Development of the flagellar apparatus during the cell cycle in unicellular algae

Summary Recent evidence has shown that algal cells acquire different flagella and a heterogeneous basal apparatus through the prolonged development of these structures over more than one cell cycle. A system for numbering algal flagella and basal bodies, which is based on developmental studies, is discussed along with the various means by which the flagellar/basal body developmental cycle can be determined. We review the information now available on development of the separate components of the flagellar apparatus-this comes particulary from the Chlorophyta and the Chromophyta-and attempt to elucidate any information which may help in phylogenetic comparisons. New data is provided on developmental changes in the cartwheel part of the basal body and basal body-associated connecting fibrils in green algae.Abbreviations Bb basal body - d right (dexter) root - df right fibrils connecting Bb triplets to microtubular and/or fibrous roots - EM electron microscopy - F flagellum - IMF immunofluorescence microscopy - LM light microscopy - NBBC nucleus-basal body connector - s left (sinister) root - sf 3left fibrils connecting Bb triplets to microtubular and/or fibrous roots. See Nomenclature section of Introduction for the numbering of basal bodies and their flagella; the same numbers apply to Bb-associated d and s roots, and df and sf fibrils     

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.     

Distribution of polyglutamylated tubulin in the flagellar apparatus of green flagellates

Polyglutamylation is a widely distributed posttranslational modification of tubulin that can be demonstrated either by biochemical analysis or by the use of specific antibodies like GT335. Western blotting using GT335 demonstrated that polyglutamylated tubulin is enriched in isolated basal apparatus of Spermatozopsis similis. Single- and double-labeling experiments, using indirect immunofluorescence and immunogold electron microscopy of isolated cytoskeletons of S. similis and Chlamydomonas reinhardtii, revealed that polyglutamylated tubulin was predominately present in the basal bodies and the proximal part of the axonemes. Using immunogold labeling of whole mounts of Spermatozopsis cytoskeletons, we obtained evidence for a predominant occurrence of polyglutamylated tubulin in the B-tubule of the axonemal doublets. Polyglutamylation occurs early during premitotic basal body assembly in S. similis, whereas the probasal bodies of Chlamydomonas, which are present through interphase, showed a reduced staining with GT335 indicating that polyglutamylation is involved in basal body maturation. During flagella regeneration of C. reinhardtii, polyglutamylation preceded detyrosination and became visible shortly after the onset of flagellar regeneration. In C. reinhardtii and S. similis polyglutamylated tubulin was absent or highly reduced in the flagellar transition region, a specialized part of the flagellum linking the basal body to the axoneme. Furthermore, the transition region and the neighboring part of the axoneme showed reduced staining with L3, an antibody directed against detyrosinated tubulin. The results indicate that differences in the modification pattern can occur in a confined area of individual microtubules. The deficiency of polyglutamylated and detyrosinated tubulin in the transition region could have functional implications for flagellar turnover or excision.     

Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii

Chlamydomonas cells excise their flagella in response to a variety of experimental conditions (e.g., extremes of temperature or pH, alcohol or detergent treatment, and mechanical shear). Here, we show that flagellar excision is an active process whereby microtubules are severed at select sites within the transition zone. The transition zone is located between the flagellar axoneme and the basal body; it is characterized by a pair of central cylinders that have an H shape when viewed in longitudinal section. Both central cylinders are connected to the A tubule of each microtubule doublet of the transition zone by fibers (approximately 5 nm diam). When viewed in cross section, these fibers are seen to form a distinctive stellate pattern characteristic of the transition zone (Manton, I. 1964. J. R. Microsc. Soc. 82:279-285; Ringo. D. L. 1967. J. Cell Biol. 33:543-571). We demonstrate that at the time of flagellar excision these fibers contract and displace the microtubule doublets of the axoneme inward. We believe that the resulting shear force and torsional load act to sever the axonemal microtubules immediately distal to the central cylinder. Structural alterations of the transition zone during flagellar excision occur both in living cells and detergent-extracted cell models, and are dependent on the presence of calcium (greater than or equal to 10(-6) M). Immunolocalization using monoclonal antibodies against the calcium-binding protein centrin demonstrate the presence of centrin in the fiber-based stellate structure of the transition zone of wild-type cells. Examination of the flagellar autotomy mutant, fa-1, which fails to excise its flagella (Lewin, R., and C. Burrascano. 1983. Experientia. 39:1397-1398), demonstrates that the fa-1 lacks the ability to completely contract the fibers of the stellate structure. We conclude that flagellar excision in Chlamydomonas involves microtubule severing that is mediated by the action of calcium-sensitive contractile fibers of the transition zone. These observations have led us to question whether microtubule severing may be a more general phenomenon than previously suspected and to suggest that microtubule severing may contribute to the dynamic behavior of cytoplasmic microtubules in other cells.     

Ultrastructure of the flagellar apparatus inPyramimonas octopus (Prasinophyceae)

Summary While green algae usually lack one of the outer dynein arms in the axoneme, flagella of the octoflagellated prasinophytePyramimonas octopus possess dynein arms on all peripheral doublets. The outer dynein arm on doublet no. 1 is modified, and additional structures are associated with doublets no. 2 and 6. The flagellar scales are asymmetrically arranged. Thus the two rows of thick flagellar hairscales are displaced towards doublet no. 6,i.e., in the direction of the effective stroke of each flagellum. The underlayer of small scales includes two nearly opposite double rows scales, arranged in the longitudinal direction of the flagellum. The hairscales emerge from these rows. The double rows are separated on one side by 9, on the other by 11 rows of helically arranged scales. The central pair of microtubules twists, but the axoneme itself (represented by the 9 peripheral doublets), does not seem to rotate. The flagella are arranged in two groups, showing modified 180° rotational symmetry. The effective strokes of the two central flagella are exactly opposite, while the other flagella beat in six intermediate directions.     

Three‐dimensional organization of flagellar basal apparatus in Ectocarpus gametes

The flagellar basal apparatus of the brown alga Ectocarpus siliculosus was re‐investigated in details using transmission electron microscopy and electron tomography. As a result, three‐dimensional structures with spatial arrangement of bands and microtubular flagellar rootlets were observed. Fibrous structures linking the anterior flagellar basal body to the major anterior rootlet (R3) or the bypassing rootlet was newly discovered in this study. A direct attachment from the minor anterior rootlet (R4) to the anterior and posterior basal bodies was also discovered, as were attachments from the minor posterior rootlet (R1) to the deltoid striated band and from the major posterior rootlet (R2) to the posterior fibrous band. The microtubular flagellar rootlets were connected to the bands and to the anterior or posterior basal body. These bands may have a role in maintaining the spatial arrangement of the anterior and posterior flagellar basal bodies and the microtubular flagellar rootlets. A numbering system of the basal body triplets was established by tracing axonemal doublets in the serial sections. From these observations, the precise position of two flagellar basal bodies, bands, and flagellar rootlets was determined.     

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.     

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.     

Aberrant distribution of the peri-axonemal structures in the human spermatozoon: possible role of the axoneme in the spatial organization of the flagellar components

A quantitative ultrastructural study combined with stereology was performed on semen samples from four men selected for apparently isolated anomalies of the peri-axonemal structures. Comparison of the results with those of a control group revealed a decrease: in the mean length of the principal piece; in the mean length of the 9 dense fibres and in the difference in length between the 9 dense fibres, all the fibres being approximately as long as the medium length fibres of the normal spermatozoon. In addition, longitudinal columns were single and/or in abnormal position. However, the extent of the dense fibres (along 60% of the principal piece) and their proportion within the flagellum (35.1% of the principal piece per fibre on average) were normal. Analysis of the results suggests that: A-tubules of the axonemal doublets are involved in the spatial arrangement of the peri-axonemal structures; axonemal microtubule-associated proteins (MAPs) may be responsible for this structural function; and three different types of longitudinal doublet differentiations may exist along the flagellar axoneme.     

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae)

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.     

Trypanosoma cruzi: antigenic composition of axonemes and flagellar membranes of epimastigotes cultured in vitro

Trypanosoma cruzi epimastigotes were sonicated in a medium containing sucrose, albumin, and calcium as stabilizers, to yield mainly unbroken parasites and free flagella. The latter were separated, first by differential centrifugation and finally by an isopicnic centrifugation, in a discontinuous sucrose gradient. The flagella obtained in the $\text{1.66}\text{1.84}\text{M}$ interphase show, by electron microscopy, the typical axonemal structure surrounded by the flagellar membrane and are completely free of extraneous subcellular components. They are also very homogeneous by polyacrylamide gel electrophoresis and enzyme marker criteria. The purified flagella were further subfractionated into well-preserved axonemes and a soluble flagellar membrane preparation. In order to detect in these fractions only the parasite immunogens that elicit a humoral response in humans, sera of chagasic patients were exclusively used. Indirect immunofluorescence reveals that both intact and membrane-free flagella are reactive. Passive hemagglutination and complement fixation of the flagellar membrane and axonemal fractions show a 21- and 8-fold purification, respectively, over a standard (Maekelt) antigen used for diagnostic purposes. Approximately 10% of the antigenicity of the total parasite is found in the flagellum, and two-thirds of this in the membrane. Double-immunodiffusion tests reveal the presence of two antigens in the axonemes and four in the flagellar membranes, one of which is common with one of the three antigens detected in a total parasite membrane fraction. The high degree of flagellar purification achieved here and the use of chagasic sera allow to conclude that at least six antigenic determinants for humoral response in humans are present in the flagellum of T. cruzi epimastigotes, two of them localized in the axoneme and four in the flagellar membrane.     

Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii

Previously, we reported that flagellar excision in Chlamydomonas reinhardtii is mediated by an active process whereby microtubules are severed at select sites within the flagellar-basal body transition zone (Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751- 1760). At the time of flagellar excision, stellate fibers of the transition zone contract and displace the microtubule doublets of the axoneme inward. The resulting shear force and torsional load generated during inward displacement leads to microtubule severing immediately distal to the central cylinder of the transition zone. In this study, we have used a detergent-extracted cell model of Chlamydomonas that allows direct experimental access to the molecular machinery responsible for microtubule severing without the impediment of the plasma membrane. We present four independent lines of experimental evidence for the essential involvement of centrin-based stellate fibers of the transition zone in the process of flagellar excision: (a) Detergent-extracted cell models excise their flagella in response to elevated, yet physiological, levels of free calcium. (b) Extraction of cell models with buffers containing the divalent cation chelator EDTA leads to the disassembly of centrin-based fibers and to the disruption of transition zone stellate fiber structure. This treatment results in a complete loss of flagellar excision competence. (c) Three separate anti-centrin monoclonal antibody preparations, which localize to the stellate fibers of the transition zone, specifically inhibit contraction of the stellate fibers and block calcium-induced flagellar excision, while control antibodies have no inhibitory effect. Finally, (d) cells of the centrin mutant vfl-2 (Taillon, B., S. Adler, J. Suhan, and J. Jarvik. 1992. J. Cell Biol. 119:1613-1624) fail to actively excise their flagella following pH shock in living cells or calcium treatment of detergent-extracted cell models. Taken together, these observations demonstrate that centrin-based fiber contraction plays a fundamental role in microtubule severing at the time of flagellar excision in Chlamydomonas.     

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.     

Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly

The principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. We show that these two isoelectric variants of alpha-tubulin are related to one another since posttranslational modification of the cell body precursor form converts it to the axonemal form. During flagellar assembly, precursor alpha-tubulin enters the flagella and is posttranslationally modified within the flagellar matrix fraction prior to or at the time of its addition to the growing axonemal microtubules. Experiments designed to identify the nature of this posttranslational modification have also been conducted. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo and, under these conditions, no other flagellar polypeptides exhibit detectable labeling.     

Ultrastructure of the biflagellate spermatozoon of tomopteris helgolandica greef, 1879 (annelida,polychaeta)

The spermatozoon of the polychaete Tomopteris helgolandica is of an aberrant type with two flagella, each measuring about 40μm. The nucleus is roughly conical and weakly bent. At the anterior end it is rounded and covered only by the nuclear and plasma membranes. Membraneous, electron-dense structures are applied laterally to the nucleus. These structures may have a helical arrangement. The middle piece contains about ten mitochondria, two centrioles, and two centriolar satellite complexes. The centriolar regions are connected with the posterior part of the nucleus. The axonemes of the two tail flagella lack the usual central complex with central tubules, radial spokes, or related structures. No arms seem to be present on the A tubules of the doublets. In the middle piece the tail flagella are surrounded by invaginations of the plasma membrane forming flagellar canals. The sperm has a bilateral symmetry whereas the primitive sperm has a radial symmetry. The occurrence of two tail flagella in this spermatozoon has no phylogenetical connection with biflagellate spermatozoa in other animal groups. A series of mutations has resulted in the development of two flagella emerging from the two centrioles, the lack of a central complex in the axoneme, and the lack of a typical acrosome. In the Polychaeta, sperm structure is generally more related to function that to phylogenetics. During swimming the spermatozoon of Tomopteris rotates around its longitudinal axis.     

[A functional flagella with a 6 + 0 pattern]

The male gamete of the Gregarine Lecudina tuzetae has been studied with transmission electron microscopy and microcinematography. It is characterized by a flagellar axoneme of 6 + 0 pattern, a reduction of the chondriome, and the abundance of storage polysaccharide or lipid bodies. The movements of the flagella are of the undulating type and they are performed in the three dimensions of space. They are very slow, with a cycle time of about 2s. The structure of the axoneme components are similar to those of flagella with a 9 + 2 pattern. Each doublet has overall dimensions of 350 x 220 A; the space between the adjacent doublets is about 160 A. The A subfiber bears arms like dynein arms. The diameter of the axoneme is about 1,000 A. The basal body consists of a cylinder of dense material 2,500 A long and 1,300-1,400 A in diameter; a microtubule 200 A in diameter is present in the axis. This study shows that a 6 + 0 pattern can generate a flagellar movement. The mechanism of the flagellar movement of the male gamete of L. tuzetae does not require the presence of central microtubules and it would include molecular interactions of the dynein-tubulin type between the adjacent peripheric doublets. The slowness of the movements is discussed in terms of the axoneme's structure and its energy supply. Finally, the phylogenetic significance of this flagella is examined on the basis of the morphopoietic potentialities of the centriolar structures.     

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.     

Calcium control of waveform in isolated flagellar axonemes of chlamydomonas

The effect of Ca(++) on the waveform of reactivated, isolated axonemes of chlamydomonas flagella was investigated. Flagella were detached and isolated by the dibucaine procedure and demembranated by treatment with the detergent Nonidet; the resulting axomenes lack the flagellar membrane and basal bodies. In Ca(++)-buffered reactivation solutions containing 10(-6) M or less free Ca(++), the axonemes beat with a highly asymmetrical, predominantly planar waveform that closely resembled that of in situ flagella of forward swimming cells. In solutions containing 10(-4) M Ca(++), the axonemes beat with a symmetrical waveform that was very similar to that of in situ flagella during backward swimming. In 10(-5) M Ca(++), the axonemes were predominantly quiescent, a state that appears to be closely associated with changes in axomenal waveform or direction of beat in many organisms. Experiments in which the concentrations of free Ca(++), not CaATP(--) complex were independently varied suggested that free Ca(++), not CaATP(--), was responsible for the observed changes. Analysis of the flagellar ATPases associated with the isolated axonemes and the nonidet- soluble membrane-matrix fraction obtained during preparation of the axonemes showed that the axonemes lacked the 3.0S Ca(++)-activated ATPase, almost all of which was recovered in the membrane-matrix fraction. These results indicate that free Ca(++) binds directly to an axonemal component to alter flagellar waveform, and that neither the 3.0S CaATPase nor the basal bodies are directly involved in this change.     

Ultrastructure of distal flagellar swellings in spermatocytes and spermatids of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Development of flagella was investigated by transmission electron microscopy in spermatocytes and spermatids of the Mediterranean mealmoth, Ephestia kuehniella Z. Growing flagella displayed voluminous distal swellings. In short flagella the apical portion of the swellings contained an amorphous, dense accumulation. In more developed flagella a less dense proximal extension of the apical accumulation was formed, which in turn was in contact with the elongating flagellar microtubules. The material of the flagellar tip is interpreted as being a precursor of the axoneme containing mainly tubulin. The material may be converted into the axoneme.