The actin filaments on the surface of echinoderm oocytes and eggs readily undergo massive reorganization during meiotic maturation and fertilization. In sea urchin eggs, the actin cytoskeletal response to the fertilizing sperm is fast enough to accompany Ca2+ signals and to guide sperm’s entry into the egg. Although recent work using live cell imaging technology confirmed changes in the actin polymerization status in fertilized eggs, as was previously shown using light and electron microscopy, it failed to provide experimental evidence of F-actin depolymerization a few seconds after insemination, which is concurrent with the sperm-induced Ca2+ release. In the present study, we applied Raman microspectroscopy to tackle this issue by examining the spectral profiles of the egg’s subplasmalemmal regions before and after treating the eggs with actin drugs or fertilizing sperm. At both early (15 s) and late (15 min) time points after fertilization, specific peak shifts in the Raman spectra revealed change in the actin structure, and Raman imaging detected the cytoskeletal changes corresponding to the F-actin reorganization visualized with LifeAct-GFP in confocal microscopy. Our observation suggests that the application of Raman spectroscopy, which does not require microinjection of fluorescent probes and exogenous gene expression, may serve as an alternative or even advantageous method in disclosing rapid subtle changes in the subplasmalemmal actin cytoskeleton that are difficult to resolve.

The obligate root parasite Cistanche armena is a recently rediscovered, extremely rare endangered species endemic to Armenia, specifically parasitizing camelthorn (Alhagi maurorum, Fabaceae) and saltwort (Salsola dendroides, Chenopodiaceae). Its populations are reputedly declining due to habitat destruction and biotic impacts. Since the only known means of its reproduction is via the seeds, understanding the mechanisms of breaking C. armena seed dormancy and germination along with the related aspects of the species’ biology is highly important both from fundamental (functional ecology and evolution) and applied (conservation and management) perspectives. Here, we present the first in vitro seed germination protocol for C. armena involving fluridone, a systemic herbicide targeting the carotenoid biosynthetic pathway. In addition, the seed micromorphology of C. armena is described using both light microscopy and lignin autofluorescence visualized by confocal laser scanning microscopy. The actin cytoskeleton in radicle cells of germinated C. armena seedlings is described for the first time, being the proof of seed viability. Further elaboration and application of the proposed germination protocol with the cultivation of C. armena on susceptible hosts are altogether seen as a valuable tool for the conservation of this species.

In this paper, with the aim to find new genes involved in mammalian spermatogenesis, we isolated, for the first time in the rat testis, a partial cDNA clone that encoded EH domain binding protein 1-like 1 (Ehbp1l1), a protein that has a single calponin homology domain (CH). Bioinformatic analysis showed that EHBP1l1 contains three domains: the N-terminal C2-like, the CH and the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domains, which are evolutionarily conserved in vertebrates. We found that Ehbp1l1 mRNA was expressed in several rat tissues, including the liver, intestine, kidney and also in the testis during its development, with a higher level in testis from 12-month-old animals. Interestingly, in situ hybridization experiments revealed that Ehbp1l1 is specifically expressed by types I and II spermatocytes, this result was validated by RT-PCR performed on total RNA obtained from enriched fractions of different testicular cell types. As EHBP1l1 has been described as linked to vesicular transport to the actin cytoskeleton and as an effector of the small GTPase Rab8, we hypothesized that it could participate both in cytoskeletal remodelling and in the regulation of vesicle sorting from the trans-Golgi network to the apical plasma membrane. Our findings provide a better understand of the molecular mechanisms of the differentiation process of spermatogenesis; Ehbp1l1 may also be used as a new marker of testicular activity.

During sea urchins fertilization, the activating spermatozoon triggers a series of physiological changes that transforms the quiescent egg into a dynamic zygote. It has been suggested that several of these egg activation events, e.g. sperm-induced plasma membrane depolarization and the Ca2+-linked cortical reaction, play additional roles to prevent the entry of supernumerary spermatozoa. In particular, the abrupt shift in egg membrane potential at fertilization, which is sustained by a Na+ influx, has been considered as a fast mechanism to block polyspermy. To test the relevance of the Na+-mediated fast electrical block to polyspermy, we fertilized sea urchin eggs in artificial seawater with a low concentration of Na+; nearly all the eggs were still monospermic, as judged by the number of Hoechst 33422-stained sperm. When fertilized in normal seawater, eggs that were pre-incubated in the low Na+ medium exhibited impaired elevation of the fertilization envelope. Nevertheless, these eggs manifested entry of a single spermatozoon, suggesting that the fertilization envelope was not the primary determinant of the block to polyspermy. Furthermore, we showed that the abnormal cleavage patterns displayed by eggs pre-incubated in low Na+, which were often considered a hallmark of polyspermy, were due to the alterations in the cortical actin filaments dynamics following fertilization, and not to the formation of multipolar spindles associated with supernumerary sperm centrosomes. Hence, our results suggested that Paracentrotus lividus eggs do not utilize Na+ to rapidly prevent additional spermatozoa from entering the egg, at variance with the hypothesis of an electrical fast block to polyspermy.

Apicomplexan parasites have unconventional actins that play a central role in important cellular processes such as apicoplast replication, motility of dense granules, endocytic trafficking and force generation for motility and host cell invasion. In this study, we investigated the actin of the apicomplexan Neospora caninum – a parasite associated with infectious abortion and neonatal mortality in livestock. Neospora caninum actin was detected and identified in two bands by one-dimensional (1D) western blot and in nine spots by the 2D technique. The mass spectrometry data indicated that N. caninum has at least nine different actin isoforms, possibly caused by post-translational modifications. In addition, the C4 pan-actin antibody detected specifically actin in N. caninum cellular extract. Extracellular N. caninum tachyzoites were treated with toxins that act on actin, jasplakinolide and cytochalasin D. Both substances altered the peripheric cytoplasmic localization of actin on tachyzoites. Our findings add complexity to the study of the apicomplexan actin in cellular processes, since the multiple functions of this important protein might be regulated by mechanisms involving post-translational modifications.

The migration pattern of sperm-specific phospholipase C-ζ (PLC-ζ) was followed and the role of this migration in actin cytoskeleton dynamics was determined. We investigated whether PLC-ζ exits sperm, opening the possibility that PLC-ζ is the ‘spermatozoidal activator factor’ (SOAF). As capacitation progresses, the highly dynamic actin cytoskeleton bound different proteins to regulate their location and activity. PLC-ζ participation at the start of fertilization was established. In non-capacitated spermatozoa, PLC-ζ is in the perinuclear theca (PT) and in the flagellum, therefore it was decided to determine whether bovine sperm actin interacts with PLC-ζ to direct its relocation as it progresses from non-capacitated (NC) to capacitated (C) and to acrosome-reacted (AR) spermatozoa. PLC-ζ interacted with actin in NC spermatozoa (100%), PLC-ζ levels decreased in C spermatozoa to 32% and in AR spermatozoa to 57% (P < 0.001). The level of actin/PLC-ζ interaction was twice as high in G-actin (P < 0.001) that reflected an increase in affinity. Upon reaching the AR spermatozoa, PLC-ζ was partially released from the cell. It was concluded that actin cytoskeleton dynamics control the migration of PLC-ζ during capacitation and leads to its partial release at AR spermatozoa. It is suggested that liberated PLC-ζ could reach the egg and favour fertilization.

Strongyloides venezuelensis is a parasitic nematode of rodents that is frequently used to obtain heterologous antigens for immunological diagnosis of human strongyloidiasis. The aim of this study was to identify antigens from filariform larvae of S. venezuelensis for immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from filariform larvae of S. venezuelensis were obtained in phosphate saline (SS and SM) and in Tris–HCl buffer (TS and TM), and were analysed by Western blotting. Different antigenic components were recognized by IgG antibodies from the sera of strongyloidiasis patients. Highest recognition was observed for a 30–40 kDa mass range present in all antigenic fractions. The band encompassing this mass range was then excised and subjected to mass spectrometry for protein identification. Immunoreactive proteins identified in the soluble fractions corresponded to metabolic enzymes, whereas cytoskeletal proteins and galectins were more abundant in the membrane fractions. These results represent the first approach towards identification of S. venezuelensis antigens for use in immunodiagnostic assays for human strongyloidiasis.

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

Bonamia exitiosa is an intracellular parasite (Haplosporidia) that has been associated with mass mortalities in oyster populations in the Southern hemisphere. This parasite was recently detected in the Northern hemisphere including Europe. Some representatives of the Bonamia genus have not been well categorized yet due to the lack of genomic information. In the present work, we have applied Whole-Genome Amplification (WGA) technique in order to characterize the actin gene in the unculturable protozoan B. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed that B. exitiosa actin is more similar to Bonamia ostreae actin gene-1. Actin phylogeny placed the Bonamia sp. infected oysters in the same clade where the herein described B. exitiosa actin resolved, offering novel information about the classification of the genus. Our results showed that WGA methodology is a promising and valuable technique to be applied to unculturable protozoans whose genomic material is limited.

To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling ‘beads on a string’. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

Cryptosporidium host cell interaction remains fairly obscure compared with other apicomplexans such as Plasmodium or Toxoplasma. The reason for this is probably the inability of this parasite to complete its life cycle in vitro and the lack of a system to genetically modify Cryptosporidium. However, there is a substantial set of data about the molecules involved in attachment and invasion and about the host cell pathways involved in actin arrangement that are altered by the parasite. Here we summarize the recent advances in research on host cell infection regarding the excystation process, attachment and invasion, survival in the cell, egress and the available data on omics.

Nosema ceranae is a widespread honeybee parasite, considered to be one of the pathogens involved in the colony losses phenomenon. To date, little is known about its intraspecific genetic variability. The few studies on N. ceranae variation have focused on the subunits of ribosomal DNA, which are not ideal for this purpose and have limited resolution. Here we characterized three single copy loci (Actin, Hsp70 and RPB1) in three N. ceranae isolates from Hungary and Hawaii. Our results provide evidence of unexpectedly high levels of intraspecific polymorphism, the coexistence of a wide variety of haplotypes within each bee colony, and the occurrence of genetic recombination in RPB1. Most haplotypes are not shared across isolates and derive from a few frequent haplotypes by a reduced number of singletons (mutations that appear usually just once in the sample), which suggest that they have a fairly recent origin. Overall, our data indicate that this pathogen has experienced a recent population expansion. The presence of multiple haplotypes within individual isolates could be explained by the existence of different strains of N. ceranae infecting honeybee colonies in the field which complicates, and must not be overlooked, further analysis of host–parasite interactions.

Dermatocarpon luridum is a subaquatic lichen which is distributed within temperate climatic zones around the world. It colonizes rock substrata along the shoreline of lakes and rivers of watersheds that regularly experience water level fluctuations. The mycobiont produces perithecia with small, simple spores that are thought to be wind dispersed. The photobiont, Diplosphaera chodatii, occurs both free-living and lichenized but little is known about its distribution and dispersal. The goal of this study was to compare the population structure of the photobiont from lakes and rivers in central North America with those of Europe. Specimens were collected in Manitoba, Canada and Austria. Population structure of the algal symbiont was assessed using the internal transcribed spacer (ITS) of ribosomal DNA (rDNA) and actin gene sequences. Results showed that genetic diversity and gene flow was high within local populations, but gene flow was low between continental populations. Low levels of gene flow between the most distant populations support the isolation-by-distance theory. The photobiont on both continents is also reported to be the photobiont for other lichen species contributing to photobiont availability for D. luridum.

During mitosis nucleation-promoting factors (NPFs) bind to the Arp2/3 complex and activate actin assembly. JMY and WAVE2 are two critical members of the NPFs. Previous studies have demonstrated that NPFs promote multiple processes such as cell migration and cytokinesis. However, the role of NPFs in development of mammalian embryos is still unknown. Results of the present study show that the NPFs JMY and WAVE2 are critical for cytokinesis during development of mouse embryos. Both JMY and WAVE2 are expressed in mouse embryos. After injection of JMY or WAVE2 siRNA, all embryos failed to develop to the morula or blastocyst stages. Moreover, using fluorescence intensity analysis, we found that the expression of actin decreased, and multiple nuclei were observed within a single cell indicating that NPFs-induced actin reduction caused the failure of cell division. In addition, injection of JMY and WAVE2 siRNA also caused ARP2 degradation, indicating that involvement of NPFs in development of mouse embryos is mainly through regulation of ARP2/3-induced actin assembly. Taken together, these data suggested that WAVE2 and JMY are involved in development of mouse embryos, and their regulation may be through a NPFs-Arp2/3-actin pathway.

The large and optically clear embryos of the zebrafish provide an excellent model system in which to study the dynamic assembly of the essential contractile band components, actin and myosin, via double fluorescent labelling in combination with confocal microscopy. We report the rapid appearance (i.e. within <2 min) of a restricted arc of F-actin patches along the prospective furrow plane in a central, apical region of the blastodisc cortex. These patches then fused with each other end-to-end forming multiple actin cables, which were subsequently bundled together forming an F-actin band. During this initial assembly phase, the F-actin-based structure did not elongate laterally, but was still restricted to an arc extending ~15° either side of the blastodisc apex. This initial assembly phase was then followed by an extension phase, where additional F-actin patches were added to each end of the original arc, thus extending it out to the edges of the blastodisc. The dynamics of phosphorylated myosin light chain 2 (MLC2) recruitment to this F-actin scaffold also reflect the two-phase nature of the contractile apparatus assembly. MLC2 was not associated with the initial F-actin arc, but MLC2 clusters were recruited and assembled into the extending ends of the band. We propose that the MLC2-free central region of the contractile apparatus acts to position and then extend the cleavage furrow in the correct plane, while the actomyosin ends alone generate the force required for furrow ingression. This biphasic assembly strategy may be required to successfully divide the early cells of large embryos.

Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are most often identified by staining with phalloidin, which binds F-actin and therefore visualizes the core. However, not only podosomes, but also many other cytoskeletal structures contain actin, which makes podosome segmentation by automated image processing difficult. Here, we have developed a quantitative image analysis algorithm that is optimized to identify podosome cores within a typical sample stained with phalloidin. By sequential local and global thresholding, our analysis identifies up to 76% of podosome cores excluding other F-actin-based structures. Based on the overlap in podosome identifications and quantification of podosome numbers, our algorithm performs equally well compared to three experts. Using our algorithm we show effects of actin polymerization and myosin II inhibition on the actin intensity in both podosome core and associated actin network. Furthermore, by expanding the core segmentations, we reveal a previously unappreciated differential distribution of cytoskeletal adaptor proteins within the podosome ring. These applications illustrate that our algorithm is a valuable tool for rapid and accurate large-scale analysis of podosomes to increase our understanding of these characteristic adhesion structures.

Although clam populations in France are known to be infected with protozoans of the genus Perkinsus, no molecular characterization was previously performed on these parasites. Considering that several members of this genus have been associated with mortalities of molluscs worldwide, a study was undertaken in order to characterize these parasites in France. For that purpose, clams, Ruditapes philippinarum and R. decussatus, collected from different production areas and found to be infected with Perkinsus sp. in thioglycolate culture medium, were selected for PCR-RFLP tests and sequencing. Perkinsus olseni was detected in all the investigated areas and results also suggested the presence of P. chesapeaki in Leucate, a lagoon on the Mediterranean coast and in Bonne Anse in Charente Maritime, on the Atlantic coast. Clonal cultures from both detected species were produced in order to describe and compare in vitro stages. Differences in size between both Perkinsus spp. were noticed especially for schizonts and zoosporangia. Lastly, in situ hybridization tests allowed confirmation of the presence of both species in the same R. decussatus population and even in same clams. This is the first detection of P. chesapeaki in Ruditapes species and outside North America, which questions its introduction into Europe.

When invading the tissue, malignant tumour cells (i.e. cancer cells) need to detach fromneighbouring cells, degrade the basement membrane, and migrate through the extracellularmatrix. These processes require loss of cell-cell adhesion and enhancement of cell-matrixadhesion. In this paper we present a mathematical model of an intracellular pathway forthe interactions between a cancer cell and the extracellular matrix. Cancer cells usesimilar mechanisms as with normal cells for their interactions with the extracellularmatrix. We develop a model of cell-matrix adhesion that accounts for reactions between thecell surface receptor integrins, the matrix glycoprotein fibronectin, and the actinfilaments in the cytoskeleton. Each represents components for an intermediate compartment,the extracellular compartment, and the intracellular compartment, respectively. Binding offibronectin with integrins triggers a clustering of protein complexes, which thenactivates and phosphorylates regulatory proteins that are involved in actin reorganisationcausing actin polymerization and stress fibre assembly. Rearrangement of actin filamentswith integrin/fibronectin complexes near adhesion sites and interaction with fibrillarfibronectin produces the force necessary for cell migration, accounting for cell-matrixadhesion.

The description of Plasmodium ookinete surface proteins and their participation in the complex process of mosquito midgut invasion is still incomplete. In this study, using phage display, a consensus peptide sequence (PWWP) was identified in phages that bound to the Plasmodium berghei ookinete surface and, in selected phages, bound to actin and enolase in overlay assays with ookinete protein extracts. Actin was localized on the surface of fresh live ookinetes by immunofluorescence and electron microscopy using specific antibodies. The overall results indicated that enolase and actin can be located on the surface of ookinetes, and suggest that they could participate in Plasmodium invasion of the mosquito midgut.