Caterina Mencarelli

Unusual axonemes of hexapod spermatozoa

Hexapod spermatozoa exhibit a great variation in their axoneme structure. The 9+2 pattern organization is present in a few basal taxa and in some derived groups. In most hexapods, a crown of nine accessory microtubules surrounds the 9+2 array, giving rise to the so-called 9+9+2 pattern. This general organization, however, displays a number of modifications in several taxa. In this review, the main variations concerning the number and localization of the accessory tubules, microtubular doublets, central microtubules, dynein arms, and axonemal length are summarized. We discuss the phylogenetic significance of all this structural information as well as the current hypotheses relating the sperm size and sperm polymorphism with reproductive success of some hexapod species. Also described are the biochemical data and the motility patterns which are currently known on some peculiar aberrant axonemes, in light of the contribution these models may give to the comprehension of the general functioning of the conventional 9+2 axoneme. Finally, we summarize methodological developments for the study of axoneme ultrastructure and the new opportunities for the molecular analysis of hexapod axonemes.

New insights into the cell biology of insect axonemes.

Insects do not possess ciliated epithelia, and cilia/flagella are present in the sperm tail and--as modified cilia--in mechano- and chemosensory neurons. The core cytoskeletal component of these organelles, the axoneme, is a microtubule-based structure that has been conserved throughout evolution. However, in insects the sperm axoneme exhibits distinctive structural features; moreover, several insect groups are characterized by an unusual sperm axoneme variability. Besides the abundance of morphological data on insect sperm flagella, most of the available molecular information on the insect axoneme comes from genetic studies on Drosophila spermatogenesis, and only recently other insect species have been proposed as useful models. Here, we review the current knowledge on the cell biology of insect axoneme, including contributions from both Drosophila and other model insects.

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

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.

Two classes of short intraflagellar transport train with different 3D structures are present in Chlamydomonas flagella

ABSTRACT 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. Highlighted article: In-depth analyses of IFT train ultrastructure in regenerating Chlamydomonas flagella shows the occurrence of three train types with different 3D structure, implicating differences in function.

Sperm ultrastructure and spermiogenesis of Coniopterygidae (Neuroptera, Insecta)

The spermiogenesis and the sperm ultrastructure of several species of Coniopterygidae have been examined. The spermatozoa consist of a three-layered acrosome, an elongated elliptical nucleus, a long flagellum provided with a 9+9+3 axoneme and two mitochondrial derivatives. No accessory bodies were observed. The axoneme exhibits accessory microtubules provided with 13, rather than 16, protofilaments in their tubular wall; the intertubular material is reduced and distributed differently from that observed in other Neuropterida. Sperm axoneme organization supports the isolated position of the family previously proposed on the basis of morphological data.

A comparative analysis of the evolution of the egg envelopes and the origin of the yolk

Abstract In this paper, the evolution of egg envelope is examined from a structural point of view. The structurally simplest eggs are found in Sponges and Cnidarians where reproduction is ensured by external fertilization. In these instances, the oocyte plasma membrane is protected by only a thin glycocalyx. A more structurally complex envelope is present in triblastic Metazoa where the vitelline membrane makes its first appearance. With the emergence of internal fertilization, the egg envelope is modified as to face the constraints imposed by the terrestrial environment. However the pursuit of such defensive strategy would have made eggs practically impermeable to spermatozoa Eggs with a thick chorion are thus endowed with a proper micropyle to allow fertilization. With the acquisition of viviparity egg envelopes have undergone marked reduction. In this paper particular attention has also been paid to the relationship of the oocyte with accessory cells and to the nature and function of the reserve materi...

Drosophila melanogaster kl-3 and kl-5 Y-loops harbor triple-stranded nucleic acids

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.

Accessory tubules and axonemal microtubules of Apis mellifera sperm flagellum differ in their tubulin isoform content.

In the insect sperm flagellum, an extra set of nine additional microtubules, named accessory tubules, is present surrounding the axoneme. Using a sarcosyl/urea extraction, we were able to fractionate the microtubular cytoskeleton of the sperm flagellum of the insect Apis mellifera resulting in the dissociation of the axonemal microtubule protein components and the accessory tubules. This has allowed us to compare the tubulin isoform content of axonemal microtubules and accessory tubules by immunoelectron microscopy and immunoblotting using a panel of monoclonal antibodies directed against different tubulin post-translational modifications (PTMs). All the PTMs occurring in axonemal tubulin are also present in accessory tubules, which indicates the close relativeness of accessory tubules to axonemal rather than to cytoplasmic microtubules. However, our results demonstrate the presence of significant differences in the tubulin isoform content of axonemal microtubules and accessory tubules. First, the tubulin tyrosination extent of accessory tubules is far lower than that of axonemal microtubules, thus confirming at the molecular level their morphogenetic origin as outgrowths from the B-subtubule of each microtubular doublet. Second, although polyglycylation seems to occurr at the same extent in both microtubular systems, alpha-tubulin exhibits a larger amount of monoglycylated sites in axonemal microtubules than in accessory tubules. Third, a greater amount of beta-tubulin molecules is glutamylated in axonemal microtubules than in accessory tubules. Moreover, highly acidic isoforms, likely molecules with longer polyglutamate side chains, are present only in axonemal microtubules. Taken together, our data are indicative of a higher level of tubulin heterogeneity in axonemal microtubules than in accessory tubules. They also show a segregation of post-translationally modified isoforms between accessory tubules and axonemal microtubules and suggest the implication of PTMs in the functional specialization of the two microtubular systems.

Evolutionary trends of neurofilament proteins in fish

1. Neurofilament complement was studied in an early chordate (Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. The anti-NF-H and anti-NF-M antisera were characterized as strictly specific for phosphorylated epitopes located in the carboxyterminal domain. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation-dependent epitopes. 5. Primitive fish (Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species (Mugil saliens, Perca fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution.

Phosphorylated epitopes of neurofilaments have been conserved during chordate evolution.

We have characterized some rabbit polyclonal responses as strictly specific for phosphorylated epitopes located in the carboxyterminal (tail) domain of the H or the M subunits of mammalian neurofilaments. These antibodies have been used to confirm the occurrence in lizard neurofilaments of a single heavy subunit cross-reacting with both H and M from mammals. A heavy subunit with similar cross-reactivity has been detected in neurofilaments preparations from fishes, whereas more primitive Chordata possess a HMW polypeptide cross-reacting with only the M subunit. We could also demonstrate in frog spinal cord two distinct heavy subunits cross-reacting with either the M or the H subunit from mammals, a fact which suggests a convergent evolution for phosphorylated epitopes of neurofilaments.