Tektin 4 is located on outer dense fibers, not associated with axonemal tubulins of flagella in rodent spermatozoa

Tektins, which are thought to be the constitutive proteins of microtubules in cilia, flagella, basal bodies, and centrioles, have been reported to be involved in the stability and structural complexity of axonemal microtubules. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin 2 and Tektin 4, have been verified to be present in sperm flagella. To elucidate the molecular localization of Tektin 4 in flagella of rodent spermatozoa, we performed immunocytochemistry, fractionation study followed by immunoblot analysis, and immunogold electron microscopy. Confocal laser scanning microscopy and immunogold electron microscopy indicated that Tektin 4 was associated with outer dense fibers (ODFs) in both the middle and principal piece of flagella in rat and mouse spermatozoa. Tektin 4 in rat spermatozoa is completely released by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Pre-embedding immunoelectron microscopy demonstrated that Tektin 4 located on the abaxial (convex) surface of ODFs in flagella, not associate with axonemal microtubules. Our data strongly suggested that Tektin 4 is not associated with axonemal tubulins but an ODFs-affiliated molecule in rodent spermatozoa.     

Tektin5, a new Tektin family member, is a component of the middle piece of flagella in rat spermatozoa

Tektins are composed of a family of filament-forming proteins localized in cilia and flagella. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin2 and Tektin4, have been verified to be present in sperm flagella. A new member of the TEKTIN gene family, which was designated as rat Tektin5, was obtained by PCR technique. Rat Tektin5 cDNA consists of 1,674 bp encoding a 62.8 kDa protein of 558 amino acids. Tektin5 protein contains a Tektin domain as well as a nonapeptide signature sequence that is a prominent feature of Tektin proteins. RT-PCR analysis indicated that Tektin5 was predominantly expressed in testis and that its expression was up-regulated during testis development. Immunoblot analyses revealed that Tektin5 is present in sperm flagella but not in heads and that it is completely released from rat spermatozoa by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Confocal laser scanning microscopy revealed that Tektin5 was located in the middle piece of flagella in rat spermatozoa with no immunolabeling in the heads and the principal piece. Immunogold electron microscopy adopting pre-embedding method discovered that Tektin5 is predominantly associated with the inner side of the mitochondrial sheath. Tektin5 might work as a middle piece component requisite for flagellar stability and sperm motility.     

Characterization of Spetex‐1, a new component of satellite fibrils associated with outer dense fibers in the middle piece of rodent sperm flagella

Spetex‐1, which has been isolated by differential display as a haploid spermatid‐specific gene, encodes a protein with two coiled‐coil motifs located in the middle piece of flagella in rodent spermatozoa. The middle piece of flagella is composed of axoneme and peri‐axonemal elements including outer dense fibers (ODFs) and satellite fibrils. Pre‐embedding immunoelectron microscopy clearly demonstrated that Spetex‐1 is located at satellite fibrils associated with ODFs in the middle piece of flagella of rat spermatozoa. Extraction of Spetex‐1 from spermatozoa by SDS or urea required dithiothreitol, suggesting crosslinking by disulfide bond is involved in the assembly of satellite fibrils containing Spetex‐1. We identified putative Spetex‐1 orthologs in many animal species, and both cysteine residues and coiled‐coil motifs were well conserved in mammalian orthologs of Spetex‐1. When Spetex‐1 was co‐transfected into COS‐7 cells with myc‐tagged Tektin4, another filamentous protein associated with ODFs, the two molecules were co‐localized in various sizes of aggregates in the cells. These data suggested that Spetex‐1, a new component of satellite fibrils, might be involved in the structural stability of the sperm flagellar middle piece and functions in co‐operation with Tektin4. Mol. Reprod. Dev. 77: 363–372, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular Cloning and Subcellular Localization of Tektin2-Binding Protein 1 (Ccdc 172) in Rat Spermatozoa

Tektins (TEKTs) are composed of a family of filament-forming proteins localized in cilia and flagella. Five types of mammalian TEKTs have been reported, all of which have been verified to be present in sperm flagella. TEKT2, which is indispensable for sperm structure, mobility, and fertilization, was present at the periphery of the outer dense fiber (ODF) in the sperm flagella. By yeast two-hybrid screening, we intended to isolate flagellar proteins that could interact with TEKT2, which resulted in the isolation of novel two genes from the mouse testis library, referred as a TEKT2-binding protein 1 (TEKT2BP1) and -protein 2 (TEKT2BP2). In this study, we characterized TEKT2BP1, which is registered as a coiled-coil domain-containing protein 172 (Ccdc172) in the latest database. RT-PCR analysis indicated that TEKT2BP1 was predominantly expressed in rat testis and that its expression was increased after 3 weeks of postnatal development. Immunocytochemical studies discovered that TEKT2BP1 localized in the middle piece of rat spermatozoa, predominantly concentrated at the mitochondria sheath of the flagella. We hypothesize that the TEKT2-TEKT2BP1 complex might be involved in the structural linkage between the ODF and mitochondria in the middle piece of the sperm flagella.     

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.     

The restructuring of the flagellar base and the flagellar necklace during spermatogenesis of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Transmission electron microscopy was used to study the development of the flagellar base and the flagellar necklace during spermatogenesis in a moth (Ephestia kuehniella Z.). Until mid-pachytene, two basal body pairs without flagella occur per cell. The basal bodies, which contain a cartwheel complex, give rise to four flagella in late prophase I. The cartwheel complex appears to be involved in the nucleation of the central pair of axonemal microtubules. In spermatids, there is one basal body; this is attached to a flagellum. At this stage, the nine microtubular triplets of the basal body do not terminate at the same proximal level. The juxtanuclear triplets are shifted distally relative to the triplets distant from the nuclear envelope. Transition fibrils and a flagellar necklace are formed at the onset of axoneme elongation. The flagellar necklace includes Y-shaped elements that connect the flagellar membrane and the axonemal doublets. In spindle-containing spermatocytes, the flagellar necklace is no longer detectable. During spermatid differentiation, the transition fibrils move distally along the axoneme and a prominent middle piece appears. Our observations and those in the literature indicate certain trends in sperm structure. In sperms with a short middle piece, we expect the presence of a flagellar necklace. The distal movement of the transition fibrils or equivalent structures is prevented by the presence of radial linkers between the flagellar membrane and the axonemal doublets. On the other hand, the absence of a flagellar necklace at the initiation of spermiogenesis enables the formation of a long middle piece. Thus, in spermatozoa possessing an extended middle piece, a flagellar necklace may be missing.     

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.     

Application of stains-all staining to the analysis of axonemal tubulins: identification of beta-tubulin and beta-isotubulins.

The cationic dye, Stains-all, is known to stain brain beta-tubulin blue and alpha-tubulin red (Serrano, L. et al. (1986) J. Biochem. Biophys. Methods 12, 281-287; Serrano, L. et al. (1989) Biochem. Int., 19, 235-246). The present experiments show that this stain can also be applied to detect beta-tubulin in axonemal tubulins from various sources such as cilia of protozoa, sperm flagella of echinoderm, and sperm flagella of mollusc. Furthermore, these experiments showed that it selectively stains isoforms of axonemal beta-tubulin blue following isoelectric focusing, whereas those of alpha-tubulin are stained red. These results indicate that Stains-all staining is a useful tool for electrophoretic analysis of axonemal tubulins.     

Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP‐dependent hyperactivation in the spermatozoa of Japanese Black bulls

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen‐stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell‐permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine‐phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen‐stored spermatozoa were classified as having a high‐grade acrosome condition before incubation, sperm tyrosine‐phosphorylated proteins, including the 33‐kDa tyrosine‐phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41‐ and 33‐kDa sperm tyrosine‐phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine‐phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen‐stored sperm were classified as having a low‐grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine‐phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine‐phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP‐dependent hyperactivation for the spermatozoa of individual Japanese Black bulls. Mol. Reprod. Dev. 77:910–921, 2010. © 2010 Wiley‐Liss, Inc.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.     

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.     

Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility

Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein (Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J. Y., and Gibbons, B. H. (1987) J. Biol. Chem. 262, 2780-2786). The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility. It is concluded that loss of the five axonemal polypeptides upon irradiation results from a Vi-sensitized photocleavage similar to that which occurs in the alpha and beta heavy chains of outer arm dynein and that these polypeptides represent Vi-inhibitable ATPase subunits of dyneins located in the inner arms and possibly elsewhere in the flagellar axoneme.     

Inhibition of tyrosine phosphorylation of sperm flagellar proteins,outer dense fiber protein‐2 and tektin‐2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, tyrphostin‐A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin‐A47‐treated spermatozoa exhibited circular motility, which was associated with a distinct hypo‐tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000–60,000. In this study, we provide evidence on the localization of capacitation‐associated tyrosine‐phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo‐tyrosine phosphorylated major proteins of tyrphostin‐A47‐treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein‐2 and the 51 kDa protein as tektin‐2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo‐tyrosine‐phosphorylation status of outer dense fiber protein‐2 and tektin‐2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR‐tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. 77: 182–193, 2010. © 2009 Wiley‐Liss, Inc.     

A novel neuronal calcium sensor family protein, calaxin, is a potential Ca(2+)-dependent regulator for the outer arm dynein of metazoan cilia and flagella

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca²⁺ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca²⁺‐binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized. Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca²⁺‐binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF‐hand Ca²⁺‐binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross‐linking experiment showed that calaxin binds to β‐tubulin in situ. Overlay experiments further indicated that calaxin binds the β‐dynein heavy chain of outer arm dynein in the presence of Ca²⁺. Conclusions. These results suggest that calaxin is a potential Ca²⁺‐dependent modulator of outer arm dynein in metazoan cilia and flagella.     

Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae)

A comparative analysis of the distribution of tubulin types in apyrene and eupyrene sperm of Euptoieta hegesia butterflies was carried out, also verifying the presence of tubulin in lacinate appendages of the eupyrene sperm. Ultrathin sections of LR White embedded spermatids and spermatozoa were labeled for alpha, beta, gamma, alpha-acetylated and alpha-tyrosinated tubulins. Apyrene and eupyrene spermatids show the same antibody recognition pattern for tubulins. All tubulin types were detected in axonemal microtubules. Alpha and gamma tubulins were also detected on the cytoplasmic microtubules. However, for beta and tyrosinated tubulins only scattered labeling was detected on cytoplasmic microtubules and acetylated tubulin was not detected. In apyrene and eupyrene spermatozoa only the axoneme labeling was analyzed since cytoplasmic microtubules no longer exist in these cells. Alpha, beta and tyrosinated tubulins were easily detected on the apyrene and eupyrene axoneme; gamma tubulin was strongly marked on eupyrene axonemes but was scattered on the apyrene ones. Acetylated tubulin appeared with scattered labeling on the axoneme of both sperm types. Our results demonstrate significant differences in tubulin distribution in apyrene and eupyrene axonemal and cytoplasmic microtubules. Extracellular structures, especially the lacinate appendages, were not labeled by antibodies for any tubulin.     

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.     

Tektin 3 is required for progressive sperm motility in mice

Tektins are evolutionarily conserved flagellar (and ciliary) filamentous proteins present in the axoneme and peri-axonemal structures in diverse metazoan species. We have previously shown that tektin 3 (TEKT3) and tektin 4 (TEKT4) are male germ cell-enriched proteins, and that TEKT4 is essential for coordinated and progressive sperm motility in mice. Here we report that male mice null for TEKT3 produce sperm with reduced motility (47.2% motility) and forward progression, and increased flagellar structural bending defects. Male TEKT3-null mice however maintain normal fertility in two different genetic backgrounds tested, in contrast to TEKT4-null mice. Furthermore, male mice null for both TEKT3 and TEKT4 show subfertility on a mixed B6;129 genetic background, significantly different from either single knockouts, suggesting partial nonredundant roles for these two proteins in sperm physiology. Our results suggest that tektins are potential candidate genes for nonsyndromic asthenozoospermia in humans.     

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.