Computer simulation of bend propagation by axoplasmic microtubules

The generation of bending waves by microtubules in squid nerve axoplasm has been modelled using appropriately modified versions of computer programs developed previously for simulation of flagellar bending waves. The results confirm that a constant longitudinal force directed along the axis of the microtubule is sufficient to cause the generation of regular oscillations and propagated bending waves when the forward gliding movement of the microtubule is obstructed. No control mechanism is required to modulate the active force-generating system. In order to obtain bending waves similar to those observed experimentally, it was necessary to use a model for the force-generating system in which the active force decreases with increasing sliding velocity. If the elastic bending resistance of axoplasmic microtubules is similar to that of microtubules in sperm terminal filaments, the longitudinal force per unit length generated by the axoplasmic microtubules must be of the same order of magnitude as the force generated by dynein arms along the doublet microtubules of eukaryotic flagella.     

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.     

Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.     

Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro

Our goal was to assess the microtubule translocating ability of individual ATPase subunits of outer arm dynein. Solubilized outer arm dynein from sea urchin sperm (Stronglocentrotus purpuratus) was dissociated into subunits by low ionic strength buffer and fractionated by zonal centrifugation. Fractions were assessed by an in vitro functional assay wherein microtubules move across a glass surface to which isolated dynein fractions had been absorbed. Microtubule gliding activity was coincident with the 12-S beta-heavy chain-intermediate chain 1 ATPase fractions (beta/IC1). Neither the alpha-heavy chain nor the intermediate chains 2 and 3 fractions coincided with microtubule gliding activity. The beta/IC1 ATPase induced very rapid gliding velocities (9.7 +/- 0.88 micron/s, range 7-11.5 micron/s) in 1 mM ATP-containing motility buffers. In direct comparison, isolated intact 21-S outer arm dynein, from which the beta/IC1 fraction was derived, induced slower microtubule gliding rates (21-S dynein, 5.6 +/- 0.7 micron/s; beta/IC1, 8.7 +/- 1.2 micron/s). These results demonstrate that a single subdomain in dynein, the beta/IC1 ATPase, is sufficient for microtubule sliding activity.     

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.     

Clockwise translocation of microtubules by flagellar inner-arm dyneins in vitro

Cilia and flagella are equipped with multiple species of dyneins that have diverse motor properties. To assess the properties of various axonemal dyneins of Chlamydomonas, in vitro microtubule translocation by isolated dyneins was examined with and without flow of the medium. With one inner-arm dynein species, dynein c, most microtubules became aligned parallel to the flow and translocated downstream after the onset of flow. When the flow was stopped, the gliding direction was gradually randomized. In contrast, with inner-arm dyneins d and g, microtubules tended to translocate at a shallow right angle to the flow. When the flow was stopped, each microtubule turned to the right, making a curved track. The clockwise translocation was not accompanied by lateral displacement, indicating that these dyneins generate torque that bends the microtubule. The torque generated by these dyneins in the axoneme may modulate the relative orientation between adjacent doublet microtubules and lead to more efficient functioning of total dyneins.     

Structural and functional characterization of paramecium dynein: initial studies.

Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N + 1) tipward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. The 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg2+, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.     

The velocity of microtubule sliding: its stability and load dependency

It is now well understood that ATP-driven active sliding between the doublet microtubules in the sperm axoneme generates flagellar movement. However, much remains to be learned about how this movement is controlled. Detailed analyses of the flagellar beating of the mammalian spermatozoa revealed that there were two beating modes at a constant rate of microtubule sliding: that is, a nearly constant-curvature beating in nonhyperactivated spermatozoa and a nearly constant-frequency beating in hyperactivated spermatozoa. The constant rate of microtubule sliding suggests that the beat frequency and waveform of the flagellar beating are dependently regulated. Comparison of the sliding velocity of several mammalian and sea urchin sperm flagella with their mechanical property clarified that the sliding velocity of the microtubule was determined by the stiffness of the flagellum at its base, and that its relationship was expressed by a logarithmic equation that is similar to the classical force-velocity equation of the muscle contraction. Data from sea urchin spermatozoa also satisfied the equation, suggesting that the same microtubule sliding system functions in both the mammalian and echinoderm spermatozoa.     

Structural and Functional Characterization of Paramecium Dynein: Initial Studies

ABSTRACT Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N+1) upward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. the 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg^2-, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.     

The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond

We used in vitro translocation and cosedimentation assays to study the microtubule binding properties of sea urchin sperm outer arm dynein and its beta/IC1 subunit. Microtubules glided on glass-absorbed sea urchin dynein for a period of time directly proportional to the initial MgATP2- concentration and then detached when 70-95% of the MgATP2- was hydrolyzed. Detachment resulted from MgATP2- depletion, because (a) perfusion with fresh buffer containing MgATP2- reconstituted binding and gliding, (b) microtubules glided many minutes with an ATP-regenerating system at ATP concentrations which alone supported gliding for only 1-2 min, and (c) microtubules detached upon total hydrolysis of ATP by an ATP-removal system. The products of ATP hydrolysis antagonized binding and gliding; as little as a threefold excess of ADP/Pi over ATP resulted in complete loss of microtubule binding and translocation by the beta/IC1 subunit. In contrast to the situation with sea urchin dynein, microtubules ceased gliding but remained bound to glass-absorbed Tetrahymena outer arm dynein when MgATP2- was exhausted. Cosedimentation assays showed that Tetrahymena outer arm dynein sedimented with microtubules in an ATP-sensitive manner, as previously reported (Porter, M.E., and K. A. Johnson. J. Biol. Chem. 258: 6575-6581). However, the beta/IC1 subunit of sea urchin dynein did not cosediment with microtubules in the absence of ATP. Thus, this subunit, while capable of generating motility, lacks both structural and rigor-type microtubule binding.     

Structural-functional relationships of the dynein,spokes, and central-pair projections predicted from an analysis of the forces acting within a flagellum

In the axoneme of eukaryotic flagella the dynein motor proteins form crossbridges between the outer doublet microtubules. These motor proteins generate force that accumulates as linear tension, or compression, on the doublets. When tension or compression is present on a curved microtubule, a force per unit length develops in the plane of bending and is transverse to the long axis of the microtubule. This transverse force (t-force) is evaluated here using available experimental evidence from sea urchin sperm and bull sperm. At or near the switch point for beat reversal, the t-force is in the range of 0.25-1.0 nN/ micro m, with 0.5 nN/ micro m the most likely value. This is the case in both beating and arrested bull sperm and in beating sea urchin sperm. The total force that can be generated (or resisted) by all the dyneins on one micron of outer doublet is also approximately 0.5 nN. The equivalence of the maximum dynein force/ micro m and t-force/ micro m at the switch point may have important consequences. Firstly, the t-force acting on the doublets near the switch point of the flagellar beat is sufficiently strong that it could terminate the action of the dyneins directly by strongly favoring the detached state and precipitating a cascade of detachment from the adjacent doublet. Secondly, after dynein release occurs, the radial spokes and central-pair apparatus are the structures that must carry the t-force. The spokes attached to the central-pair projections will bear most of the load. The central-pair projections are well-positioned for this role, and they are suitably configured to regulate the amount of axoneme distortion that occurs during switching. However, to fulfill this role without preventing flagellar bend formation, moveable attachments that behave like processive motor proteins must mediate the attachment between the spoke heads and the central-pair structure.     

Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation

It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.     

One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved Structures

The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1–2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.     

Ap58: a novel in situ outer dynein arm-binding protein

Outer arm dynein is a molecular motor that is positioned at 24 nm intervals on outer doublet microtubules in cilia and flagella. In the present paper, we report identification of a 58 kDa novel protein with a tetratricopeptide repeat (TPR), referred to as ap58 (for 58 kDa axonemal protein) in sea urchin sperm axonemes. Ap58 is extracted along with the outer arm dynein by a high salt solution from axonemes. Sucrose density gradient centrifugation or gel filtration of the extract separates the outer arm dynein core from ap58. Most ap58 sediments to the lower density fraction or elutes in fractions of smaller molecules. However, immunogold localization reveals that ap58 is distributed at approximately 25 nm intervals on doublet microtubules, suggesting that in situ it is associated with the outer dynein arm. Thus, ap58 with the TPR motif is a new member of outer dynein arm-binding proteins distinct from the outer dynein arm-docking complex.     

Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.     

Inhibition of gliding movement by calcium in doublet microtubules on Tetrahymena ciliary dyneins in vitro.

We examined the effects of Ca ions on the gliding movement of Tetrahymena ciliary doublet microtubules induced by 14S or 22S dyneins in an in vitro motility assay system. The doublet microtubule appeared as circular-arc in solution, about 5 to 6 microns in length [1]. The doublet microtubules glided distal-end first on a 14S or 22S dynein-coated glass surface either clockwise or counterclockwise following the addition of ATP. The diameter of the circular path changed according to Ca concentration in the solution. Gliding velocity was from 1 to 5 microns/s. The addition of 0.1% Nonidet P-40 was necessary to induce the gliding movement on 22S dynein. This movement on 22S dynein was strongly inhibited above 0.5 mM ATP in the presence of 10(-9) M Ca, and at 0.05 to 1 mM ATP in the presence of 10(-3) M Ca. Many studies have indicated that Ca ions regulate ciliary movement [2-8] in which dyneins and doublet microtubule in the axoneme may play an essential role. The inhibition of the gliding movement of doublet microtubule on dyneins at appropriate concentrations of Ca and ATP as observed in this study may be the key for understanding Ca regulation of ciliary motility.     

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein".     

Polarized microtubule gliding and particle saltations produced by soluble factors from sea urchin eggs and embryos

In this report, we describe an in vitro system for analyzing microtubule-based movements in supernatants of sea urchin egg and embryo homogenates. Using video enhanced DIC microscopy, we have observed bidirectional saltatory particle movements on native taxol-stabilized microtubules assembled in low speed supernatants of Lytechinus egg homogenates, and gliding of these microtubules across a glass surface. A high speed supernatant of soluble proteins, depleted of organelles, microtubules, and their associated proteins supports the gliding of exogenous microtubules and translocation of polystyrene beads along these microtubules. The direction of microtubule gliding has been determined directly by observation of the gliding of flagellar axonemes in which the (+) and (-) ends could be distinguished by biased polar growth of microtubules off the ends. Microtubule gliding is toward the (-) end of the microtubule, is ATP sensitive, and inhibited only by high concentrations of vanadate. These characteristics suggest that the transport complex responsible for microtubule gliding in S2 is kinesin-like. The implications of these molecular interactions for mitosis and other motile events are discussed.     

The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena

Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.     

A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.