Isomin: a novel cytoplasmic intermediate filament protein from an arthropod species.

BACKGROUND: The 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. RESULTS: Here, 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. CONCLUSIONS: Our 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.

Tubulin glycylation and glutamylation deficiencies in unconventional insect axonemes.

Though the 9+2 axonemal organization has generally been conserved throughout metazoan evolution, insect spermatozoa possess a substantial variety in axoneme ultrastructure, displaying different axonemal patterns. Therefore, insects provide a wide range of models that may be useful for the study of the mechanisms of axoneme assembly. We have used antibodies specific for glutamylated, monoglycylated, and polyglycylated tubulin to investigate the tubulin isoform content expressed in the unorthodox sperm axonemes of four insect species belonging to both of the superorders Palaeoptera and Neoptera. Each one of these axonemal models exhibits distinctive structural features, either showing the typical radial organization endowed with a ninefold symmetry or consisting of an helical arrangement with up to 200 microtubular doublets, but in all cases these axonemes share the absence of a microtubule central pair. Our results showed that all these atypical patterns are characterized by a dramatic decrease in both tubulin glycylation and glutamylation levels or even lack of both polymodifications. These data provide the first examples of a simultaneous extreme reduction or even absence of both polymodifications in axonemal tubulin. Given the unrelated positions of the analyzed species in the insect phylogenetic tree, this common feature is probably not due to evolutionary relationships. Therefore, our findings support the hypothesis of the existence of a correlation between the low level of polymodifications and the lack of a microtubule central pair in these peculiar insect flagellar axonemes, similarly as was previously proposed for cilia of Tetrahymena glycylation site mutants.

Glutamylated and glycylated tubulin isoforms in the aberrant sperm axoneme of the gall-midge fly, Asphondylia ruebsaameni.

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.