Flagellar Doublet Microtubules Research Articles

Structures of sperm flagellar doublet microtubules expand the genetic spectrum of male infertility

Sperm motility is crucial for successful fertilization. Highly decorated doublet microtubules (DMTs) form the sperm tail skeleton, which propels the movement of spermatozoa. Using cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based modeling, we determined the structures of mouse and human sperm DMTs and built an atomic model of the 48-nm repeat of the mouse sperm DMT. Our analysis revealed 47 DMT-associated proteins, including 45 microtubule inner proteins (MIPs). We identified 10 sperm-specific MIPs, including seven classes of Tektin5 in the lumen of the A tubule and FAM166 family members that bind the intra-tubulin interfaces. Interestingly, the human sperm DMT lacks some MIPs compared with the mouse sperm DMT. We also discovered variants in 10 distinct MIPs associated with a subtype of asthenozoospermia characterized by impaired sperm motility without evident morphological abnormalities. Our study highlights the conservation and tissue/species specificity of DMTs and expands the genetic spectrum of male infertility.

Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules: TEKTINS, Ca2+-BINDING PROTEINS, AND STABLE PROTOFILAMENTS

Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.

Chlamydomonas Flagellar Outer Row Dynein Assembly Protein Oda7 Interacts with Both Outer Row and I1 Inner Row Dyneins

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.

Tektin3 encodes an evolutionarily conserved putative testicular microtubules-related protein expressed preferentially in male germ cells.

Tektins are microtubule-associated cytoskeletal proteins that are expressed primarily in the male germ cell-lineage in centrioles and basal bodies and within ciliary and flagellar doublet microtubules. They are proposed to be important for axonemal architecture and microtubule stability in the sperm tail and in other ciliated and flagellar structures. Using an in silico (electronic database) subtractive approach to identify germ cell-specific genes in vertebrates, we isolated a new member of the Tektin gene family from mice, Tektin3. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses confirm that Tektin3 encodes a 1.7 kb transcript detectable preferentially in the testes of adult mice. In situ hybridization analysis in the testes revealed Tektin3 mRNA expression exclusively in late pachytene spermatocytes and early round spermatids. The putative TEKTIN3 protein shares 83.5% overall sequence identity with the human ortholog and includes a fully conserved carboxy terminal nonapeptide signature sequence present in all TEKTIN family members. In addition, using database mining, highly conserved TEKTIN3 orthologs were identified in puffer fish (Fugu rubripes) and rats (Rattus norvegicus) which shared 60.9 and 91.4% identity, respectively, with mouse TEKTIN3. The Tekt3 gene maps to murine chromosome 11 in a region that is syntenic to the human 17p12 chromosomal region containing the human TEKTIN3 gene. Our studies demonstrate that TEKTIN3 is a novel evolutionarily conserved male germ cell-enriched protein and suggest that it might perform important roles in male reproductive development and physiology.

The Mr 140,000 intermediate chain of Chlamydomonas flagellar inner arm dynein is a WD-repeat protein implicated in dynein arm anchoring.

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on beta-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/beta-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo.

The Chlamydomonas reinhardtii ODA3 gene encodes a protein of the outer dynein arm docking complex.

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the uter ynein rm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737- 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83. 4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.

The Propagation of a Zone of Activation along Groups of Flagellar Doublet Microtubules

The complexity of the 9 + 2 flagellar axoneme has made it difficult to discover the mechanism of bend propagation. We have studied a simplified preparation of mechanically "opened-out" groups of doublet microtubules that adopt a helical, ribbon-like form. From the very long sperm of the quail, ribbons of doublets up to 130 μm long have been obtained. We reactivated them photolytically by releasing ATP from caged ATP thus observing the reactivation from the beginning. The response to ATP was a reduction in the pitch and diameter of the helix, in what we refer to as an "active zone" (AZ). Ahead of the AZ was a short region of increase in helical pitch and diameter, the pre-AZ. We ascribe these two altered geometries to the development of tension between the doublets, actively (in the AZ) and passively (in the pre-AZ). The AZ/pre-AZ complex established itself at one end of a helix—almost certainly the proximal end-then it propagated toward the other end of the helix at a mean velocity of 15 μm s-1 (using 1 mM caged ATP), maintaining or increasing its length as it traveled. This is the same velocity as that for bend propagation on cylindrical axonemes detached from their basal structures. Successive propagations on the same helix were seen. Thus, active and inactive segments of the same doublet assembly can coexist, even though all parts are exposed to ATP. The motor response is seen to be a localized event that is transmitted metachronally. The propagation of activation is an intrinsic property of structures in the interdoublet gap and does not require constituents of the cytosol other than ATP and Mg2+. Since it occurs in helical ribbons (3 + 0, 4 + 0, etc.), the propagation of activity must be independent of central axonemal structures; furthermore, it cannot be dependent on the integrity of the 9 + 2 cylinder nor on any feedback from large-scale features of the waveform.

The microtubule lattice — 20 years on

In 1974, optical diffraction and image analysis indicated that tubulin dimers in the cylindrically complete A-tubule of flagellar doublet microtubules are arranged with helical symmetry, while those in the incomplete B-tubule associate differently. Recently, electron micrographs of reassembled brain microtubules decorated with kinesin heads have shown that the tubulin dimers there are arranged as in the B-tubule. The lack of symmetry of microtubules assembled in vitro prompts Linda Amos to speculate here that the assembly process in vitro may differ from that occurring in the cell.

Chapter 51 Methods for the Isolation of Tektins and Sarkosyl-Insoluble Protofilament Ribbons

This chapter discusses the methods of fractionating ciliary and flagellar microtubules into a stable subset of protofilaments (known as pf-ribbons) and to subfractionate these pf-ribbons into filaments composed of the proteins tektins. Flagellar doublet microtubules from Chlamydomonas reinhardtii could be fractionated by Sarkosyl detergent into stable ribbons of three protofilaments. Similar observations were reported for sea urchin sperm flagellar microtubules. Stability of the pf-ribbons is because of their polypeptide composition. Based on the synergistic effects of detergents and urea to disrupt protein-protein interactions, a Sarkosyl-urea extraction was devised that provided a remarkably clean fractionation of the pf-ribbons. Under optimal conditions of 0.5% Sarkosyl and 2 M urea, flagellar microtubules could be fractionated into 2- to 3-nm-diameter filaments composed almost exclusively of equimolar amounts of three proteins named tektins A (∼55 kDa), B (∼51 kDa), and C (∼47 kDa). It has become possible to isolate filaments composed of only tektins A and B. Methods for isolating pf-ribbons and tektin filaments are presented in the chapter, followed by a discussion of the characterization of tektins. These methods were developed for sperm flagellar axonemes from the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus. The methods are isolation and purification of pf-ribbons and tektin filaments, to prepare tektin filaments from pf-ribbons, and glycerination. The tektin polypeptide chains are predicted to be α-helical, and evidence indicates that tektins exist as longitudinal, heterodimeric protofilaments in the pf-ribbon and thus in flagellar A-tubules. Tektin heterodimers are linear, rodlike molecules, measuring∼48 nm long, giving the tektin filament the potential to interact with and stabilize adjacent tubulin protofilaments, as well as providing longitudinal binding sites for axonemal components with periodicities that are multiples of the 8-nm tubulin dimer and the 48-nm tektin spacing. Studies of cilia and flagella have contributed to understanding microtubule motility, structure, polarity, and assembly.

Chapter 50 In Vitro Polymerization of Tubulin from Echinoderm Sperm Flagellar Microtubules

Publisher Summary The purpose of this chapter is to outline two basic methods for the purification and in vitro polymerization of tubulin from echinoderm sperm flagellar microtubules and to summarize the uses and advantages of this system. Methods were developed for the purification and assembly of tubulin and microtubule-associated proteins (MAPS), by depolymerization at 0°C and repolymerization at 37°C, in Mg 2+ /GTP-containing buffers. Relative to the basal body (minus) and the distal (plus) ends of cilia and flagella, microtubules were defined kinetically as having plus and minus ends; the plus end corresponds to the fast-growing end with a higher on rate for tubulin assembly and it is distal relative to the cell center, whereas the minus end is a slow-growing end generally associated with the centrosomes/spindle poles in both interphase and mitotic cells. Flagellar doublet microtubules could be solubilized by sonication in Mg 2+ /GTP-containing buffers at 0°C and repolymerized by elevating the temperature to 37°C; the conditions and kinetic parameters of flagellar tubulin assembly in vitro were essentially identical to those for mammalian brain tubulin. A method developed by combining the fractionation procedure used to selectively solubilize tubulin from the B-subfiber and the need to protect the GTP binding site during solubilization to answer the question of how tubulin isoforms might influence tubulin assembly and microtubule structure. The methods described in the chapter are solubilization of doublet tubules by sonication, thermal fractionation of doublet tubules into crude, soluble B-Tubulin, and cycled polymerization and depolymerization of soluble doublet tubulin. Native doublet microtubules are complex structures consisting of an A-tubule with 13 protofilaments and an incomplete B-tubule with 10 protofilaments that are attached to the A-tubule in a specific pattern. Soluble doublet tubulin polymerizes in vitro to give singlet microtubules with variable numbers of protofilaments and these microtubules are cold labile.

Direct Evidence for Tension Development between Flagellar Doublet Microtubules

In this study of isolated ribbons of flagellar doublet microtubules, we demonstrate that a resistance to sliding exists in the interdoublet gap. By photolytically releasing ATP from caged ATP, it has been possible to follow closely the responses of individual specimens. Distortion of the helical superstructure of the doublets, most often by a reduction in helical pitch, is interpreted as revealing the development of tension between doublets. Tension does not develop in the presence of vanadate.

Tubulin tyrosination in Crithidia: modifying enzymes and modification states of tubulin.

An enzyme that adds C-terminal tyrosine to tubulin has been identified in Crithidia fasciculata. It tyrosinates Crithidia, but not brain, tubulin and is specific for the alpha chain. Crithidia cells could not be shown to fix tyrosine in the absence of protein synthesis, which is consistent with the pattern of distribution of C-terminal tyrosine in tubulin from different subcellular compartments of this protozoan. Terminal tyrosine was present in about 5% of flagellar alpha chain from cells in stationary phase and 20% from cells from midlog phase; none was detected in tubulin from cytosol or the subpellicular corset. In contrast to mammalian cells, in which a higher state of tyrosinolation characterizes recently assembled or unstable microtubules, terminal tyrosine was present only in the most stable polymer, the flagellar doublet microtubules.

Characterization of antibodies as probes for structural and biochemical studies of tektins from ciliary and flagellar microtubules.

Rabbit antibodies raised and purified against three tektins, proteins of flagellar doublet microtubules from sea-urchin sperm (Lytechinus pictus and Strongylocentrotus purpuratus), were used to study tektin biochemistry and their structural localization. Doublet microtubules were fractionated into tektin filaments and separated by SDS-PAGE into three major tektin polypeptide bands (Mr = 47, 51 and 55 (X 10(3)), which were used to immunize rabbits. Antibodies against each tektin (anti-tektins) were affinity-purified and then characterized by two-dimensional isoelectric focusing/SDS-PAGE immunoblotting and by immunofluorescence microscopy. In two-dimensional immunoblots of 0.5% Sarkosyl-resistant fractions of flagellar microtubules, the antibody against the 55 X 10(3) Mr tektin (anti-55) stained one major polypeptide of 55 X 10(3) Mr and pI 6.9, anti-51 stained two polypeptides of 51 X 10(3) Mr and pI approximately 6.15, and anti-47 stained one major polypeptide of 47 X 10(3) Mr and pI 6.15. The anti-tektins also stained several minor neighbouring polypeptides, which may be isoelectric variants, novel tektins or unrelated proteins. Furthermore, anti-47 crossreacted with the major 55 X 10(3) Mr polypeptide. By immunofluorescence microscopy all three anti-tektins stained methanol-fixed echinoderm sperm flagella and embryonic cilia. In addition, anti-47 and anti-55 stained unfixed, demembranated axonemes. Besides staining axonemes, all anti-tektins labelled the basal body region, and anti-51 labelled the sperm head envelope. These results indicate that the tektins are a complex family of proteins that are components of axonemal microtubules and possibly other cytoplasmic and nuclear structures.

Studies of tektin filaments from flagellar microtubules by immunoelectron microscopy.

Chemically resistant 2-3 nm filaments with a high alpha-helical content, isolated from sea-urchin sperm flagellar doublet microtubules, consist of proteins that have been named tektins. Polyclonal affinity-purified antibodies to tektins labelled sperm tails all along their lengths, as shown by indirect immunofluorescence microscopy, provided the specimens were not too well fixed. Results obtained for unfixed specimens studied by immunoelectron microscopy suggested the tektins are normally masked by tubulin. A monoclonal anti-tektin antibody labelled bare tektin filaments at longitudinal intervals of approximately 48 nm, which fits in well with the 96 nm longitudinal repeat of axonemes. We discuss a possible scheme for the regular interaction of tubulin monomers with an alpha-helical coiled coil.

Cytoplasmic dynein of the sea urchin egg. II. Purification, characterization and interactions with microtubules and Ca-calmodulin.

Purification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised several chromatographies, including a calmodulin-Sepharose 4B affinity column chromatography. This is the first report of the purification of cytoplasmic dynein to near homogeneity. The purified fraction was composed of a single high molecular weight polypeptide and some minor low molecular weight polypeptides. The high molecular weight polypeptide comigrated with flagellar dynein A beta chain from sperm on SDS-polyacrylamide gels. There was no polypeptide stainable with periodic acid-Schiff reagent (PAS) in the purified cytoplasmic dynein fraction. Cytoplasmic dynein showed characteristics quite similar to those of axonemal dynein, i.e. high substrate specificity for ATP and inhibition by low concentrations of vanadate, though its Ca-ATPase activity showed almost the same dependence on the concentration of either divalent cations or KC1 as the Mg-ATPase activity. The purified enzyme seemed to possess functional form as judged from its properties: 1) pH dependence of the ATPase activity, 2) dependence of the ATPase activity on MgCl2 and KCl concentration, 3) Km for Mg-ATP, and 4) binding to flagellar doublet microtubules. Cytoplasmic dynein bound to calmodulin-Sepharose 4B only in the presence of Ca2+, and was eluted with EGTA. Furthermore, the ATPase activity was enhanced 6-fold by calmodulin in a Ca2+-dependent manner. The activation by calmodulin was prevented by a stoichiometric amount of trifluoperazine.

Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly.

B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication-derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.

Separate mRNAs code for tubulin subunits.

MICROTUBULES are a common feature in eukaryotic cells. They have been implicated in several functions including neurite growth, axoplasmic flow, cell division and excretion1. They are polymers of a 110,000 molecular weight heterodimer, tubulin2,3. The subunits, α and β tubulin, seem to have the same molecular weight, 55,000, but their amino acid compositions, although similar, are not identical3,4. Furthermore, the first 25 residues at the N terminus have been sequenced, and the α and β chains differ at 14 (ref 4). α Tubulin has been resolved into two components by hydroxylapatite chromatography5,6 and electrophoresis in polyacrylamide gels containing SDS and urea7. Mitotic microtubules and ciliary doublet A tubules from the sea urchin contain both forms of the α subunit, but flagellar doublet microtubules from the same organism contain only one form7. The α forms seem to differ in charge rather than size. In view of this heterogeneity of the tubulin subunits, it is reasonable to expect that eukaryotes have more than one tubulin gene. Here we present evidence that there are at least two tubulin genes. We have isolated two tubulin mRNA fractions from chick embryo brain. One codes for two α subunits, and the other codes for the β subunit.

Flagellar doublet microtubules: fractionation of minor components and alpha-tubulin from specific regions of the A-tubule.

Proteins occurring minor amounts with purified sperm flagellar doublet microtubules were identified and studied by SDS-gel electrophoresis. Methods were developed to solubilize selectively these minor components; electron microscopy (EM) of the fractionated products revealed possible locations of these proteins in the tubule. Doublet microtubules were prepared from sea-urchin (Echinus esculentus and Stronglyocentrotus droebachiensis) and scallop (Pecten maximus) sperm by dialysing flagellar axonemes against 2 mM Tris-0-2 mM EDTA-0-5 mM DTT. EM indicates that these doublet tubule preparations retain at least 70% of their radial spokes; cross-sections show a globule or fibre applied to the inside wall of the A-tubule, across from the inner B-tubule junction. On SDS-gels these preparations separate into at least 10 minor bands, accounting for 20-30% of the total protein; the remaining 75 +/- 4% migrates as tubulin. For E. esculentus the molecular weights and relative amounts of these components are: Component Ee 8 (150000 Daltons; 1%), 11 (114000; 2-5%), 15 (89000; 2%), 16 (80000; 2-5%), 17 (74000; 2%), 18 (69000; 2%), 19 (66000; 2%), 21 (48000; 4-5%), 22 (45000; 3%) and 23 (44500; 3%). Treatment of sea-urchin tubules with 0-1-0-5% sarkosyl, 0-1-0-3 M KSCN or 0-3-0-6 M KI results in the selective solubilization of: first, component 8 and some B-subfibre tubulin; second, components 11 and 23 and the remaining B-subfire tubulin; third, most of the A-subfire tubulin and components 17, 18 and 19. Thermal fractionation extracts none of these components, suggesting they are principally associated with the A-tubule. Finally 25-35% of the original protein is resistant to solubilization, and appears in the EM as ribbons of 3 protofilaments with 16-nm axial repeats. The resistant ribbons contain components 15, 16, 21 and 22 (plus component 20 in S. droebachiensis) in addition to 25 +/- 4% of the total tubulin. The data support the existence of two stable moieties in each doublet tubule: (1) a ribbon of 3 protofilaments and (2) either a second ribbon of 3 protofilaments or an equivalent amount of tubulin in some other form. EM images suggest that one ribbon forms the lateral side of the A-tubule (e.g. protofilaments A1,2,3 or A13,1,2 in the model) and that the globule applied to A13 may be a multisubunit complex of remaining minor components. Treatment of scallop tubules with 0-3 M KSCN preferentially extracts alpha-tubulin, yielding ribbons 1-4 protofilaments wide. The significance of this finding is discussed.

The hands of helical lattices in flagellar doublet microtubules.

ABSTRACT Negatively stained arrays of flagellar doublet microtubules have been tilted about an axis perpendicular to the tubule axes and imaged at angles of ± 20°. The images have been filtered to enhance the 4-nm periodicities in the tubulin lattice, and the resulting filtered images interpreted by comparison with simulated projections of tilted helical lattices. The different appearances of the edges of filtered doublet tubules for positive and negative tilt angles suggest that the 4-nm lattices of both subfibres are of the same hand, with the 3-start helices in each being left-handed. The information has been incorporated in a model proposed for the arrangement of subunits in doublet microtubules, which is described in the preceding paper.