Effects of trypsin-digested outer-arm dynein fragments on the velocity of microtubule sliding in elastase-digested flagellar axonemes

Flagellar movement is caused by the coordinated activity of outer and inner dynein arms, which induces sliding between doublet microtubules. In trypsin-treated flagellar axonemes, microtubule sliding induced by ATP is faster in the presence than in the absence of the outer arms. To elucidate the mechanism by which the outer arms regulate microtubule sliding, we studied the effect of trypsin-digested outer-arm fragments on the velocity of microtubule sliding in elastase-treated axonemes of sea urchin sperm flagella. We found that microtubule sliding was significantly slower in elastase-treated axonemes than in trypsin-treated axonemes, and that this difference disappeared after the complete removal of the outer arms. After about 95% of the outer arms were removed, however, the velocity of sliding induced by elastase and ATP increased significantly by adding outer arms that had been treated with trypsin in the presence of ATP. The increase in sliding velocity did not occur in the elastase-treated axonemes from which the outer arms had been completely removed. Among the outer arm fragments obtained by trypsin treatment, a polypeptide of about 350 kDa was found to be possibly involved in the regulation of sliding velocity. These results suggest that the velocity of sliding in the axonemes with only inner arms is similar to that in the axonemes with both inner and outer arms, and that the 350 kDa fragment, probably of the alpha heavy chains, increases the sliding activity of the intact outer and inner arms on the doublet microtubules.     

Regulation of flagellar dynein by calcium and a role for an axonemal calmodulin and calmodulin-dependent kinase

Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonas mutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15 axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway.     

Effects of imposed bending on microtubule sliding in sperm flagella

The movement of eukaryotic flagella is characterized by its oscillatory nature. In sea urchin sperm, for example, planar bends are formed in alternating directions at the base of the flagellum and travel toward the tip as continuous waves. The bending is caused by the orchestrated activity of dynein arms to induce patterned sliding between doublet microtubules of the flagellar axoneme. Although the mechanism regulating the dynein activity is unknown, previous studies have suggested that the flagellar bending itself is important in the feedback mechanism responsible for the oscillatory bending. If so, experimentally bending the microtubules would be expected to affect the sliding activity of dynein. Here we report on experiments with bundles of doublets obtained by inducing sliding in elastase-treated axonemes. Our results show that bending not only "switches" the dynein activity on and off but also affects the microtubule sliding velocity, thus supporting the idea that bending is involved in the self-regulatory mechanism underlying flagellar oscillation.     

A physical model of microtubule sliding in ciliary axonemes.

Ciliary movement is caused by coordinated sliding interactions between the peripheral doublet microtubules of the axoneme. In demembranated organelles treated with trypsin and ATP, this sliding can be visualized during progressive disintegration. In this paper, microtubule sliding behavior resulting from various patterns of dynein arm activity and elastic link breakage is determined using a simplified model of the axoneme. The model consists of a cylindrical array of microtubules joined, initially, by elastic links, with the possibility of dynein arm interaction between microtubules. If no elastic links are broken, sliding can produce stable distortion of the model, which finds application to straight sections of a motile cilium. If some elastic links break, the model predicts a variety of sliding patterns, some of which match, qualitatively, the observed disintegration behavior of real axonemes. Splitting of the axoneme is most likely to occur between two doublets N and N + 1 when either the arms on doublet N + 1 are active and arms on doublet N are inactive or arms on doublet N - 1 are active while arms on doublet N are inactive. The analysis suggests further experimental studies which, in conjunction with the model, will lead to a more detailed understanding of the sliding mechanism, and will allow the mechanical properties of some axonemal components to be evaluated.     

Regulation of flagellar dynein by the axonemal central apparatus

Numerous studies indicate that the central apparatus, radial spokes, and dynein regulatory complex form a signaling pathway that regulates dynein activity in eukaryotic flagella. This regulation involves the action of several kinases and phosphatases anchored to the axoneme. To further investigate the role of the central apparatus in this signaling pathway, we have taken advantage of a microtubule-sliding assay to assess dynein activity in central apparatus defective mutants of Chlamydomonas. Axonemes isolated from both pf18 and pf15 (lacking the entire central apparatus) and from pf16 (lacking the C1 central microtubule) have reduced microtubule-sliding velocity compared with wild-type axonemes. Based on functional analyses of axonemes isolated from radial spokeless mutants, we hypothesized that inhibitors of casein kinase 1 (CK1) and cAMP dependent protein kinase (PKA) would rescue dynein activity and increase microtubule-sliding velocity in central pairless mutants. Treatment of axonemes isolated from both pf18 and pf16 with DRB, a CK1 inhibitor, but not with PKI, a PKA inhibitor, restored dynein activity to wild-type levels. The DRB-induced increase in dynein-driven microtubule sliding was inhibited if axonemes were first incubated with the phosphatase inhibitor, microcystin. Inhibiting CK1 in pf15 axonemes, which lack the central pair as well as PP2A [Yang et al., 2000: J. Cell Sci. 113:91-102], did not increase microtubule-sliding velocity. These data are consistent with a model in which the central apparatus, and specifically the C1 microtubule, regulate dynein through interactions with the radial spokes that ultimately alter the activity of CK1 and PP2A. These data are also consistent with localization of axonemal CK1 and PP2A near the dynein arms.     

Vigorous beating of Chlamydomonas axonemes lacking central pair/radial spoke structures in the presence of salts and organic compounds

Flagella of Chlamydomonas mutants lacking the central pair of microtubules or radial spokes do not beat; however, axonemes isolated from these mutants were found to display vigorous bending movements in the presence of ATP and various salts, sugars, alcohols, and other organic compounds. For example, about 15% of the total axonemes isolated from pf18, a mutant lacking the central pair, displayed beating in the presence of 10 mM MgSO(4) and 0.2 mM ATP at about 22 Hz, while none beat with the same concentration of ATP and < or = 5 mM or > or = 25 mM MgSO(4). The beat frequency and waveform of beating pf18 axonemes were similar to those of wild type axonemes beating under the same conditions. Similarly, 10-50% of the axonemes beat in the presence of 0.5 M sucrose, 2.0 M glycerol, or 1.7 M[10% (v/v)] ethanol. The appearance of motility did not correlate with the change in axonemal ATPase; however, these substances at those concentrations commonly increased the amplitude of nanometer-scale oscillation (hyper-oscillation) in pf18 axonemes, as well as the extent of ATP-induced sliding disintegration of protease-treated axonemes. Axonemes of double mutants lacking both the central pair and various subspecies of inner-arm dynein also beat at increased MgSO(4) concentrations, but axonemes lacking outer-arm dynein in addition to the central pair did not beat. These and other observations suggest that small molecules perturb the regulation of microtubule sliding through some change in water activity or osmotic stress. Axonemes must have an intrinsic ability to beat without the central pair/radial spokes under a variety of non-physiological solution conditions, as long as the outer dynein arms are present. Apparently, the major function of the central pair/radial spoke structures is to restore this activity under physiological conditions.     

Interactions of dynein arms with b subfibers of Tetrahymena cilia: quantitation of the effects of magnesium and adenosine triphosphate

Tetrahymena 30S dynein was extracted with 0.5 M KCl and tested for retention of several functional properties associated wtih its in situ force-generating capacity. The dynein fraction will rebind to extracted outer doublets in the presence of Mg2+ to restore dynein arms. The arms attach at one end to the A subfiber and form bridges at the other end to the B subfiber of an adjacent doublet. Recombined arms retain an ATPase activity that remains coupled to potential generation of interdoublet sliding forces. To examine important aspects of the dynein- tubulin interaction that we presume are directly related to the dynein force-generating cross-bridge cycle, a simple and quantitative spectrophotometric assay was devised for monitoring the associations between isolated 30S dynein and the B subfiber. Utilizing this assay, the binding of dynein to B subfibers was found to be dependent upon divalent cations, saturating at 3 mM Mg2+. Micromolar concentrations of MgATP2- cause the release of dynein from the B subfiber; however, not all of the dynein bound under these conditions is released by ATP. ATP- insensitive dynein binding results from dynein interactions with non-B- tubule sites on outer-doublet and central-pair microtubules and from ATP-insensitive binding to sites on the B subfiber. Vanadate over a wide concentration range (10(-6)-10(-3) M) has no effect on the Mg2+- induced binding of dynein or its release by MgATP2-, and was used to inhibit secondary doublet disintegration in the suspensions. In the presence of 10 microM vanadate, dynein is maximally dissociated by MgATP2- concentrations greater than or equal to 1 microM with half- maximal release at 0.2 microM. These binding properties of isolated dynein arms closely resemble the cross-bridging behavior of in situ dynein arms reported previously, suggesting that quantitative studies such as those presented here may yield reliable information concerning the mechanism of force generation in dynein-microtubule motile systems. The results also suggest that vanadate may interact with an enzyme- product complex that has a low affinity for tubulin.     

Flagellar quiescence in Chlamydomonas: Characterization and defective quiescence in cells carrying sup-pf-1 and sup-pf-2 outer dynein arm mutations

Chlamydomonas reinhardtii can use their flagella for two distinct types of movement: swimming through liquid or gliding on a solid substrate. Cells switching from swimming to gliding motility undergo a reversible flagellar quiescence. This phenomenon appears to involve the outer dynein arms, since mutants having altered outer arm beta and gamma dyneins (sup-pf-1 and sup-pf-2) show a diminished ability to quiesce. Sup-pf-1 and sup-pf-2 were originally isolated as gain-of-function mutations that suppress the flagellar paralysis resulting from radial spoke or central pair defects. Defective quiescence is also a gain-of-function phenomenon, as cells completely lacking outer arm heavy chains show a normal quiescence phenotype. These data suggest that regulation of outer arm dynein activity is essential for flagellar quiescence and furthermore that regulation of quiescence involves a signal transduction pathway that shares elements with the radial spoke/central pair system.     

Motor apparatus in human spermatozoa that lack central pair microtubules

Electron microscopic examination of the spermatozoa from a man suffering from asthenozoospermia (poor or low sperm motility) showed that approximately 92% of the sperm flagella lacked central pair microtubules but possessed dynein arms and radial spokes while a small percentage of the spermatozoa had complete flagella. The characteristics of the motor apparatus of the spermatozoa and the effects of caffeine on the sperm motility were examined, as were the reactivation of demembranated spermatozoa and the sliding of doublet microtubules. Almost all spermatozoa were immotile in a Tyrode solution while only a small percentage of spermatozoa showed slow forward movement or feeble flagellar vibration, whereas addition of caffeine to the sperm suspension induced forward swimming of approximately half of the spermatozoa. The reactivation of demembranated spermatozoa with MgATP(2-) could not succeed because of disintegration of the demembranated flagella. However, when the demembranated spermatozoa were exposed to MgATP(2-) and then treated with elastase, the microtubular doublets of approximately half the number of the flagella slid from the end or middle of the flagella. These results suggest that the motor apparatus in the sperm flagella that lack the central pair microtubules is functionally assembled and intrinsically capable of undergoing flagellar movement but not strong enough to beat normally.     

The Pcdp1 complex coordinates the activity of dynein isoforms to produce wild-type ciliary motility

Generating the complex waveforms characteristic of beating cilia requires the coordinated activity of multiple dynein isoforms anchored to the axoneme. We previously identified a complex associated with the C1d projection of the central apparatus that includes primary ciliary dyskinesia protein 1 (Pcdp1). Reduced expression of complex members results in severe motility defects, indicating that C1d is essential for wild-type ciliary beating. To define a mechanism for Pcdp1/C1d regulation of motility, we took a functional and structural approach combined with mutants lacking C1d and distinct subsets of dynein arms. Unlike mutants completely lacking the central apparatus, dynein-driven microtubule sliding velocities are wild type in C1d- defective mutants. However, coordination of dynein activity among microtubule doublets is severely disrupted. Remarkably, mutations in either outer or inner dynein arm restore motility to mutants lacking C1d, although waveforms and beat frequency differ depending on which isoform is mutated. These results define a unique role for C1d in coordinating the activity of specific dynein isoforms to control ciliary motility.     

Structural conformation of ciliary dynein arms and the generation of sliding forces in Tetrahymena cilia

The sliding tubule model of ciliary motion requires that active sliding of microtubules occur by cyclic cross-bridging of the dynein arms. When isolated, demembranated Tetrahymena cilia are allowed to spontaneously disintegrate in the presence of ATP, the structural conformation of the dynein arms can be clearly resolved by negative contrast electron microscopy. The arms consist of three structural subunits that occur in two basic conformations with respect to the adjacent B subfiber. The inactive conformation occurs in the absence of ATP and is characterized by a uniform, 32 degrees base-directed polarity of the arms. Inactive arms are not attached to the B subfiber of adjacent doublets. The bridged conformation occurs strictly in the presence of ATP and is characterized by arms having the same polarity as inactive arms, but the terminal subunit of the arms has become attached to the B subfiber. In most instances the bridged conformation is accompanied by substantial tip-directed sliding displacement of the bridged doublets. Because the base-directed polarity of the bridged arms is opposite to the direction required for force generation in these cilia and because the bridges occur in the presence of ATP, it is suggested that the bridged conformation may represent the initial attachment phase of the dynein cross-bridge cycle. The force-generating phase of the cycle would then require a tip-directed deflection of the arm subunit attached to the B subfiber.     

The alpha subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules

Glass-adsorbed intact sea urchin outer arm dynein and its beta/IC1 subunit supports movement of microtubules, yet does not form a rigor complex upon depletion of ATP (16). We show here that rigor is a feature of the isolated intact outer arm, and that this property subfractionates with its alpha heavy chain. Intact dynein mediates the formation of ATP-sensitive microtubule bundles, as does the purified alpha heavy chain, indicating that both particles are capable of binding to microtubules in an ATP-sensitive manner. In contrast, the beta/IC1 subunit does not bundle microtubules. Bundles formed with intact dynein are composed of ribbon-like sheets of parallel microtubules that are separated by 54 nm (center-to-center) and display the same longitudinal repeat (24 nm) and cross-sectional geometry of dynein arms as do outer doublets in situ. Bundles formed by the alpha heavy chain are composed of microtubules with a center-to-center spacing of 43 nm and display infrequent, fine crossbridges. In contrast to the bridges formed by the intact arm, the links formed by the alpha subunit are irregularly spaced, suggesting that binding of the alpha heavy chain to the microtubules is not cooperative. Cosedimentation studies showed that: (a) some of the intact dynein binds in an ATP-dependent manner and some binds in an ATP-independent manner; (b) the beta/IC1 subunit does not cosediment with microtubules under any conditions; and (c) the alpha heavy chain cosediments with microtubules in the absence or presence of MgATP2-. These results suggest that the structural binding observed in the intact arm also is a property of its alpha heavy chain. We conclude that whereas force-generation is a function of the beta/IC1 subunit, both structural and ATP-sensitive (rigor) binding of the arm to the microtubule are mediated by the alpha subunit.     

The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella

The regulation of dynein activity to produce microtubule sliding in flagella has not been well understood. To gain more insight into the roles of ATP and ADP in the regulation, we examined the effects of fluorescent ATP analogues and fluorescent ADP analogues on the ATPase activity and motile activity of dynein. 21S dynein purified from the outer arms of sea urchin sperm flagella hydrolyzed BODIPY(R) FL ATP (FL-ATP) at 78% of the rate for ATP hydrolysis. FL-ATP at 0.1-1 mM, however, induced neither microtubule translocation on a dynein-coated glass surface nor sliding disintegration of elastase-treated axonemes. Direct observation of single molecules of the fluorescent analogues showed that both the ATP and ADP analogues were stably bound to dynein over several minutes (dissociation rates = 0.0038-0.0082/s). When microtubule translocation on 21S dynein was induced by ATP, the initial increase of the mean velocity was accelerated by preincubation of the dynein with ADP. Similar increase was also induced by the preincubation with the ADP analogues. Even after preincubation with ADP, FL-ATP did not induce sliding disintegration of elastase-treated axonemes. After preincubation with a nonhydrolyzable ATP analogue, AMPPNP (adenosine 5'-(beta:gamma-imido)triphosphate), however, FL-ATP induced sliding disintegration in approximately 10% of the axonemes. These results indicate that both noncatalytic ATP binding and stable ADP binding, in addition to ATP hydrolysis, are involved in the regulation of the chemo-mechanical transduction in axonemal dynein.     

Length dependence of displacement fluctuations and velocity in microtubule sliding movement driven by sea urchin sperm outer arm beta dynein in vitro

We have studied the dependence on microtubule length of sliding velocity and positional fluctuation from recorded trajectories of microtubules sliding over sea urchin sperm outer arm beta dynein in a motility assay in vitro. The positional fluctuation was quantified by calculating the mean-square displacement deviation from the average, the calculation of which yields an effective diffusion coefficient. We have found that (1) the sliding velocity depends hyperbolically on the microtubule length, and (2) the effective diffusion coefficients do not depend on the length for sufficiently long microtubules. The length dependence of the sliding velocity indicates that the duty ratio, defined as the force producing period over the total cycle time of beta dynein interaction with microtubule, is very small. The length independence of the effective diffusion coefficient indicates that there is a correlation in the sliding movement fluctuation of microtubules.     

Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes

The inner row of dynein arms contains three dynein subforms. Each is distinct in composition and location in flagellar axonemes. To begin investigating the specificity of inner dynein arm assembly, we assessed the capability of isolated inner arm dynein subforms to rebind to their appropriate positions on axonemal doublet microtubules by recombining them with either mutant or extracted axonemes missing some or all dyneins. Densitometry of Coomassie blue-stained polyacrylamide gels revealed that for each inner dynein arm subform, binding to axonemes was saturable and stoichiometric. Using structural markers of position and polarity, electron microscopy confirmed that subforms bound to the correct inner arm position. Inner arms did not bind to outer arm or inappropriate inner arm positions despite the availability of sites. These and previous observations implicate specialized tubulin isoforms or nontubulin proteins in designation of specific inner dynein arm binding sites. Further, microtubule sliding velocities were restored to dynein-depleted axonemes upon rebinding of the missing inner arm subtypes as evaluated by an ATP-induced microtubule sliding disintegration assay. Therefore, not only were the inner arm dynein subforms able to identify and bind to the correct location on doublet microtubules but they bound in a functionally active conformation.     

Bending motion of Chlamydomonas axonemes after extrusion of central- pair microtubules

Relatively little is known about the functions of central-pair microtubules (Tamm, S. L., and G. A. Horridge, 1970, Proc. Roy. Soc. Lond. B, 175: 219-233; Omoto, C. K., and C. Kung, 1979, Nature (Lond.). 279:532-534) and radial spokes (Warner, F. D., and P. Satir, 1974, J. Cell Biol., 63:35-63), although a sliding microtubule mechanism has been established for the flagellar movement (Summers, K. E., and I. R. Gibbons, 1971, Proc. Natl. Acad. Sci. USA., 68:3092-3096). In the present report, an attempt was made to determine the functions of central-pair microtubules in flagellar motility. Central-pair microtubules were found to extrude from the tips of elastase-digested axonemes of demembranated Chlamydomonas flagella after the addition of ATP. The length of the extruded central-pair microtubules was approximately 70-100% that of the axoneme. After extrusion, axonemes continued to swim slowly backwards in the reactivation medium, with a trailing central pair attached like a tail to the flagellar tip. During bending movement of the axonemes, partially extruded central pairs rotated counterclockwise about the axoneme axis, as viewed from the distal end (Kamiya, R., 1982, Cell Motil. [Suppl.]:169-173). Axonemes swam backwards with a symmetric waveform and a beat frequency of approximately 10 Hz in the reactivation medium containing 10(-9)-10(-4) M Ca ions. Even at a lower Ca++ concentration, no ciliary-type swimming was noted on the axonemes.     

Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild- type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke- deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP- mediated mechanism.     

Mutations in the "dynein regulatory complex" alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes

To understand mechanisms of regulation of dynein activity along and around the axoneme we further characterized the "dynein regulatory complex" (drc). The lack of some axonemal proteins, which together are referred to as drc, causes the suppression of flagellar paralysis of radial spoke and central pair mutants. The drc is also an adapter involved in the ATP-insensitive binding of I2 and I3 inner dynein arms to doublet microtubules. Evidence supporting these conclusions was obtained through analyses of five drc mutants: pf2, pf3, suppf3, suppf4, and suppf5. Axonemes from drc mutants lack part of I2 and I3 inner dynein arms as well as subsets of seven drc components (apparent molecular weight from 29,000 to 192,000). In the absence of ATP-Mg, dynein-depleted axonemes from the same mutants bind I2 and I3 inner arms at both ATP-sensitive and -insensitive sites. At ATP-insensitive sites, they bind I2 and I3 inner arms to an extent that depends on the drc defect. This evidence suggested to us that the drc forms one binding site for the I2 and I3 inner arms on the A part of doublet microtubules.     

The control of ciliary beat frequency

Ciliary movement is powered by axonemal dynein. This article considers how a signal transduction cascade initiated at the cell membrane may activate outer dynein arms to change the velocity of microtubule sliding and the swimming speed of ciliated cells. For Paramecium, a critical event in the cascade is the cAMP-dependent phosphorylation of a 29 kDa polypeptide that is associated with the outer dynein arm.     

Different structural states of a microtubule cross-linking molecule, captured by quick-freezing motile axostyles in protozoa

Freeze-etch preparation of the laminated bundles of microtubules in motile axostyles demonstrates that the cross-bridges populating individual layers or laminae are structurally similar to the dynein arms of cilia and flagellae. Also, like dynein, they are extracted by high salt and undergo a change in tilt upon removal of endogenous ATP (while the axostyle as a whole straightens and becomes stiff). On the other hand, the bridges running between adjacent microtubule laminae in the axostyle turn out to be much more delicate and wispy in appearance, and display no similarity to dynein arms. Thus we propose that the internal or "intra-laminar" cross-bridges are the active force- generating ATPases in this system, and that they generate overall bends or changes in the helical pitch of the axostyle by altering the longitudinal and lateral register of microtubules in each lamina individually; e.g., by "warping" each lamina and creating longitudinal shear forces within it. The cross-links between adjacent laminae, on the other hand, would then simply be force-transmitting elements that serve to translate the shearing forces generated within individual laminae into overall helical shape changes. (This hypothesis differs from the views of earlier workers who considered a more active role for the later cross-links, postulating that they cause an active sliding between adjacent layers that somehow leads to axostyle movement.) Also described here are physical connections between adjacent intra-laminar cross-bridges, structurally analogous to the overlapping components of the outer dynein arms of cilia and flagella. As with dynein, these may represent a mechanism for propagating local changes from cross-bridge to cross-bridge down the axostyle, as occurs during the passage of bends down the length of the organelle.