Bending of the “9+2” axoneme analyzed by the finite element method

Many data demonstrate that the regulation of the bending polarity of the “9+2” axoneme is supported by the curvature itself, making the internal constraints central in this process, adjusting either the physical characteristics of the machinery or the activity of the enzymes involved in different pathways. Among them, the very integrated Geometric Clutch model founds this regulation on the convenient adjustments of the probability of interaction between the dynein arms and the β-tubulin monomers of the outer doublet pairs on which they walk. Taking into consideration (i) the deviated bending of the outer doublets pairs (Cibert, C., Heck, J.-V., 2004. Cell Motil. Cytoskeleton 59, 153-168), (ii) the internal tensions of the radial spokes and the tangential links (nexin links, dynein arms), (iii) a theoretical 5 μm long proximal segment of the axoneme and (iv) the short proximal segment of the axoneme, we have reevaluated the adjustments of these intervals using a finite element approach. The movements we have calculated within the axonemal cylinder are consistent with the basic hypothesis that found the Geometric Clutch model, except that the axonemal side where the dynein arms are active increases the intervals between the two neighbor outer doublet pairs. This result allows us to propose a mechanism of bending reversion of the axoneme, involving the concerted ignition of the molecular engines along the two opposite sides of the axoneme delineated by the bending plane.     

Evidence for axonemal distortion during the flagellar beat of Chlamydomonas

In order to understand the working mechanism that governs the flagellar beat it is essential to know if the axoneme undergoes distortion during the course of the beat cycle. The rapid fixation method employed by Mitchell was able to preserve the waveform of Chlamydomonas flagella much as it appears during normal flagellar beating [Mitchell, Cell Motil Cytoskeleton 2003;56:120-129]. This conservation of the waveform suggests that the stress responsible for the production of bending is also trapped by the fixation procedure. Longitudinal sections of these well-preserved flagella were used to document variations in the relative axonemal diameter. Sections aligned to the plane of bending, showing both the central pair microtubules and outer doublets, were examined for this purpose. Micrographs were selected that continuously showed both the outer doublets and the central pair from a straight region to a curved region of the flagellum. Axoneme diameters measured from these select micrographs showed an increase in relative diameter that averaged 39 nm greater at the crest of the bent region. This constituted a 24% increase in the axoneme diameter in the bends. The transverse stress acting across the axoneme during bending was calculated from the Geometric Clutch computer model for a simulated Chlamydomonas-like flagellar beat. If we assume that this is representative of the transverse stress acting in a real flagellum, then the Young's modulus of the intact axoneme is approximately 0.02 MPa. The possibility that the distortion of the axoneme during the beat could play a significant role in regulating dynein function is discussed.     

Axonemal activity relative to the 2D/3D-waveform conversion of the flagellum

The waveform of the flagellum of the sea urchin spermatozoon is mainly planar, but its 3D-properties were evoked for dynamic reasons and described as helical. In 1975, the apparent twisting pattern of the sea urchin axoneme was described [Gibbons I. 1975. The molecular basis of flagellar motility in sea urchin spermatozoa. In: Inoué S, Stephens R, editors. Molecular and cellular movement. New York: Raven Press, p. 207-232.] and was considered to be one of the main elements involved in axonemal behaviour. Recently, planar, quasi-planar, and helical waveforms were observed when the flagellum of sea urchin sperm cells was submitted to an increase in viscosity. The quasi-planar conformation seemed to be due to the alternating torsion of the inter-bend segments [Woolley D, Vernon G. 2001. A study of helical and planar waves on sea urchin sperm flagella, with a theory of how they are generated. J. Exp. Biol. 204:1333-1345]. These three waveforms, which are due to a change in axonemal activity, are possibly used by the sperm cells to adapt their movement to variations in the physico-chemical characteristics of the medium (seawater) in which the cells normally swim. We constructed a simple model to describe qualitatively the central shear (between the axonemal doublets and the central pair) and the tangential shear (between the doublets themselves). In this model, the 3D-bending is resolved into components in two perpendicular planes and each of the nine planes of inter-doublet interaction defines a potential bending plane that is independently regulated. These shears were calculated for the three waveforms and their inter-conversion. This allowed us to propose that axoneme is resolved in successive modules delineated by abscissas where the sliding is always nil. We discuss these data concerning the axonemal machinery, and especially the alternating activity of opposite sides of (two) neutral surface(s) that seem(s) to be responsible for this inter-conversion, and for the possible twist of the axoneme during the beating.     

Elastic extension and jump of the flagellar nexin links: a theoretical mechanical cycle.

The functions of the nexin links of a flagellar axoneme have not been clearly demonstrated. Taking into account both the elastic (Hookean) characteristics and the possible jump of the nexin links, we calculated the sliding to bending conversion of a theoretical model in a tip-ward direction step by step, according to the essential principles proposed by the geometric clutch hypothesis [Lindemann, 1994: J Theoret Biol 168:175-189]: the activity of the dynein arms depends on the transverse forces induced by the axonemal curvature. In our calculations, however, the transverse forces that are involved in the regulation of the activities of the dynein arms were due to the extension of the nexin links located upstream of a given abscissa. This allowed us to define a bent segment as the axonemal portion at whose proximal and distal ends the nexin links were relaxed, and as fully extended as possible, respectively. The model creates an apparent disorder in the orientation of the nexin links as already observed [Bozkurt and Wooley, 1993: Cell Motil Cytoskeleton 24:109-118; Wooley, 1997: J Cell Sci 110:85-94]. We propose that the nexin links are involved in a mechanical cycle, whose 3 stages are (1) rapid extension, (2) slow relaxation, and (3) stand-by. The rapid extension is compatible with the mechanical interactions between the nexin links and the inner dynein arms with which they form the dynein regulatory complex. This was correlated with the oscillating properties of the nexin links along the axoneme that allow them to be one of the regulatory elements of the local ATPase activity of the dynein arms.     

Geometry drives the "deviated-bending" of the bi-tubular structures of the 9 + 2 axoneme in the flagellum

The axoneme "9 + 2" is basically a system constituted of a cylinder of 9 microtubule doublets surrounding a central pair of microtubules. These bi-tubular structures are considered as the support system of the active molecular complexes that generate and regulate the axonemal movement. Schoutens has calculated their moments of inertia [Schoutens, 1994: Journal of Theoretical Biology 171:163-177]. The results obtained allowed us to assume that these bi-tubular systems are endowed with dynamic properties that could be involved in the regulation of the axonemal machinery. For the first time, using the finite elements methods and the resistance of material principles, we have now calculated that the curvature of the axoneme induces the deviated-bending of the bi-tubular structures of the axoneme, because of their geometry only; they behave as beams in a framework. This approach is similar to the one used to measure the deflection of a single microtubule [Kasas et al., 2004: Chem Phys Chem 5:252-257]. These behaviors induce internal movement or constraints of either couples or triplets of doublets within the axonemal cylinder that could be directly involved in a constrained or a spontaneous "convergence/divergence" equilibrium of the cylindrical generatrices that they draw along the axonemal cylinder, which could apparently regulate the activity of the axonemal motors (the dynein arms). These results are discussed here, taking into consideration the dynamic propagation of the wave train along the flagellar axoneme, and the regulated balance between the activities of the two opposite sides of the axoneme during the beat. This study raises a few questions about the architecture-activity duo of the axonemal doublets.     

Mutations in genes encoding inner arm dynein heavy chains in Tetrahymena thermophila lead to axonemal hypersensitivity to Ca2+

Calcium-dependent ciliary reversals are seen in ciliated protozoans such as Tetrahymena in response to depolarizing stimuli, but the axonemal mechanisms responsible for this response are not well understood. The model is that the outer arm dyneins (OADs) control the beating frequency while the inner arm dyneins (IADs) regulate ciliary waveform. Since ciliary reversal is a type of waveform change, the model would predict that IAD mutations could affect ciliary reversal. We have used gene disruption techniques to generate several behavioral mutants of Tetrahymena with functional disruptions of various IADs. One such mutant, called KO-6, is missing I1 (the two-headed IAD) and is unable to show ciliary reversals in response to any stimuli due to a loss of axonemal Ca2+ sensitivity [Eur J Cell Biol 80 (2001) 486-497; Cell Motil Cytoskeleton 53 (2002) 281-288.]. In contrast, disruption of 3 one-headed IADs [Liu et al., Cell Motil Cytoskeleton 59 (2004), 201-214] produced mutants, which showed over-responsiveness in bioassays measuring either their depolarization-induced avoiding reactions (AR) in Na+ and Ba2+ solutions or their duration of backward swimming (continuous ciliary reversal or CCR) in K+ solutions. Detergent-extracted and reactivated mutants also showed increased probabilities of CCR at lower Ca2+ concentrations suggesting that the behavioral over-responsiveness of these three mutants in vivo is due to increased axonemal Ca2+ sensitivity. Our data suggest the possibility that the one-headed IADs and the two-headed IAD act antagonistically in vivo and that loss of any one of the one-headed IADs leads to behavioral over-responsiveness due to less resistance to I1-induced reversals.     

Axoneme specialization embedded in a "generalist" beta-tubulin

The relationship between the primary structure of the beta-tubulin C-terminal tail (CTT) and axoneme structure and function is explored using the spermatogenesis-specific beta2-tubulin of Drosophila. We previously showed that all beta-tubulins used for motile 9 + 2 axonemes contain a conserved sequence motif in the proximal part of the CTT, the beta-tubulin axoneme motif. The differential ability of tubulin isoforms and abilities of beta2-tubulin C-terminal truncations to form axonemes led us to hypothesize that the axoneme motif is essential for axoneme formation and the distal half of the CTT was less important. The studies we report here indicate that it is not that simple. Unexpectedly, some changes in the core sequence of the axoneme motif did not disrupt formation of motile axonemes. And, while deletion of the distal CTT did not disrupt the ability to produce functional sperm [Popodi et al., Cell Motil Cytoskeleton 2005;62:48-64], changing the amino acid sequence in this region can. Thus both regions are important. The deep conservation of the axoneme motif in all eukaryotic groups implies that the presence of the sequence motif confers a functional advantage. The central pair is the axoneme structure most sensitive to perturbations in tubulin molecules; we hypothesize central pair assembly is facilitated by the presence of this motif. Our data reveal that beta2-tubulin has robust properties for axoneme assembly, and that axonemal specializations are embedded in both the CTT and the body of the beta2 molecule.     

Disruption of genes encoding predicted inner arm dynein heavy chains causes motility phenotypes in Tetrahymena

The multi-dynein hypothesis [Asai, 1995: Cell Motil Cytoskeleton 32:129-132] states: (1) there are many different dynein HC isoforms; (2) each isoform is encoded by a different gene; (3) different isoforms have different functions. Many studies provide evidence in support of the first two statements [Piperno et al., 1990: J Cell Biol 110:379-389; Kagami and Kamiya, 1992: J Cell Sci 103:653-664; Gibbons, 1995: Cell Motil Cytoskeleton 32:136-144; Porter et al., 1996: Genetics 144:569-585; Xu et al., 1999: J Eukaryot Microbiol 46:606-611] and there is evidence that outer arms and inner arms play different roles in flagellar beating [Brokaw and Kamiya, 1987: Cell Motil. Cytoskeleton 8:68-75]. However, there are few studies rigorously testing in vivo whether inner arm dyneins, especially the 1-headed inner arm dyneins, play unique roles. This study tested the third tenet of the multi-dynein hypothesis by introducing mutations into three inner arm dynein HC genes (DYH8, 9 and 12) that are thought to encode HCs associated with 1-headed inner arm dyneins. Southern blots, Northern blots, and RT-PCR analyses indicate that all three mutants (KO-8, 9, and 12) are complete knockouts. Each mutant swims slower than the wild-type cells. The beat frequency of KO-8 cells is lower than that of the wild-type cells while the beat frequencies of KO-9 and KO-12 are not different from that of wild-type cells. Our results suggest that each inner arm dynein HC is essential for normal cell motility and cannot be replaced functionally by other dynein HCs and that not all of the 1-headed inner arm dyneins play the same role in ciliary motility. Thus, the results of our study support the multi-dynein hypothesis [Asai, 1995: Cell Motil Cytoskeleton 32:129-132].     

Are the local adjustments of the relative spatial frequencies of the dynein arms and the beta-tubulin monomers involved in the regulation of the "9+2" axoneme?

The “9+2” axoneme is a highly specific cylindrical machine whose periodic bending is due to the cumulative shear of its 9 outer doublets of microtubules. Because of the discrete architecture of the tubulin monomers and the active appendices that the outer doublets carry (dynein arms, nexin links and radial spokes), this movement corresponds to the relative shear of these topological verniers, whose characteristics depend on the geometry of the wave train. When an axonemal segment bends, this induces the compressed and dilated conformations of the tubulin monomers and, consequently, the modification of the spatial frequencies of the appendages that the outer doublets carry. From a dynamic point of view, the adjustments of the spatial frequencies of the elements of the two facing verniers that must interact create different longitudinal periodic patterns of distribution of the joint probability of the molecular interaction as a function of the location of the doublet pairs around the axonemal cylinder and their spatial orientation within the axonemal cylinder. During the shear, these patterns move along the outer doublet intervals at a speed that ranges from one to more than a thousand times that of sliding, in two opposite directions along the two opposite halves of the axoneme separated by the bending plane, respecting the polarity of the dynein arms within the axoneme. Consequently, these waves might be involved in the regulation of the alternating activity of the dynein arms along the flagellum, because they induce the necessary intermolecular dialog along the axoneme since they could be an element of the local dynamic stability/instability equilibrium of the axoneme. This complements the geometric clutch model [Lindemann, C., 1994. A “geometric clutch” hypothesis to explain oscillations of the axoneme of cilia and flagella. J. Theor. Biol. 168, 175-189].     

Composition and organization of tubulin isoforms reveals a variety of axonemal models

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.     

Ca/Ba/Sr-induced conformational changes of ciliary axonemes

Macrocilia of the ctenophore Bero? undergo Ca/Ba/Sr-dependent activation of ciliary beating and microtubule sliding disintegration [Tamm, J. Comp. Physiol. A163:23-31, 1988a; Tamm, Cell Motil. Cytoskeleton 11:126-138, 1988b; Tamm, Cell Motil. Cytoskeleton 12:104-112, 1989; Tamm and Tamm, Proc. Natl. Acad. Sci. U.S.A. 86:6987-6991, 1989]. Here we report that detergent-extracted macrocilia show an ATP-independent conformational change in response to high concentrations of Ca, Ba, or Sr ions. When applied locally by iontophoresis, these ions induce a rapid planar curvature of the distal end of the macrociliary shaft, followed by a slower relaxation to the rest position. Tip curling occurs in a direction opposite to the physiological Ca/Ba/Sr response. When applied uniformly in the bath, a threshold concentration of 10(-1) M Sr is required to induce curling of the tip, and the distal ends remain curved. Calmodulin antagonists do not inhibit the tip curling response. Previous workers found that Ca induces changes in the helical shape of isolated doublet microtubules [Miki-Noumura and Kamiya, Exp. Cell Res. 97:451-453, 1976; Miki-Noumura and Kamiya, J. Cell Biol. 81:355-360, 1979; Takasaki and Miki-Noumura, J. Mol. Biol. 158:317-324, 1982] and sperm axonemes [Okuno and Brokaw, Cell Motil. 1:349-362, 1981] and suggested that conformational changes in microtubules may play a role in Ca regulation of ciliary motility. We propose that the Ca/Ba/Sr-induced curling of the macrociliary tip is due to similar helical changes of doublet microtubules, some of which in macrocilia are prevented from sliding by permanent connections (compartmenting lamellae) between adjacent axonemes within the shaft. Although the tip curling response does not appear to be directly relevant to the physiological Ca response of macrocilia, it provides a novel system for investigating Ca-induced conformational changes of microtubules independent of dynein-powered active sliding.     

Synchronous triggering of trout sperm is followed by an invariable set sequence of movement parameters whatever the incubation medium.

The movement of live trout spermatozoa is very brief (25 sec at 20 degrees C) and conditions have been developed to get synchronous initiation of sperm motility which allowed quantification of the major parameters of sperm movement during the motility phase. Recorded flagellar beat frequencies decreased steadily from values of 55 Hz at the beginning to 20 Hz at the end of the motility phase. Sperm forward velocities followed a similar pattern from 250 to 20 microns.sec-1 in the same conditions and the diameters of sperm trajectories were reduced from 370 to 40 microns. Thus none of the characteristics of sperm movement was constant during the motile phase which ended abruptly by a straightening of the flagella. The decrease in flagellar beat frequencies and sperm velocities are much greater than what could be extrapolated from the decrease of intracellular ATP (Christen R. et al: Eur. J. Biochem, 166: 667-671, 1987) or from measurements of ATP-dependence of reactivated sperm velocities (Okuno M. and Morisawa N.: In Biological Functions of Microtubules and Related Structures. New York: Academic Press, pp. 151-162, 1982). Therefore, the cessation of flagellar beating at 25 sec is not directly the result of the low concentration of intracellular ATP. The decrease in the diameters of sperm trajectories which occurred during the first part of the motility phase was correlated with [Ca]i measurements (Cosson M.P. et al, Cell Motil. Cytoskeleton, 14:424-434, 1989). The effect of Ca2+ at the axonemal level does not indicates that Ca2+ influx is previous to flagellar beating but rather suggests a classical Ca2+ regulation of the flagellar assymetry. The short duration of the motility phase and the characteristics of sperm movement were very similar in various conditions (high external K+, low pH media) where increased external Ca2+ or divalent ions were shown to overcome K+ and H+ inhibition of sperm motility, both conditions which have been shown to depolarize the plasma membrane potential (Gatti J.L. et al: J. Cell Physiol., 143:546-554, 1990). The present study of the parameters of sperm movement suggests that once motility is initiated, a defined set of axonemal events will take place whatever the external conditions.     

Three-dimensional structure of the Trypanosome flagellum suggests that the paraflagellar rod functions as a biomechanical spring

Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture. Trypanosoma brucei is a uniflagellated protozoan parasite that causes African sleeping sickness. The T. brucei flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the T. brucei flagellum. We define the T. brucei axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57 nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of T. brucei cell motility.     

Flagellate spermatozoa of Protura (Insecta,Apterygota) are motile

A new model of sperm axoneme with 16 + 0 doublets is described. The spermatozoon of Acerentulus confinis (Apterygota : Protura) has a short conical acrosome, a long helicoidal nucleus, well-developed centriolar adjunct material, and a long flagellum. Using fixation with a glutaraldehyde-tannic acid mixture, without osmium post-fixation, doublet protofilaments, inner dynein arms, radial spokes, nexin bridges, and Y-links of the sperm axoneme of A. confinis and Acerentomon italicum were clearly observed. Optical observation shows that the proturan flagellate spermatozoa are motile cells. The process involving the transformation of the spermatozoa from a coiled to an elongated swimming form was studied by scanning electron microscope. The findings confirmed that flagellar motility is due to the presence of a single dynein arm on doublets in spite of the unusual axonemal pattern.     

Flagellar motility contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system

The flagellum of Trypanosoma brucei is a multifunctional organelle with critical roles in motility and other aspects of the trypanosome life cycle. Trypanin is a flagellar protein required for directional cell motility, but its molecular function is unknown. Recently, a trypanin homologue in Chlamydomonas reinhardtii was reported to be part of a dynein regulatory complex (DRC) that transmits regulatory signals from central pair microtubules and radial spokes to axonemal dynein. DRC genes were identified as extragenic suppressors of central pair and/or radial spoke mutations. We used RNA interference to ablate expression of radial spoke (RSP3) and central pair (PF16) components individually or in combination with trypanin. Both rsp3 and pf16 single knockdown mutants are immotile, with severely defective flagellar beat. In the case of rsp3, this loss of motility is correlated with the loss of radial spokes, while in the case of pf16 the loss of motility correlates with an aberrant orientation of the central pair microtubules within the axoneme. Genetic interaction between trypanin and PF16 is demonstrated by the finding that loss of trypanin suppresses the pf16 beat defect, indicating that the DRC represents an evolutionarily conserved strategy for dynein regulation. Surprisingly, we discovered that four independent mutants with an impaired flagellar beat all fail in the final stage of cytokinesis, indicating that flagellar motility is necessary for normal cell division in T. brucei. These findings present the first evidence that flagellar beating is important for cell division and open the opportunity to exploit enzymatic activities that drive flagellar beat as drug targets for the treatment of African sleeping sickness.     

Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella

Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo-electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named "radial spoke 3 stand-in," which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.     

Geometric Clutch model version 3: the role of the inner and outer arm dyneins in the ciliary beat

The Geometric Clutch model of ciliary and flagellar beating uses the transverse force (t-force) that develops between the outer doublets of the axoneme as the regulator for activating and deactivating the dynein motors and organizing the flagellar beat. The version of the model described here adds detail to the formulations used in the two previous versions as follows: (1) In place of two opposing sets of dyneins, the new model has four sets of dyneins, corresponding to two sets on each side of the axoneme acting in series. (2) The four sets of dyneins are each subdivided into two ranks representing inner and outer arm dyneins. (3) The force produced by each dynein is governed by a force-velocity relationship that is independently specified for the inner and outer arms. Consistent with the original model, the new version of the Geometric Clutch model can simulate both the effective and recovery stroke phases of the ciliary beat using a single uniform algorithm. In addition, the new version can operate with the outer arms disabled. Under this condition, the simulation exhibits a beat pattern similar to the original but the beat frequency is reduced to approximately one third. These results are contingent on using force-velocity relationships for the inner and outer arms similar to those described by Brokaw [1999: Cell Motil. Cytoskeleton 42:134-148], where the inner arms contribute most of the driving force at low shear velocities. This constitutes the first examination of the effects of the force-velocity characteristics of dynein on a cilia-like beat in a theoretical framework.     

Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body

Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.     

Accessory tubules and axonemal microtubules of Apis mellifera sperm flagellum differ in their tubulin isoform content

In the insect sperm flagellum, an extra set of nine additional microtubules, named accessory tubules, is present surrounding the axoneme. Using a sarcosyl/urea extraction, we were able to fractionate the microtubular cytoskeleton of the sperm flagellum of the insect Apis mellifera resulting in the dissociation of the axonemal microtubule protein components and the accessory tubules. This has allowed us to compare the tubulin isoform content of axonemal microtubules and accessory tubules by immunoelectron microscopy and immunoblotting using a panel of monoclonal antibodies directed against different tubulin post-translational modifications (PTMs). All the PTMs occurring in axonemal tubulin are also present in accessory tubules, which indicates the close relativeness of accessory tubules to axonemal rather than to cytoplasmic microtubules. However, our results demonstrate the presence of significant differences in the tubulin isoform content of axonemal microtubules and accessory tubules. First, the tubulin tyrosination extent of accessory tubules is far lower than that of axonemal microtubules, thus confirming at the molecular level their morphogenetic origin as outgrowths from the B-subtubule of each microtubular doublet. Second, although polyglycylation seems to occurr at the same extent in both microtubular systems, alpha-tubulin exhibits a larger amount of monoglycylated sites in axonemal microtubules than in accessory tubules. Third, a greater amount of beta-tubulin molecules is glutamylated in axonemal microtubules than in accessory tubules. Moreover, highly acidic isoforms, likely molecules with longer polyglutamate side chains, are present only in axonemal microtubules. Taken together, our data are indicative of a higher level of tubulin heterogeneity in axonemal microtubules than in accessory tubules. They also show a segregation of post-translationally modified isoforms between accessory tubules and axonemal microtubules and suggest the implication of PTMs in the functional specialization of the two microtubular systems.     

Thinking about flagellar oscillation

Bending of cilia and flagella results from sliding between the microtubular outer doublets, driven by dynein motor enzymes. This review reminds us that many questions remain to be answered before we can understand how dynein-driven sliding causes the oscillatory bending of cilia and flagella. Does oscillation require switching between two distinct, persistent modes of dynein activity? Only one mode, an active forward mode, has been characterized, but an alternative mode, either inactive or reverse, appears to be required. Does switching between modes use information from curvature, sliding direction, or both? Is there a mechanism for reciprocal inhibition? Can a localized capability for oscillatory sliding become self-organized to produce the metachronal phase differences required for bend propagation? Are interactions between adjacent dyneins important for regulation of oscillation and bend propagation? Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.