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.

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.

Functional protofilament numbering of ciliary, flagellar, and centriolar microtubules.

This article discusses the current state of knowledge about the evolutionarily conserved structure of ciliary, flagellar and centriolar microtubules, and formally proposes a functional numbering convention for their protofilaments.

Tektin interactions and a model for molecular functions.

Tektins from echinoderm flagella were analyzed for microheterogeneity, self-associations and association with tubulin, resulting in a general model of tektin filament structure and function applicable to most eukaryotic cilia and flagella. Using a new antibody to tektin consensus peptide RPNVELCRD, well-characterized chain-specific antibodies and quantitative gel densitometry, tektins A, B and C were found to be present in equimolar amounts in Sarkosyl-urea-stable filaments. In addition, two isoforms of tektin A are present in half-molar ratios to tektins B and C. Cross-linking of AB filaments indicates in situ nearest neighbor associations of tektin A1B and A2B heterodimers, -trimers, -tetramers and higher oligomers. Soluble purified tektin C is cross-linked as homodimers, trimers and tetramers, but not higher oligomers. Tektin filaments associate with both loosely bound and tightly bound tubulin, and with the latter in a 1:1 molar ratio, implying a specific, periodic association of tightly bound tubulin along the tektin axis. Similarly, in tektin-containing Sarkosyl-stable protofilament ribbons, two polypeptides ( approximately 67/73 kDa, homologues of rib72, efhc1 and efhc2) are present in equimolar ratios to each other and to individual tektins, co-fractionating with loosely bound tubulin. These results suggest a super-coiled arrangement of tektin filaments, the organization of which has important implications for the evolution, assembly and functions of cilia and flagella.

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 approximately 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.