NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein gamma-tubulin. In mammals, gamma-tubulin associates with additional proteins into a large complex, the gamma-tubulin ring complex (gammaTuRC). We characterize NEDD1, a centrosomal protein that associates with gammaTuRCs. We show that the majority of gammaTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of gamma-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to gammaTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of gamma-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of gamma-tubulin to the centrosome.     

Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry

In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The on-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, seven and nine phosphorylated serine and/or threonine residues were identified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.     

Distinct modulation of the endothelin-1 pathway in iNOS-/- and eNOS-/- mice

We hypothesized that constitutive endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) have opposite effects on the regulation of endothelin and its receptors. We therefore sought to determine whether deletions of iNOS or eNOS genes in mice modulate pressor responses to endothelin and the expression of ETA and ETB receptors in a similar fashion. Despite unchanged baseline hemodynamic parameters, anesthetized iNOS-/- mice displayed reduced pressor responses to endothelin-1, but not to that of IRL-1620, a selective ETB agonist. Protein content of cardiac ETA receptors was reduced in iNOS-/- mice compared with wild-type mice, but that of ETB receptors was unchanged. Anesthetized eNOS-/- mice presented a hypertensive state, accompanied by an enhanced pressor response to intravenous endothelin-1, whereas the pressor response to IRL-1620 was reduced. Protein levels were also found to be increased for ETA receptors, but reduced for ETB receptors, in cardiac tissues of eNOS-/- mice. In conscious animals, both strains responded equally to the hypotensive effect of an ETA antagonist, ABT-627, whereas orally administered A-192621, an ETB antagonist, increased MAP to a greater extent in eNOS-/- than in wild-type mice. Furthermore, significant levels of immunoreactive endothelin were found in mesenteric arteries in eNOS-/- but not in iNOS-/- or wild-type congeners. Our study shows that repression of iNOS or eNOS has differential effects on endothelin-1 and its receptors. We have also shown that the heart is the main organ in which iNOS or eNOS repression induces important alterations in protein content of endothelin receptors in adult mice.     

Maintenance of intercellular coupling by the antiarrhythmic peptide rotigaptide suppresses arrhythmogenic discordant alternans

Discordant action potential alternans creates large gradients of refractoriness, which are thought to be the mechanisms linking T-wave alternans to cardiac arrhythmogenesis. Since intercellular coupling acts to maintain synchronization of repolarization between cells, we hypothesized that intercellular uncoupling, such as during ischemia, would initiate discordant alternans and that restoration of intercellular coupling by the gap junction opener rotigaptide may provide a novel approach for suppressing arrhythmogenic discordant alternans. Optical mapping was used to record action potentials from ventricular epicardium of Langendorff-perfused guinea pig hearts. Threshold for spatially synchronized (i.e., concordant) alternans and discordant alternans was determined by increasing heart rate step-wise during 1) baseline, 2) treatment with rotigaptide or vehicle, and 3) global low-flow ischemia + rotigaptide or vehicle. Ischemia reduced the threshold for concordant alternans in both groups from 362 +/- 8 to 305 +/- 9 beats/min (P < 0.01) and for discordant alternans from 423 +/- 6 to 381 +/- 7 beats/min (P < 0.01). Interestingly, rotigaptide also increased the threshold for discordant alternans relative to vehicle both before (438 +/- 7 vs. 407 +/- 8 beats/min, P < 0.05) and during (394 +/- 7 vs. 364 +/- 9 beats/min, P < 0.05) ischemia. Rotigaptide increased conduction velocity and prevented conduction slowing and dispersion of repolarization during ischemia. Confocal immunofluorescence revealed that total connexin43 quantity and cellular distribution were unchanged before or after low-flow ischemia, with and without rotigaptide. However, connexin43 dephosphorylation in response to low-flow ischemia was significantly prevented by rotigaptide (15.9 +/- 7.0 vs. 0.3 +/- 6.4%, P < 0.001). These data suggest that intercellular uncoupling plays an important role in the transition from concordant to discordant alternans. By suppressing discordant alternans, repolarization gradients, and connexinx43 dephosphorylation, rotigaptide may protect against ischemia-induced arrhythmias. Drugs that selectively open gap junctions offer a novel strategy for antiarrhythmic therapy.     

Could a defective epithelial sodium channel lead to bronchiectasis

Background

Bronchiectasis is defined as a permanent dilation of the airways arising from chronic bronchial inflammation/infection. In 50% of cases, no etiology can be identified. Recently, the role of the epithelial sodium channel ENaC has been pointed out in the pathophysiology of cystic fibrosis, a disease due to mutations in the CFTR gene and causing bronchiectasis in the airways. Moreover, it was found that transgenic mice overexpressing ENaCβ present cystic fibrosis-like lung disease symptoms. Our aim was to evaluate if a defective ENaC protein could be involved in the development of bronchiectasis.

Methods

We extensively analysed ENaCβ and γ genes in 55 patients with idiopathic bronchiectasis and without two mutations in the coding regions of CFTR. Thirty-eight patients presented functional abnormalities suggesting impaired sodium transport (abnormal sweat chloride concentration or nasal potential difference measurement), and 17 had no such evidence.

Results

Sequencing of the exons and flanking introns of the ENaCβ and γ gene identified five different amino-acid changes (p.Ser82Cys, p.Pro369Thr, p.Asn288Ser in ENaCβ ; and p.Gly183Ser, p.Glu197Lys in ENaCγ) in heterozygous state in 8 patients. The p.Ser82Cys amino-acid change was found in 3 unrelated patients who were also heterozygous for a CFTR mutation or variant (1 p.F508del, 1 IVS8-5T, and 1 IVS8-5T:1716G>A (p.E528E)). The other mutations were found in patients without CFTR mutation, the p.Glu197Lys mutation in 2 patients and the other variants in single patients. Among the 8 patients bearing an ENaC mutation, 5 had functional abnormalities suggesting impaired sodium transport.

Conclusion

Our results suggest that several variants in ENaCβ and γ genes might be deleterious for ENaC function and lead to bronchiectasis, especially in patients who are trans-heterozygotes for ENaCβ/CFTR mutations or variants.     

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (K_d = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.     

Functional Diversity of Plant–Pollinator Interaction Webs Enhances the Persistence of Plant Communities

Pollination is exclusively or mainly animal mediated for 70% to 90% of angiosperm species. Thus, pollinators provide an essential ecosystem service to humankind. However, the impact of human-induced biodiversity loss on the functioning of plant–pollinator interactions has not been tested experimentally. To understand how plant communities respond to diversity changes in their pollinating fauna, we manipulated the functional diversity of both plants and pollinators under natural conditions. Increasing the functional diversity of both plants and pollinators led to the recruitment of more diverse plant communities. After two years the plant communities pollinated by the most functionally diverse pollinator assemblage contained about 50% more plant species than did plant communities pollinated by less-diverse pollinator assemblages. Moreover, the positive effect of functional diversity was explained by a complementarity between functional groups of pollinators and plants. Thus, the functional diversity of pollination networks may be critical to ecosystem sustainability.     

The Golgi Protein ACBD3, an Interactor for Poliovirus Protein 3A,Modulates Poliovirus Replication

We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence.     

Experimental study of the impacts of silver carp on plankton communities of eutrophic Villerest reservoir (France)

We examined the impact of five silver carp biomass levels (0, 8, 16, 20, and 32 g m⁻³) on plankton communities and water quality of Villerest eutrophic reservoir (France). We realized the experiments using outdoor mesocosms. The presence of silver carp led to changes in zooplankton and phytoplankton assemblages. High fish biomass strongly reduced cladoceran abundance (through predation). Silver carp inefficiently grazed down particles < 20 μm. More importantly, however, the suppression of herbivorous cladocerans resulted in the increase of small size algae which were relieved from grazing and benefit from high nutrient concentrations. In contrast, in mesocosms without fish, the dominance of cladocerans (mainly Daphnia) controlled small size algae and probably also larger size algae (colonial chlorophytes, cyanobacteria). Thus, the Secchi disc transparency increased markedly. Through cascade effects, the modification of grazers communities led to changes in the utilization patterns of the added nutrients by phytoplankton communities. In high fish biomass treatments, nutrients were more efficiently accumulated into particulate fractions compared with no-fish and low-fish biomass treatments that were characterized by higher dissolved nutrients concentrations. Zooplankton was an essential source of food for silver carp. The productivity of zooplankton sustained a moderate silver carp biomass (up to 16 g m⁻³). In the presence of the highest fish biomass, the productivity of zooplankton was not large enough and silver carps fed on additional phytoplankton. Although mesocosms with high fish biomass were characterized by a slight cyanobacteria development compared with other fish mesocosms, silver carp was not effective in reducing cyanobacteria dominance. This revised version was published online in August 2006 with corrections to the Cover Date.