1 from sea urchin sperm flagella

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.     

The effect of antidynein 1 serum on the movement of reactivated sea urchin sperm

Rabbit antiserum prepared against an ATPase-containing tryptic fragment of dynein by Ogawa and Mohri (J. Biol. Chem. 250: 6476-6483) specifically inhibited the ATPase activity of dynein 1 and not that of dynein 2. Varying amounts of this antidynein 1 serum were added to demembranated sperm while they were swimming in reactivating solution containing 1 mM ATP. The sperm continued to form regularly propagated flagellar bending waves, but the beat frequency decreased gradually with time, the greater part of the change occurring in the first 15 min. The beat frequency after 1 h was a function of the amount of antiserum used, and could be as low as 1 Hz. The waveforms of the treated sperm resembled those of normal reactivated sperm except that the bend angles of both the principal and reverse bends were larger in the proximal portion of flagellum. The ATPase activity and corresponding beat frequency of sperm which had been pretreated with varying amounts of antidynein 1 serum for 15 min at 0 degrees C and then diluted were both decreased as a function of the amount of antiserum added, the ATPase activity of homogenized, nonmotile sperm also decreased upon pretreatment with antiserum, but the percentage decrease was less than for motile sperm. For moderate to low concentrations of antiserum, the rates of reaction with motile and with rigor sperm were almost identical. The overall results suggest that antidynein 1 inhibits the functioning of the dynein arms, probably by blocking the ATPase sites of the dynein 1.     

Motor function and regulation of myosin X.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.     

A dynein ATPase inhibitor isolated from a commercial ATP preparation.

Preparations of ATP from equine muscle contained an inhibitor of dynein Mg2+-activated ATPase. The inhibitory material was separated from the ATP by molecular sieve filtration. The several molecular species of dynein extracted from three different axonemal sources were all inhibited; myosin ATPase was not. With increasing amounts of inhibitor the inhibition did not go to completion but reached a plateau when the rate had been reduced to 1/5 the uninhibited rate. A plot of 1/[S] against 1/v at several inhibitor concentrations yielded parallel lines. There was little inhibition of dynein ATPase when Mg2+ was replaced by Ca2+. The inhibitor appeared slightly smaller in molecular size than ATP, had anionic character, and was not adsorbed to charcoal.     

Metal cation controls myosin and actomyosin kinetics

We have perturbed myosin nucleotide binding site with magnesium‐, manganese‐, or calcium‐nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time‐resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation‐specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number.     

Partial characterization of a carotenoid droplet ATPase and its possible significance in carotenoid droplet dispersion in goldfish xanthophores

The dispersion of carotenoid droplets in permeabilized goldfish xanthophores is dependent on ATP, F-actin, and cytosol. We report here that the motor (ATPase, translocator) resides with the permeabilized cell remnants and not in the cytosol. We also report that the carotenoid droplets have an ATPase that is not conventional myosin, dynein, or an ion pump. Its activity appears to correlate with the actin content of the carotenoid droplet preparation. A carotenoid droplet protein of Mr 72,000 (p72) is shown to be labeled by irradiation with 8-azido-ATP with concomitant loss of ATPase activity of the carotenoid droplets. We propose that this protein may be the ATPase responsible for carotenoid droplet dispersion.     

Kinetics of steady state ATPase activity and rigor complex formation of acto-heavy meromyosin

1. Actin and heavy meromyosin, initially mixed in a Mg-ATP solution, began to form the rigor complex slowly after ATP in the solution had been completely hydrolyzed.

2. This was because the heavy meromyosin-product complex formed via ATP hydrolysis was almost completely dissociated from actin even in the absence of ATP and as soon as this heavy meromyosin-product complex was decomposed, the heavy meromyosin combined with actin forming the rigor complex.

3. Linear plots were obtained when the reciprocal of the excess rate of the actin-accelerated rigor complex formation was plotted against the reciprocal of the added actin concentration as similar with those made on the steady acto-heavy meromyosin ATPase.

4. The V of the rigor complex formation process was about 1/5 of that of the steady acto-heavy meromyosin ATPase activity, showing that the actomyosin ATPase activity could not be explained merely by the actin-accelerated decomposition of the heavy meromyosin-product complex.

5. The same analyses were carried out on myosin subfragment 1.

6. Our results could be explained by considering the two non-identical active sites of myosin, and we propose the following scheme for the actomyosin ATPase.

7. Actin accelerates the rate-limiting bond hydrolysis in the ATPase occurring at one active site of myosin, as well as the rate-limiting decomposition of the heavy meromyosin-product complex formed at another site.     

Actomyosin interactions in the presence of ATP and the N-terminal segment of actin.

The binding of myosin subfragment 1 (S-1) to actin in the presence of ATP and the acto-S-1 ATPase activities of acto-S-1 complexes were determined at 5 degrees C under conditions of partial saturation of actin, up to 90%, by antibodies against the first seven N-terminal residues on actin. The antibodies [Fab(1-7)] inhibited strongly the acto-S-1 ATPase and the binding of S-1 to actin in the presence of ATP at low concentrations of S-1, up to 25 microM. Further increases in S-1 concentration resulted in a partial and cooperative recovery of both the binding of S-1 to actin and the acto-S-1 ATPase while causing only limited displacement of Fab(1-7) from actin. The extent to which the binding and the ATPase activity were recovered depended on the saturation of actin by Fab(1-7). The combined amounts of S-1 and Fab binding to actin suggested that the activation of the myosin ATPase activity was due to actin free of Fab. Examination of the acto-S-1 ATPase activities as a function of S-1 bound to actin at different levels of actin saturation by Fab(1-7) revealed that the antibodies inhibited the activation of the bound myosin. Thus, the binding of antibodies to the N-terminal segment of actin can act to inhibit both the binding of S-1 to actin in the presence of ATP and a catalytic step in ATP hydrolysis by actomyosin. The implications of these results to the regulation of actomyosin interaction are discussed.     

The phosphorylated L2 light chain of skeletal myosin is a modifier of the actomyosin ATPase

Incubation of rabbit skeletal myosin with an extract of light chain kinase plus ATP phosphorylated the L2 light chain and modified the steady state kinetics of the actomyosin ATPase. With regulated actin, the ATPase activity of phosphorylated myosin (P-myosin) was 35 to 181% greater than that of unphosphorylated myosin when assayed with 0.05 to 5 micro M Ca2+. Phosphorylation had no effect on the Ca2+ concentration required for half-maximal activity, but it did increase the ATPase activity at low Ca2+. With pure actin, the percentage of increase in the actomyosin ATPase activity correlated with the percentage of phosphorylation of myosin. Steady state kinetic analyses of the actomyosin system indicated that 50 to 82% phosphorylation of myosin decreased significantly the Kapp of actin for myosin with no significant effect on the Vmax. Phosphorylaton of heavy meromyosin similarly modified the steady state kinetics of the acto-heavy meromyosin system. Both the K+/EDTA- and Mg-ATPase activities of P-myosin and phosphorylated heavy meromyosin were within normal limits indicating that phosphorylaiion had not altered significantly the hydrolytic site. Phosphatase treatment of P-myosin decreased both the level of phosphorylation of L2 and the actomyosin ATPase activity to control levels for unphosphorylated myosin. It is concluded levels for unphosphorylated myosin. It is concluded from these results that the ability of P-myosin to modify the steady state kinetics of the actomyosin ATPase was: 1) specific for phosphorylation; 2) independent of the thin filament regulatory proteins.     

Effect of spin-labeled maleimide on 14S and 30S dyneins in solution and on demembranated ciliary axonemes.

The effects of N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide(SLM) on the pellet height response and ATPase activity of glycerinated Triton X-100 extracted cilia of Tetrahymena pyriformis have been studied. Preincubation of cilia with SLM caused complete inhibition of the pellet height response and an initial increase in ATPase activity followed upon longer exposure to SLM by inhibition of ATPase. The effect of SLM on extracted 30S dynein was the reverse of that for whole cilia: ATPase activity was increased when 30S dynein was added to a mixture of ATP and SLM and inhibited when the 30S dynein was preincubated with SLM. The activity of 14S dynein was only inhibited by SLM. Electron spin resonance spectra of ciliary axonemes that had reacted with SLM for various times showed that much of the covalently bound SLM was strongly immobilized even after 1 min of reaction, when ATPase activity increased twofold. The proportion of strongly immobilized label increased with longer times of reaction. Addition of ATP to SLM-labeled axonemes caused a small decrease in the height of the spectral peak corresponding to strongly immobilized label as compared with that of weakly immobilized label, indicating an increase in rotational freedom of some covalently bound label. The results suggest that ATP causes a conformation change affecting a sulfhydryl group(s) involved in the mechanochemical system. It was also shown that beta,gamma-methylene ATP(AMP-PCP) is an inhibitor of dynein ATPase. This analogue of ATP is not hydrolyzed by whole cilia or by the extracted dyneins and does not cause a pellet height response. With Mg2+ as divalent cation, AMP-PCP inhibits 30S dynein more than it inhibits 14S dynein; with Ca2+, the inhibition of 30S dynein is reduced, and there is no inhibition of 14S dynein. Under conditions where AMP-PCP inhibited 30S dynein ATPase it was much less effective than ATP in protecting against the loss of ATPase activity by SLM. Although SLM inhibited Mg2+-activated 14S and 30S dyneins in solution, it did not inhibit ciliary ATPase activity. These results support the view that at least 2 SH groups are involved in ciliary motility and that their reactivity to SH reagents depends on whether the dyneins are in situ or have been extracted.     

Oxygen exchange between phosphate and water accompanies calcium-regulated ATPase activity of skinned fibers from rabbit skeletal muscle

The extent of oxygen exchange between phosphate and water has been measured for the calcium-regulated magnesium-dependent ATPase activity of chemically skinned fibers from rabbit skeletal muscle. The oxygen exchange was determined for isometrically held fibers by measuring with a mass spectrometer the distribution of 18O atoms in the product inorganic phosphate when ATP hydrolysis was carried out in H2(18)O. The extent of exchange was much greater in relaxed muscle (free Ca2+ less than 10(-8) M) than in calcium-activated muscle (free Ca2+ approximately equal to 3 X 10(-5) M). Activated fibers had an ATPase activity at least 30-fold greater than the relaxed fibers. These results correlate well with the extents of oxygen exchange accompanying magnesium-dependent myosin and unregulated actomyosin ATPase activities, respectively. In relaxed fibers, comparison of the amount of exchange with the ATPase activity suggests that the rate constant for the reformation of myosin-bound ATP from the myosin products complex is about 10 s-1 at 20 degrees C and pH 7.1. In each experiment the distribution of 18O in the Pi formed was incompatible with a single pathway for ATP hydrolysis. In the case of the calcium-activated fibers, the multiple pathways for ATP hydrolysis appeared to be an intrinsic property of the actomyosin ATPase in the fiber. These results indicate that in muscle fibers, as in isolated actomyosin, cleavage of protein-bound ATP is readily reversible and that association of the myosin products complex with actin promotes Pi release.     

Characterization of the motor activity of mammalian myosin VIIA

Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg(2+)-ATPase activity that was markedly activated by actin with K(actin) of 33 microm and V(max) of 0.53 s(-1) head(-1). Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K(actin) of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor.     

Evidence suggesting the presence of common antigenic determinant between dynein and tubulin.

The present experiments showed that the guinea pig antiserum prepared against the main polypeptides of 14 S dynein from Tetrahymena cilia reacted with sea urchin sperm flagellar dynein and with bovine brain high molecular weight protein to give rise to a precipitin line confluent with that formed between the antiserum and Tetrahymena dynein. Furthermore, it was found that this antiserum also reacted with tubulins from Tetrahymena cilia, sea urchin sperm flagella and bovine brain to give rise to the confluent precipitin line. Among muscle proteins, only actin preparation from rabbit skeletal muscle reacted with the anti-Tetrahymena dynein serum, whereas neither rabbit skeletal muscle myosin, chicken skeletal muscle tropomyosin nor chicken skeletal muscle troponin reacted with the antiserum. These results suggest that dynein and tubulin and probably actin share an antigenic determinant regardless of different protein species and of different animal species. The common antigenic determinant was detected only when the proteins denatured with urea/sodium dodecyl sulfate/beta-mercaptoethanol/N-ethylmaleimide were used, but it was not detected at all when the native proteins were used. This implies that a certain common antigenic determinant which is involved in the precipitin line formation exists in the primary structures of dyneins and tubulins and probably actin, and is hidden inside the tertiary structures of the native protein molecules.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

The substrate specificity of dynein from Tetrahymena cilia

The substrate specificity of the 22S dynein ATPase from Tetrahymena cilia was investigated. The 22S dynein exhibited a high specificity for ATP in terms of both apparent Km and Vmax: naturally occurring nucleoside triphosphates other than ATP were hydrolyzed slowly with an apparent Km of 0.25-1 mM, a sharp contrast to that of ATP hydrolysis (1-4 microM). Pyrophosphate was a poor inhibitor for the dynein ATPase, indicating weak affinity. Since dynein binds ATP tightly and hydrolyzes it at a high rate, a method to determine a trace amount of ATP in the presence of other nucleoside triphosphates has been developed by taking advantage of this enzymatic characteristic of dynein. The effect of P1,P5-di(adenosine-5'-)-pentaphosphate (Ap5A) on the 22S dynein ATPase was also investigated. Ap5A acted as a weak competitive inhibitor of the ciliary 22S dynein ATPase and the nonlinearity of the double-reciprocal plot of the ATPase was confirmed in the presence of Ap5A.     

Activation of the Adenosine Triphosphatase of Limulus polyphemus Actomyosin by Tropomyosin

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.     

Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.