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Diagnosis by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification using previously published primers specific for the capsid protein gene of fish nodaviruses detected an isolate of sea bass, Dicentrarchus labrax L., nervous necrosis (SBNNV) from the French Mediterranean coast, but did not detect an isolate from the Atlantic coast. The capsid protein coding sequence of the nodavirus of both isolates was cloned. Sequence analysis revealed that the Mediterranean isolate was identical to the sequence previously published for SBNNV, while the Atlantic isolate is related, but carried numerous substitutions, in particular in the region used for RT-PCR diagnosis. A new primer set was tested which detected the viral genome of the Atlantic isolate. Using the new primer set, PCR amplification of a range of 10-fold dilutions of a plasmid containing the capsid protein gene of the Atlantic isolate showed that the limit of detection of the assay was between 10 and 100 copies of plasmid. [ABSTRACT FROM AUTHOR]

Tubulin post-translational modifications regulate microtubule properties and functions. Mitotic spindle microtubules are highly modified. While tubulin detyrosination promotes proper mitotic progression by recruiting specific microtubule-associated proteins motors, tubulin acetylation that occurs on specific microtubule subsets during mitosis is less well understood. Here, we show that siRNA-mediated depletion of the tubulin acetyltransferase ATAT1 in epithelial cells leads to a prolonged prometaphase arrest and the formation of monopolar spindles. This results from collapse of bipolar spindles, as previously described in cells deficient for the mitotic kinase PLK1 . ATAT1 -depleted mitotic cells have defective recruitment of PLK1 to centrosomes, defects in centrosome maturation and thus microtubule nucleation, as well as labile microtubule-kinetochore attachments. Spindle bipolarity could be restored, in the absence of ATAT1 , by stabilizing microtubule plus-ends or by increasing PLK1 activity at centrosomes, demonstrating that the phenotype is not just a consequence of lack of K-fiber stability. We propose that microtubule acetylation of K-fibers is required for a recently evidenced cross talk between centrosomes and kinetochores.

Mature megakaryocytes extend long processes called proplatelets from which platelets are released in the blood stream. The Rho GTPases Cdc42 and Rac as well as their downstream target, p21-activated kinase 2 (PAK2), have been demonstrated to be important for platelet formation. Here we address the role, during platelet formation, of PAK1, another target of the Rho GTPases. PAK1 decorates the bundled microtubules (MTs) of megakaryocyte proplatelets. Using a validated cell model which recapitulates proplatelet formation, elongation and platelet release, we show that lack of PAK1 activity increases the number of proplatelets but restrains their elongation. Moreover, in the absence of PAK1 activity, cells have hyperacetylated MTs and lose their MT network integrity. Using inhibitors of the tubulin deacetylase HDAC6, we demonstrate that abnormally high levels of MT acetylation are not sufficient to increase the number of proplatelets but cause loss of MT integrity. Taken together with our previous demonstration that MT acetylation is required for proplatelet formation, our data reveal that MT acetylation levels need to be tightly regulated during proplatelet formation. We identify PAK1 as a direct regulator of the MT acetylation levels during this process as we found that PAK1 phosphorylates the MT acetyltransferase MEC-17 and inhibits its activity.

Background: Upon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation., Results: Here, we show that in proplatelets from mouse megakaryocytes, microtubules are both acetylated and polyglutamylated. To bypass the difficulties of working with differentiating megakaryocytes, we used a cell model that allowed us to test the functions of these modifications. First, we show that α2bβ3integrin signaling in D723H cells is sufficient to induce β1tubulin expression and recapitulate the specific microtubule behaviors observed during proplatelet elongation and platelet release. Using this model, we found that microtubule acetylation and polyglutamylation occur with different spatio-temporal patterns. We demonstrate that microtubule acetylation, polyglutamylation, and β1tubulin expression are mandatory for proplatelet-like elongation, swelling formation, and cytoplast severing. We discuss the functional importance of polyglutamylation of β1tubulin-containing microtubules for their efficient bundling and coiling during platelet formation., Conclusions: We characterized and validated a powerful cell model to address microtubule behavior in mature megakaryocytes, which allowed us to demonstrate the functional importance of microtubule acetylation and polyglutamylation for platelet release. Furthermore, we bring evidence of a link between the expression of a specific tubulin isotype, the occurrence of microtubule post-translational modifications, and the acquisition of specific microtubule behaviors. Thus, our findings could widen the current view of the regulation of microtubule behavior in cells such as osteoclasts, spermatozoa, and neurons, which express distinct tubulin isotypes and display specific microtubule activities during differentiation.

While our knowledge of bivalve gametogenesis has progressed in recent times, more molecular markers are needed in order to develop tissue imaging. Here, we identified stem cell and mitotic markers to further characterize oyster early gametogenesis, mainly through immunofluorescence microscopy. Intense alkaline phosphatase activity, a non-specific marker for stem cells, was detected on the outer edge of the gonad ducts at the post-spawning stage, suggesting an abundance of undifferentiated cells very early during the sexual cycle. This observation was confirmed using an antibody against Sox2, a transcription factor specific for stem or germline cells, which labeled cells in the gonad duct inner mass and ciliated epithelium early during the initial oyster sexual cycle. Moreover, Vasa, a cytoplasmic marker for germline cells, was also detected in the gonad acini and duct cells, thus confirming that germline cells were abundant early on. In addition, the binding of the minichromosome maintenance MCM6 protein to chromatin indicated the gonad acini and duct cells were engaged in the cell cycle. DNA replication was indeed confirmed by an abundant in vivo incorporation of BrdU into the duct cell chromatin. Finally, proliferation of acini and duct cells was demonstrated by the chromatin-bound Ser10-phosphorylated histone H3, a mitotic marker. The markers for the cell cycle and mitosis used here thus indicate that acini and duct cells were already actively dividing early during the oyster sexual cycle. In addition, together with the stem cell markers, these data reveal that the epithelium delimiting the duct outer edge contains a dynamic population of undifferentiated cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Long-lived animals show a non-observable age-related decline in immune defense, which is provided by blood cells that derive from self-renewing stem cells. The oldest living animals are bivalves. Yet, the origin of hemocytes, the cells involved in innate immunity, is unknown in bivalves and current knowledge about mollusk adult somatic stem cells is scarce. Here we identify a population of adult somatic precursor cells and show their differentiation into hemocytes. Oyster gill contains an as yet unreported irregularly folded structure (IFS) with stem-like cells bathing into the hemolymph. BrdU labeling revealed that the stem-like cells in the gill epithelium and in the nearby hemolymph replicate DNA. Proliferation of this cell population was further evidenced by phosphorylated-histone H3 mitotic staining. Finally, these small cells, most abundant in the IFS epithelium, were found to be positive for the stemness marker Sox2. We provide evidence for hematopoiesis by showing that co-expression of Sox2 and Cu/Zn superoxide dismutase, a hemocyte-specific enzyme, does not occur in the gill epithelial cells but rather in the underlying tissues and vessels. We further confirm the hematopoietic features of these cells by the detection of Filamin, a protein specific for a sub-population of hemocytes, in large BrdU-labeled cells bathing into gill vessels. Altogether, our data show that progenitor cells differentiate into hemocytes in the gill, which suggests that hematopoiesis occurs in oyster gills., (© 2014. Published by The Company of Biologists Ltd.)

Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle.

Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (Deltavsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Deltavsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Deltavsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.

Tumorhead (TH) regulates neural plate cell proliferation during Xenopus early development, and gain or loss of function prevents neural differentiation. TH shuttles between the nuclear and cytoplasmic/cortical cell compartments in embryonic cells. In this study, we show that subcellular distribution of TH is important for its functions. Targeting TH to the cell cortex/membrane potentiates a TH gain of function phenotype and results in neural plate expansion and inhibition of neuronal differentiation. We have found that TH subcellular localization is regulated, and that its shuttling between the nucleus and the cell cortex/cytoplasm is controlled by the catalytic activity of p21-activated kinase, X-PAK1. The phenotypes of embryos that lack, or have excess, X-PAK1 activity mimic the phenotypes induced by loss or gain of TH functions, respectively. We provide evidence that X-PAK1 is an upstream regulator of TH and discuss potential functions of TH at the cell cortex/cytoplasmic membrane and in the nucleus.

The p21-activated kinase (PAK) proteins regulate many cellular events including cell cycle progression, cell death and survival, and cytoskeleton rearrangements. We previously identified X-PAK5 that binds the actin and microtubule networks, and could potentially regulate their coordinated dynamics during cell motility. In this study, we investigated the functional importance of this kinase during gastrulation in Xenopus. X-PAK5 is mainly expressed in regions of the embryo that undergo extensive cell movements during gastrula such as the animal hemisphere and the marginal zone. Expression of a kinase-dead mutant inhibits convergent extension movements in whole embryos and in activin-treated animal cap by modifying behavior of cells. This phenotype is rescued in embryo by adding back X-PAK5 catalytic activity. The active kinase decreases cell adhesiveness when expressed in animal hemisphere and inhibits the calcium-dependent reassociation of cells, while dead X-PAK5 kinase localizes to cell-cell junctions and increases cell adhesion. In addition, endogenous X-PAK5 colocalizes with adherens junction proteins and its activity is regulated by extracellular calcium. Taken together, our results suggest that X-PAK5 regulates convergent extension movements in vivo by modulating the calcium-mediated cell-cell adhesion.

To date, the functions of most neural intermediate filament (IF) proteins have remained elusive. Peripherin is a type III intermediate filament (IF) protein that is expressed in developing and in differentiated neurons of the peripheral and enteric nervous systems. It is also the major IF protein expressed in PC12 cells, a widely used model for studies of peripheral neurons. Dramatic increases in peripherin expression have been shown to coincide with the initiation and outgrowth of axons during development and regeneration, suggesting that peripherin plays an important role in axon formation. Recently, small interfering RNAs (siRNA) have provided efficient ways to deplete specific proteins within mammalian cells. In this study, it has been found that peripherin-siRNA depletes peripherin and inhibits the initiation, extension, and maintenance of neurites in PC12 cells. Furthermore, the results of these experiments demonstrate that peripherin IF are critical determinants of the overall shape and architecture of neurons.

Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. Here, we demonstrate that a novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and that its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions.

A novel intron-length polymorphism at the actin gene locus mac-1 is here reported and used as a genetic marker for population studies in mussels of the genus Mytilus. Two closely related genes subsequently identified as alleles, mac-1a1 and mac-1b1, from a genomic library of M. galloprovincialis were partially cloned and sequenced. They mainly differed from each other by a 65-bp insertion within their first intron. Polymerase chain reaction (PCR) primers were designed outside the insertion. The PCR analysis of 166 individual mussels from M. galloprovincialis and M. edulis populations revealed three size-classes of alleles or allelomorphs, two of which were of the expected sizes for mac1a1 and mac-1b1. One allelomorph was absent from M. edulis samples, although it was present at substantial frequencies in M. galloprovincialis populations. The frequencies of the two other allelomorphs significantly differed between M. galloprovincialis and M. edulis populations. The comparison of six mac-1 intron sequences over 277 bp showed at once that allelomorphs encompassed alleles differing from one another by substantial numbers of mutations, and that identical alleles were present in both M. galloprovincialis and M. edulis individuals, a probable result of the recent introgression between the two species.

Sequencing of HIV-1(GER) long terminal repeat (LTR) has demonstrated, for the first time in an HIV-1 primary isolate, a TAR duplication referred to as TAR1 (nucleotides +1 through +68) and TAR2 (nucleotides +69 through +136). This TAR duplication is stable during replication of HIV-1(GER) isolate in CEM cells. Analysis of LTR-CAT reporter constructs demonstrated that under Tat transactivation the HIV-1(GER)/LTR (containing TAR1 and TAR2) was expressed at a higher level than a similar construct (HIV-1(GER)DeltaTAR) containing a single TAR sequence. Among the two transcription initiation sites found in the HIV-1(GER)/LTR, only the most 5' start site was shown to be functionally active. The predicted secondary structure of the 5'-end mRNAs of HIV-1(GER) suggests it may fold into a double TAR hairpin which resembles that of HIV-2. Finally, HIV-1(GER) Tat protein shows primary sequence similarity with Tat proteins from other isolates of HIV-1 and is apparently unrelated to HIV-2 Tat proteins. This work provides the first evidence of a TAR sequence duplication in HIV-1 which increases the efficiency of transactivation by Tat. Copyright 1996 S. Karger AG, Basel

Twelve microsatellites were isolated from a partial genomic library of Ostrea edulis using (GA/TC)n and (AC/GT)n probes and were subsequently sequenced. We estimate that, on average, 12,700 (GA/TC)n and 3900 (AC/GT)n microsatellites could be found in the genome assuming a random distribution. These estimates are high enough for the construction of a saturated genetic map. Primers were designed for three microsatellite loci, and analyses of polymorphism in a wild cohort revealed that one was suitable for population genetics studies (5 alleles), while the other two were highly polymorphic (between 17 and 48 alleles) and thus were more useful for paternity testing. Mendelian inheritance was tested on two full-sib families, and significant distortions of genotypic frequencies were found, although the gametic distributions seemed to be in agreement with Mendelian expectations. We interpret this as evidence for zygotic selection.

Sequencing of the dnapol promoter region of Autographa californica nuclear polyhedrosis virus (AcNPV) revealed an overlapping open reading frame (ORF) in an antisense orientation, referred to as ORF-2. Analysis of the ORF-2 deduced amino-acid sequence revealed two short regions of homology with a similar ORF from Lymantria dispar nuclear polyhedrosis virus (LdNPV). Two 3' processing signals of this gene, expressed late during infection, were shown to be located on the orf-2 stop codon and 162 nucleotides further downstream.

Based on a correlation between expression patterns of an abundant tissue-specific small nuclear ribonucleoprotein (N protein) and the calcitonin gene-related protein (CGRP) splicing choice, a small nuclear ribonucleoprotein (the N factor) has been hypothesized to potentially function as a trans-acting factor involved in the regulation of the alternative splicing of the calcitonin/CGRP transcript. RNA analysis indicated that most rat, human, and simian cell lines and tissues making the CGRP mRNA splicing choice expressed the N factor mRNA. These data led us to address the effect of ectopic expression of the N factor in HeLa cells, which exhibit a calcitonin splicing choice when expressing the calcitonin/CGRP gene. Expression of the N factor exerts no effect on the calcitonin/CGRP splicing choice in HeLa cells. Furthermore, several cell lines such as the human 293 cell line make the CGRP mRNA splicing choice in the absence of any detectable level of the mRNA encoding the N factor. Together, these data reveal that the N protein is neither sufficient nor required for the tissue-specific CGRP splicing decision and that the N protein is not the trans-acting factor regulating the alternative splicing of the calcitonin/CGRP gene.

The retinoic acid receptor (RAR) requires coregulators to bind effectively to response elements in target genes. A strategy of sequential screening of expression libraries with a retinoic acid response element and RAR identified a cDNA encoding a coregulator highly related to RXR alpha. This protein, termed RXR beta, forms heterodimers with RAR, preferentially increasing its DNA binding and transcriptional activity on promoters containing retinoic acid, but not thyroid hormone or vitamin D, response elements. Remarkably, RXR beta also heterodimerizes with the thyroid hormone and vitamin D receptors, increasing both DNA binding and transcriptional function on their respective response elements. RXR alpha also forms heterodimers with these receptors. These observations suggest that retinoid X receptors meet the criteria for biochemically characterized cellular coregulators and serve to selectively target the high affinity binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate DNA response elements.

Most simian cells are permissive for SV40 and adenovirus-SV40 hybrids but nonpermissive for human adenoviruses, and the defect has been shown to take place at the level of processing of late viral mRNAs (Klessig and Grodzicker, 1979). Viral DNA synthesis and virus progeny production were studied in simian cells infected with different adenovirus serotypes. Adenoviruses belonging to oncogenic subgroups A and B (Ad31 and Ad3) failed to replicate their DNA in CV1 cells, whereas DNA replication occurred for all the other serotypes. Co-infection of CV1 cells with SV40 and Ad3 (or Ad31) resulted in the inhibition of SV40 DNA synthesis, as well as cellular DNA synthesis. The inhibition was not related to adenovirus DNA replication, since SV40 did not complement the Ad3/Ad31 replication defective function. Similar results were obtained in coinfected BSC and MK2 simian cell lines. Inhibition of Ad2ND1 DNA synthesis and gene expression also occurred in co-infection of simian cells with nondefective Ad2ND1 hybrid and defective Ad3/Ad31. In permissive human cell lines (HeLa or KB) co-infected with Ad2 and Ad3 (or Ad31), a dominant, inhibitory effect of Ad3 (or Ad31) over Ad2 was also observed. The inhibition appeared to function stoichiometrically and not catalytically, and to involve early adenovirus gene products. In both simian and human cells a hierarchy of dominance appeared between serotypes belonging to different subgroups. The degree of inhibitory effect occurred in the following decreasing order: Ad3 and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad31 (A), Ad2 and Ad5 (C).