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Intraflagellar transport (IFT) is responsible for the bidirectional trafficking of molecular components required for the elongation and maintenance of eukaryotic cilia and flagella. Cargo is transported by IFT 'trains', linear rows of multiprotein particles moved by molecular motors along the axonemal doublets. We have previously described two structurally distinct categories of 'long' and 'short' trains. Here, we analyse the relative number of these trains throughout flagellar regeneration and show that long trains are most abundant at the beginning of flagellar growth whereas short trains gradually increase in number as flagella elongate. These observations are incompatible with the previous hypothesis that short trains are derived solely from the reorganization of long trains at the flagellar tip. We demonstrate with electron tomography the existence of two distinct ultrastructural organizations for the short trains, we name these 'narrow' and 'wide', and provide the first 3D model of the narrow short trains. These trains are characterized by tri-lobed units, which repeat longitudinally every 16 nm and contact protofilament 7 of the B-tubule. Functional implications of the new structural evidence are discussed.

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The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed.

We present here for the first time a 3D reconstruction of in situ axonemal outer dynein arms. This reconstruction has been obtained by electron tomography applied to a series of tilted images collected from metal replicas of rapidly frozen, cryofractured, and metal-replicated sperm axonemes of the cecidomid dipteran Monarthropalpus flavus. This peculiar axonemal model consists of several microtubular laminae that proved to be particularly suitable for this type of analysis. These laminae are sufficiently planar to allow the visualization of many dynein molecules within the same fracture face, allowing us to recover a significant number of equivalent objects and to improve the signal-to-noise ratio of the reconstruction by applying advanced averaging protocols. The 3D model we obtained showed the following interesting structural features: First, each dynein arm has two head domains that are almost parallel and are obliquely oriented with respect to the longitudinal axis of microtubules. The two heads are therefore positioned at different distances from the surface of the A-tubule. Second, each head domain consists of a series of globular subdomains that are positioned on the same plane. Third, a stalk domain originates as a conical region from the proximal head and ends with a small globular domain that contacts the B-tubule. Fourth, the stem region comprises several globular subdomains and presents two distinct points of anchorage to the surface of the A-tubule. Finally, and most importantly, contrary to what has been observed in isolated dynein molecules adsorbed to flat surfaces, the stalk and the stem domains are not in the same plane as the head.

BackgroundThe expression of intermediate filaments (IFs) is a hallmark feature of metazoan cells. IFs play a central role in cell organization and function, acting mainly as structural stress-absorbing elements. There is growing evidence to suggest that these cytoskeletal elements are also involved in the integration of signalling networks. According to their fundamental functions, IFs show a widespread phylogenetic expression, from simple diblastic animals up to mammals, and their constituent proteins share the same molecular organization in all species so far analysed. Arthropods represent a major exception in this scenario. Only lamins, the nuclear IF proteins, have so far been identified in the model organisms analysed; on this basis, it has been considered that arthropods do not express cytoplasmic IFs.ResultsHere, we report the first evidence for the expression of a cytoplasmic IF protein in an arthropod - the basal hexapod Isotomurus maculatus. This new protein, we named it isomin, is a component of the intestinal terminal web and shares with IFs typical biochemical properties, molecular features and reassembly capability. Sequence analysis indicates that isomin is mostly related to the Intermediate Filament protein C (IFC) subfamily of Caenorhabditis elegans IF proteins, which are molecular constituents of the nematode intestinal terminal web. This finding is coherent with, and provides further support to, the most recent phylogenetic views of arthropod ancestry. Interestingly, the coil 1a domain of isomin appears to have been influenced by a substantial molecular drift and only the aminoterminal part of this domain, containing the so-called helix initiation motif, has been conserved.ConclusionsOur results set a new basis for the analysis of IF protein evolution during arthropod phylogeny. In the light of this new information, the statement that the arthropod phylum lacks cytoplasmic IFs is no longer tenable.See commentary article: http://www.biomedcentral.com/1741-7007-9-16.

The axonemal organization expressed in the sperm flagella of the cecidomyiid dipteran Asphondylia ruebsaameni is unconventional, being characterized by the presence of an exceedingly high number of microtubular doublets and by the absence of both the inner dynein arms and the central pair/radial spoke complex. Consequently, its motility, both in vivo and in vitro, is also peculiar. Using monoclonal antibodies directed against posttranslational modifications, we have analyzed the presence and distribution of glutamylated and glycylated tubulin isoforms in this aberrant axonemal structure, and compared them with those of a reference insect species (Apis mellifera), endowed with a conventional axoneme. Our results have shown that the unorthodox structure and motility of the Asphondylia axoneme are concomitant with: (1). a very low glutamylation extent in the alpha-tubulin subunit, (2). a high level of glutamylation in the beta-subunit, (3). an extremely low total extent of glycylation, with regard to both monoglycylated and polyglycylated sites, either in alpha- or in beta-tubulin, (4). the presence of a strong labeling of glutamylated tubulin isoforms at the proximal end of the axoneme, and (5). a uniform distribution of glutamylated as well as glycylated isoforms along the rest of the axoneme. Thus, our data indicate that tubulin molecular heterogeneity is much lower in the Asphondylia axoneme than in the conventional 9+2 axoneme with regard to both isoform content and isoform distribution along the axoneme.

Primary spermatocyte nuclei of Drosophila melanogaster contain three prominent lampbrush-like loops. The development of these structures has been associated with the transcription of three fertility factors located on the Y chromosome, named kl-5, kl-3 and ks-1. These loci have huge physical dimensions and contain extremely long introns. In addition, kl-3 and kl-5 were shown to encode two putative dynein subunits required for the correct assembly of the sperm axoneme. Here, we show that both the kl-5 and kl-3 loops are intensely decorated by monoclonal antibodies recognizing triple-stranded nucleic acids, and that each loop presents a peculiar molecular organization of triplex structures. Moreover, immunostaining of Drosophila hydei primary spermatocytes revealed that also in this species -which diverged from D. melanogaster 58 million years ago -Y-loops are decorated by anti-triplex antibodies, strongly suggesting a conserved role of loop-associated triplexes. Finally, we showed that in D. melanogaster wild-type lines that are raised at the non-permissive temperature of 31±0.5°C (which is known to induce male sterility in flies) both the triplex immunostaining and the axonemal dynein heavy chains encoded by kl-3 and kl-5 are no longer detectable, which suggests a functional correlation between loop-associated triplexes, the presence of axonemal proteins and male fertility in fly. Journal of Cell Science 1606 triplexes, the most stable triads are those involving a protonated cytosine (C + ) formed by C + *GC triad and T*AT nucleotides (the asterisks mark the nucleotides on the third strand). Notably, these sequences are overrepresented in all eukaryotic genomes (Behe, 1987; Behe, 1995), as well as in eukaryotic viruses (Beasty and Behe, 1988). It is also possible to have triplex formation without homopurine stretches, and these triplexes seem to be as stable as the others at least under certain conditions (Dayn et al., 1992). Although triplexes are well characterized in vitro, their biological significance in living organisms is still under discussion (Zain and Sun, 2003). It has been demonstrated that, upon formation, these structures can frequently downregulate (Cooney et al., 1988; Birg et al., 1990; Faria et al., 2000;Faria et al., 2001) and sometimes upregulate (reviewed in Faria and Giovannangeli, 2001) gene expression, a fact which demonstrates their potential in gene control and suggests their use in gene therapy (Wang et al., 1995;Vasquez et al., 2000) (for a review, see Rogers et al., 2005). The structures are also able to impair DNA polymerization (Dayn et al., 1992), and can influence DNA recombination and repair (Faruqi et al., 1996;Wang et al., 1996;Faruqi et al., 2000;Vasquez et al., 2002;Kalish et al., 2005;Raghavan et al., 2005) (for reviews, see Seidman and Glazer, 2003; Chin et al., 2007). Triplexes might also have a role in chromatin organization of both interphase nuclei (Agazie et al., 1996;Ohno et al., 2002) and mitotic chromosomes . Recently, it has been demonstrated that a triplestran...