Richard W. Linck

At least one of the protofilaments in flagellar microtubules is not composed of tubulin

BACKGROUND The core of the eukaryotic flagellum is the axoneme, a complex motile organelle composed of approximately 200 different polypeptides. The most prominent components of the axoneme are the central pair and nine outer doublet microtubules. Each doublet microtubule contains an A and a B tubule; these are composed, respectively, of 13 and 10-11 protofilaments, all of which are thought to be made of tubulin. The mechanisms that control the assembly of the doublet microtubules and establish the periodic spacings of associated proteins, such as dynein arms and radial spokes, are unknown. Tektins, a set of microtubule-associated proteins, are present in the axoneme as stable filaments that remain after the extraction of doublet microtubules; they are localized near to where the B tubule attaches to the A tubule and near to the binding sites for radial spokes, inner dynein arms and nexin links. Tektin filaments may contribute in an interesting way to the structural properties of axonemes. RESULTS We have fractionated doublet microtubules from sea urchin sperm flagella into ribbons of stable protofilaments, which can be shown to originate from the A tubule. Using cryo-electron microscopy, conventional electron microscopy, scanning transmission electron microscopy, three-dimensional reconstruction and kinesin decoration, we have found that one protofilament in the ribbon is not composed of tubulin. This protofilament is an integral protofilament of the A tubule wall, has less mass per unit length than tubulin and does not bind kinesin. CONCLUSION Contrary to what is generally assumed, at least one protofilament in the wall of the A tubule is not composed of tubulin. Our data suggest that this nontubulin protofilament is primarily composed of tektins, proteins that show some structural similarity to intermediate filament proteins. A 480 A axial periodicity within these ribbons, revealed by scanning transmission electron microscopy, can be related to the structure of tektin, and may determine the large-scale structure of the axoneme in terms of the binding of dynein, nexin and radial spokes to the doublet microtubule.

Cryo-electron tomography reveals conserved features of doublet microtubules in flagella

The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the “beak” structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.

Rib72, a Conserved Protein Associated with the Ribbon Compartment of Flagellar A-microtubules and Potentially Involved in the Linkage between Outer Doublet Microtubules

Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized “ribbon” of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an ∼96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis- and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility.

The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. Recently we described the sequence of the first mammalian tektin protein, Tekt1 (from mouse testis), which is most homologous with sea urchin tektin C. We have now investigated the temporal and spatial expression of Tekt1 during mouse male germ cell development. By in situ hybridization analysis TEKT1 RNA expression is detected in spermatocytes and in round spermatids in the mouse testis. Immunofluorescence microscopy analysis with anti-Tekt1 antibodies showed no distinct labeling of any subcellular structure in spermatocytes, whereas in round spermatids anti-Tekt1 antibodies co-localize with anti-ANA antibodies to the centrosome. At a later stage, elongating spermatids display a larger area of anti-Tektl staining at their caudal ends; as spermiogenesis proceeds, the anti-Tekt1 staining disappears. Together with other evidence, these results provide the first intraspecies evidence that Tekt1 is transiently associated with the centrosome, and indicates that Tekt1 is one of several tektins to participate in the nucleation of the flagellar axoneme of mature spermatozoa, perhaps being required to assemble the basal body.

Expression of Ciliary Tektins in Brain and Sensory Development

Many types of neural tissues and sensory cells possess either motile or primary cilia. We report the first mammalian (murine testis) cDNA for tektin, a protein unique to cilia, flagella, and centrioles, which we have used to identify related proteins and genes in sensory tissues. Comparison with the sequence database reveals that tektins are a gene family, spanning evolution from Caenorhabditis elegans (in which they correlate with touch receptor cilia) andDrosophila melanogaster, to Mus musculusand Homo sapiens (in which they are found in brain, retina, melanocytes, and at least 13 other tissues). The peptide sequence RPNVELCRD, or a variant of it, is a prominent feature of tektins and is likely to form a functionally important protein domain. Using the cDNA as a probe, we determined the onset, relative levels, and locations of tektin expression in mouse for several adult tissues and embryonic stages by Northern blot analysis and in situ hybridization. Tektin expression is significant in adult brain and in the choroid plexus, the forming retina (primitive ependymal zone corresponding to early differentiating photoreceptor cells), and olfactory receptor neurons of stage embryonic day 14 embryos. There is a striking correlation of tektin expression with the known presence of either motile or primary cilia. The evolutionary conservation of tektins and their association with tubulin in cilia and centriole formation make them important and useful molecular targets for the study of neural development.

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

Background: Ciliary microtubules contain hyperstable Ribbons of adjoining protofilaments. Results: Using echinoderm flagella, the locations of Ribbons, tektins, and Ca2+-binding proteins (related to human epilepsy) are studied biochemically and by immuno-cryo-electron tomography. Conclusion: The locations of these proteins create a biochemically, structurally unique region of ciliary A-microtubules. Significance: The results indicate specialized functions for Ribbons, with potential roles in assembly, motility, and/or signal transduction. 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.

The axoneme: the propulsive engine of spermatozoa and cilia and associated ciliopathies leading to infertility.

This review article provides a critical analysis of the structure and molecular mechanisms of the microtubule axoneme of cilia and sperm flagella and their associated elements required for male fertility.A broad range of genetic and molecular defects (ciliopathies) are considered in the context of human diseases involving impaired motility in cilia and sperm flagella, providing provocative thought for future research in the area of male infertility.

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

Publisher Summary 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.

Visualization of three-dimensional microtubule structure

An animated visualization of the 3-D structure of a microtubule for an expensive structural analysis using state-of-the-art technology is reported. The use of a mathematical model for 3-D reconstruction and a hierarchical data structure provides a simple but concrete representation of a microtubule on 16-mm film and videotape. The modeling, motion control, and rendering of the 3-D image are discussed.<<ETX>>