Role of the sub-cellular localization of RasGAP fragment N2 for its ability to sensitize cancer cells to genotoxin-induced apoptosis.

The specific sensitization of tumor cells to the apoptotic response induced by genotoxins is a promising way of increasing the efficacy of chemotherapies. The RasGAP-derived fragment N2, while not regulating apoptosis in normal cells, potently sensitizes tumor cells to cisplatin- and other genotoxin-induced cell death. Here we show that fragment N2 in living cells is mainly located in the cytoplasm and only minimally associated with specific organelles. The cytoplasmic localization of fragment N2 was required for its cisplatin-sensitization property because targeting it to the mitochondria or the ER abrogated its ability to increase the death of tumor cells in response to cisplatin. These results indicate that fragment N2 requires a spatially constrained cellular location to exert its anti-cancer activity.

Role of the transcriptional factor C/EBPbeta in free fatty acid-elicited beta-cell failure.

Fatty acids can favour the development of Type 2 diabetes by reducing insulin secretion and inducing apoptosis of pancreatic beta-cells. Here, we show that sustained exposure of the beta-cell line MIN6 or of isolated pancreatic islets to the most abundant circulating fatty acid palmitate increases the level of C/EBPbeta, an insulin transcriptional repressor. In contrast, two unsaturated fatty acids, oleate and linoleate were without effect. The induction of C/EBPbeta elicited by palmitate was prevented by inhibiting the ERK1/2 MAP kinase pathway or by reducing mitochondrial fatty acid oxidation with an inhibitor of Carnitine Palmitoyl Transferase-1. Overexpression of C/EBPbeta mimicked the detrimental effects of palmitate and resulted in a drastic reduction in insulin promoter activity, impairment in the capacity to respond to secretory stimuli and an increase in apoptosis. Our data suggest a potential involvement of C/EBPbeta as mediator of the deleterious effects of unsaturated free fatty acids on beta-cell function.

Binding of d-methadone, l-methadone, and dl-methadone to proteins in plasma of healthy volunteers: role of the variants of alpha 1-acid glycoprotein.

The plasma concentrations of alpha 1-acid glycoprotein (AAG), albumin, triglycerides, cholesterol, and total proteins, as well as the plasma binding of racemic, d-methadone, and l-methadone were measured in 45 healthy subjects. The AAG phenotypes and the concentrations of AAG variants were also determined. The measured free fractions for racemic, d-methadone, and l-methadone were, respectively, 12.7% +/- 3.3%, 10.0% +/- 2.9%, and 14.2% +/- 3.2% (mean +/- SD). A significant correlation was obtained between the binding ratio (B/F) for dl-methadone and the total AAG concentration (r = 0.724; p less than 0.001). A multiple stepwise regression analysis showed that AAG was the main explanatory variable for the binding of the racemate. When concentrations of AAG variants were considered, a significant correlation was obtained between the binding ratio of dl-methadone and orosomucoid2 A concentration (r = 0.715; p less than 0.001), a weak correlation between dl-methadone and orosomucoid1 S concentration (r = 0.494; p less than 0.001), and no correlation between dl-methadone and orosomucoid1 F1 concentration (r = 0.049; not significant). Similar findings were obtained with the enantiomers. This study shows the importance of considering not only total AAG but also concentrations of AAG variants when measuring the binding of methadone and possibly of other drugs in plasma.

Role of the chemokine receptor CX3CR1 in the mobilization of phagocytic retinal microglial cells.

We recently showed that subretinal CX3CR1-dependent microglial cell (MC) accumulation may lead to age-related macular degeneration. The fate of MC after engulfing retinal debris is poorly understood. Severe photoreceptor degeneration was observed 40days after exposure to bright light in CX3CR1-deficient but not control mice, and more MCs accumulated in the subretinal space of the former than the latter. To study the fate of subretinal MCs in CX3CR1 competent animals, we used a dystrophic rat model in which abundant subretinal MC accumulation is observed secondary to retinal degeneration. In dystrophic rats, MCs containing rhodopsin or rod outer segment (ROS) debris were found outside the outer retina at sites suggesting choroidal and ciliary egress. In conclusion, our data indicate that MC accumulation at injury sites is independent of CX3CR1 and precedes photoreceptor degeneration. The ectopic presence of rhodopsin-positive MCs suggests that CX3CR1 participates in MC egress from the outer retina.

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit.

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.

Role of the microenvironment on epithelial stem cell fate

Stratified epithelia of mammals contain adult stem/progenitor cells that are instrumental for renewal, regeneration and repair. We have recently demonstrated, using clonal and functional analysis, that all stratified epithelia contain clonogenic stem cells that can respond to skin morphogenetic signals, while cells obtained from simple or pseudo-stratified epithelia cannot. A genome-wide expression analysis favors multilineage priming rather than reprogramming. Collectively, these observations are reminiscent of epithelial metaplasia, a phenomenon in which a cell adopts the phenotype of another epithelial cell, often in response to repeated environmental stress, e.g. smoking, alcohol and micro-traumatisms. Furthermore, they support the notion that metaplasia results from the expression of an unseen potency, revealed by an environmental deficiency. The thymus supposedly contains only progenitor epithelial cells but no stem cells. We have demonstrated that the thymus also contains a small population of clonogenic cells that can function as bona fide multipotent hair follicle stem cells in response to an inductive skin microenvironment and a genome-wide expression analysis indicates that it correlates with robust changes in the expression of genes important for thymus identity. Hence, multilineage priming or reprogramming can account for the fate change of epithelial stem/progenitor cells in response to a varying microenvironment.

Role of the host cell's unfolded protein response in arenavirus infection.

Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.

Role of the gene Miniature in Drosophila wing maturation.

Miniature is an extracellular zona pellucida domain-containing protein, required for flattening of pupal wing epithelia in Drosophila. Here, we show that Miniature also plays an important role in the post-eclosion wing maturation processes triggered by the neurohormone bursicon. Wing expansion and epithelial apoptosis are drastically delayed in miniature loss-of-function mutants, and sped up upon overexpression of the protein in wings. Miniature acts upstream from the heterotrimeric Gs protein transducing the bursicon signal in wing epithelia. We propose that Miniature interacts with bursicon and regulates its diffusion through or stability within the wing tissue.

Role of the HCF-1 basic region in sustaining cell proliferation.

BACKGROUND: The human herpes simplex virus-associated host cell factor 1 (HCF-1) is a conserved human transcriptional co-regulator that links positive and negative histone modifying activities with sequence-specific DNA-binding transcription factors. It is synthesized as a 2035 amino acid precursor that is cleaved to generate an amino- (HCF-1(N)) terminal subunit, which promotes G1-to-S phase progression, and a carboxy- (HCF-1(C)) terminal subunit, which controls multiple aspects of cell division during M phase. The HCF-1(N) subunit contains a Kelch domain that tethers HCF-1 to sequence-specific DNA-binding transcription factors, and a poorly characterized so called "Basic" region (owing to a high ratio of basic vs. acidic amino acids) that is required for cell proliferation and has been shown to associate with the Sin3 histone deacetylase (HDAC) component. Here we studied the role of the Basic region in cell proliferation and G1-to-S phase transition assays. METHODOLOGY/PRINCIPAL FINDINGS: Surprisingly, much like the transcriptional activation domains of sequence-specific DNA-binding transcription factors, there is no unique sequence within the Basic region required for promoting cell proliferation or G1-to-S phase transition. Indeed, the ability to promote these activities is size dependent such that the shorter the Basic region segment the less activity observed. We find, however, that the Basic region requirements for promoting cell proliferation in a temperature-sensitive tsBN67 cell assay are more stringent than for G1-to-S phase progression in an HCF-1 siRNA-depletion HeLa-cell assay. Thus, either half of the Basic region alone can support G1-to-S phase progression but not cell proliferation effectively in these assays. Nevertheless, the Basic region displays considerable structural plasticity because each half is able to promote cell proliferation when duplicated in tandem. Consistent with a potential role in promoting cell-cycle progression, the Sin3a HDAC component can associate independently with either half of the Basic region fused to the HCF-1 Kelch domain. CONCLUSIONS/SIGNIFICANCE: While conserved, the HCF-1 Basic region displays striking structural flexibility for controlling cell proliferation.

Role of the epithelial sodium channel (ENaC) and its positive regulator, the channel-activating protease 1 (CAP1) in skin

Summary: The mammalian epidermis is a pluristratified epithelium composed of 90% keratinocytes, and its main function is to serve as barrier for the body. The epithelial sodium channel (ENaC), formed by three homologous subunits α, β and γ is found in a variety of epithelia including epidermis. Previous studies showed that ENaC modulates different aspects of epidermal differentiation, such as synthesis of differentiation-specific proteins and lipid secretion. ENaC plays also a critical role in sodium homeostasis of renal and pulmonary epithelia, and its activity is thereby well controlled by hormones and non-hormonal factors, such as the serine protease CAP1 (channel-activating protease 1), also termed prostasin encoded by Prss8 gene. Serine proteases are proteolytic enzymes involved in numerous physiological and pathological processes in the epidermis. In order to evaluate the role of β and γENaC in epidermis, we analyzed the skin phenotype of β and γENaC null mutant (βENaC-/- and γENaC-/-) mice in comparison with the phenotype of αENaC-deficient mice. Furthermore, keratin14-specific CAP1-deficient mice (Prss8lox/Δ /K14-Cre) were generated in order to unveil the role of the serine protease CAP1 in epidermal development and function. This study reveals that the skin phenotype of βENaC and γENaC null mutant mice is less severe than the one of αENaC-deficient mice. However, all these mice present a common premature lipid secretion in the mid-granular layer of the epidermis. Further, the composition of the lipids of the stratum corneum in αENaC-deficient mice is strongly altered, suggesting that epidermal barrier function is compromised. K14-specific CAP1-deficient newborn mice are born at the expected Mendelian ratio, but die soon after birth, showing that CAP1 is required for postnatal survival. The epidermis of these mice exhibits striking malformations of the stratum corneum showing hyperkeratosis. These defects seriously affect both inward and outward epidermal barrier function, leading to rapid and fatal dehydration. As in αENaC-deficient mice, the lipid composition of the stratum corneum of K14-specific CAP1-deficient mice is disturbed. Furthermore, lack of CAP1 leads to the selective loss of filaggrin monomers, important for keratins aggregation and skin moisturization, and to an increased of aberrant profilaggrin precursors. In conclusion, both ENaC and CAP1 expression in the epidermis are crucial for keratinocyte differentiation processes and/or barrier function. Since the abnormalities in K14-specific CAP1-deficient mice resemble key features of human skin ichthyosis, in particular Harlequin ichthyosis, the study of ENaC and CAP1 mutant mice might allow new insights into mechanisms underlying skin diseases. Résumé: L'épiderme des mammifères est un épithélium pluristratifié, protégeant le corps contre les perturbations extérieures et la déshydratation. Le canal épithélial à sodium (ENaC), formé de trois sous-unités α, β et γ, est exprimé dans de nombreux épithélia, comme l'épiderme. Des études ont montré que l'absence de la sous-unité αENaC modulait différents aspects de la différenciation des kératinocytes de l'épiderme, comme la synthèse de protéines spécifiques ou la sécrétion de lipides dans la couche granulaire de l'épiderme. ENaC joue également un rôle crucial dans l'homéostasie du sodium dans les épithélia électriquement étanches, comme l'épithélium rénal ou pulmonaire. L'activité de ENaC est par conséquent finement régulée, en partie par des hormones, mais aussi par des facteurs non-hormonaux, telle que la sérine protéase CAP1 (« channel-activating protease 1 >>) (nommée également prostasine et codée par le gène Prss8). Le but de ce travail a donc été d'étudier le rôle des sous-unités β et γENaC dans l'épiderme en comparaison avec celui de la sous-unité α en utilisant des souris mutantes βENaC-/- et γENaC-/-. Dans un deuxième temps, le phénotype d'une souris chez qui CAP1 a été spécifiquement invalidé dans l'épiderme (Prsslox/Δ/K14-Cre) a été analysé, dans le but de mettre en évidence le rôle de cette protéase dans l'épiderme. Comme déjà montré pour les souris αENaC-/-, la sécrétion des lipides dans la couche granulaire de l'épiderme des souris βENaC-/- et γENaC-/- est prématurée. Cependant, l'hyperplasie et l'expression anormale des protéines marqueurs de la différenciation présents chez les souris αENaC-/- n'ont pas été observés dans l'épiderme des souris βENaC-/- et γENaC-/-. La composition lipidique de la couche cornée des souris αENaC-/- est fortement altérée suggérant que la fonction de barrière de l'épiderme de ces souris est compromise. Les souris mutantes CAP1 ont quant à elles révélé des malformations sévères de leur couche cornée, affectant la fonction de barrière de leur épiderme et conduisant à la mort de ces souris par déshydratation quelques jours après leur naissance. De plus, la composition en lipides de la couche cornée ainsi que la taille des cellules cornées, les cornéocytes, de ces souris sont modifiées par rapport aux souris contrôles. L'invalidation de la protéine CAP1 dans l'épiderme conduit aussi à la perte de la filaggrine, une protéine cruciale pour l'agrégation des kératines dans la couche cornée et le maintien du niveau d'hydratation de la peau, et à l'accumulation de ses précurseurs. En conclusion, l'expression de ENaC et de CAP1 est cruciale pour la différenciation de l'épiderme et/ou sa fonction de barrière. De plus, le phénotype des souris mutantes CAP1 présente des caractéristiques qui ressemblent à celles observées dans certaines pathologies humaines cutanées, comme l'ichthyose d'Harlequin. L'étude des souris mutantes ENaC et CAP1 pourrait donc apporter de nouvelles connaissances dans les mécanismes impliqués dans l'ichthyose d'Harlequin ou d'autres maladies de la peau chez l'homme. Résumé tout public: La peau est le plus grand organe vital du corps humain. Sa fonction principale est de protéger le corps comme une barrière, contre les agressions extérieures et la déshydratation. De nombreuses maladies de la peau résultent d'une perte de fonction de cette barrière. Bien que les pathologies cutanées soient très bien décrites, leur cause génétique n'est en général pas encore connue. La souris est alors un modèle de choix pour la recherche fondamentale. En effet, grâce aux progrès récents de la science, le génome de la souris peut aujourd'hui être modifié dans le but d'étudier le rôle de nombreuses protéines. Dans différents organes, comme le rein et le poumon, le canal épithélial à sodium (ENaC), composé de trois sous-unités protéiques homologues (α, β, et γ), joue un rôle essentiel dans la réabsorption du sodium. L'activité de ENaC est régulée par de nombreux facteurs hormonaux et non-hormonaux, telle que la protéase CAP1 (« channel-activating protease 1 »). L'invalidation de la sous-unité αENaC chez la souris a permis de montrer que dans la peau, le canal ENaC est impliqué dans la différenciation des cellules de l'épiderme et la croissance des poils. Durant ce travail, le phénotype des souris chez qui la protéine βENaC, γENaC ou CAP1 a été invalidée (souris mutantes), a été étudié dans le but de mieux comprendre le rôle des sous-unités du canal ENaC et de son régulateur CAP1 dans la peau. Les résultats de ce projet ont montré que les souris mutantes βENaC et γENaC présentent un épiderme anormal avec une synthèse prématurée de lipides dans la couche granulaire, suggérant l'implication de ENaC dans la fonction de barrière de la peau. De plus, quand CAP1 est invalidé de manière totale chez les souris, le développement embryonnaire est perturbé et ces souris meurent avant la naissance. CAP1 a donc été invalidé spécifiquement dans l'épiderme des souris. Ces souris mutantes « épiderme-spécifique » naissent normalement, mais meurent peu après la naissance de déshydratation. La couche superficielle de l'épiderme, appelée couche cornée, de ces souris est malformée et ne confère plus à la peau sa fonction de barrière. De plus, les composants de la couche cornée, les cellules cornées entourées de lipides, sont sévèrement altérés. Le phénotype de ces souris ressemble aux caractéristiques présentes chez les patients atteints d'ichthyoses, en particulier l'ichthyose d'Harlequin. En conclusion, le canal ENaC ainsi que son régulateur CAP1 jouent un rôle clé dans les processus de différenciation de l'épiderme et/ou de sa fonction de barrière. De plus, les souris mutantes pour CAP1 et ENaC se révéleront peut-être comme des modèles appropriés dans l'étude de l'ichthyose d'Harlequin ou d'autres maladies cutanées.

Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.

Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells.

Consensus paper: the role of the cerebellum in perceptual processes.

Various lines of evidence accumulated over the past 30 years indicate that the cerebellum, long recognized as essential for motor control, also has considerable influence on perceptual processes. In this paper, we bring together experts from psychology and neuroscience, with the aim of providing a succinct but comprehensive overview of key findings related to the involvement of the cerebellum in sensory perception. The contributions cover such topics as anatomical and functional connectivity, evolutionary and comparative perspectives, visual and auditory processing, biological motion perception, nociception, self-motion, timing, predictive processing, and perceptual sequencing. While no single explanation has yet emerged concerning the role of the cerebellum in perceptual processes, this consensus paper summarizes the impressive empirical evidence on this problem and highlights diversities as well as commonalities between existing hypotheses. In addition to work with healthy individuals and patients with cerebellar disorders, it is also apparent that several neurological conditions in which perceptual disturbances occur, including autism and schizophrenia, are associated with cerebellar pathology. A better understanding of the involvement of the cerebellum in perceptual processes will thus likely be important for identifying and treating perceptual deficits that may at present go unnoticed and untreated. This paper provides a useful framework for further debate and empirical investigations into the influence of the cerebellum on sensory perception.

Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory.

Immune protection from intracellular pathogens depends on the generation of terminally differentiated effector and of multipotent memory precursor CD8 T cells, which rapidly regenerate effector and memory cells during recurrent infection. The identification of factors and pathways involved in CD8 T cell differentiation is of obvious importance to improve vaccination strategies. Here, we show that mice lacking T cell factor 1 (Tcf-1), a nuclear effector of the canonical Wingless/Integration 1 (Wnt) signaling pathway, mount normal effector and effector memory CD8 T cell responses to infection with lymphocytic choriomeningitis virus (LCMV). However, Tcf-1-deficient CD8 T cells are selectively impaired in their ability to expand upon secondary challenge and to protect from recurrent virus infection. Tcf-1-deficient mice essentially lack CD8 memory precursor T cells, which is evident already at the peak of the primary response, suggesting that Tcf-1 programs CD8 memory cell fate. The function of Tcf-1 to establish CD8 T cell memory is dependent on the catenin-binding domain in Tcf-1 and requires the Tcf-1 coactivators and Wnt signaling intermediates beta-catenin and gamma-catenin. These findings demonstrate that the canonical Wnt signaling pathway plays an essential role for CD8 central memory T cell differentiation under physiological conditions in vivo. They raise the possibility that modulation of Wnt signaling may be exploited to improve the generation of CD8 memory T cells during vaccination or for therapies designed to promote sustained cytotoxic CD8 T cell responses against tumors.

Extended family structure in the ant Formica paralugubris: the role of the breeding system

In populations of various ant species, many queens reproduce in the same nest (polygyny), and colony boundaries appear to be absent with individuals able to move fi eely between nests (unicoloniality). Such societies depart strongly from a simple family structure and pose a potential challenge to kin selection theory, because high queen number coupled with unrestricted gene flow among nests should result in levels of relatedness among nestmates close to zero. This study investigated the breeding system and genetic structure of a highly polygynous and largely unicolonial population of the wood ant Formica paralugubris. A microsatellite analysis revealed that nestmate workers, reproductive queens and reproductive males (the queens' mates) are all equally related to each other, with relatedness estimates centring around 0.14. This suggests that most of the queens and males reproducing in the study population had mated within or close to their natal nest, and that the queens did not disperse far after mating. We developed a theoretical model to investigate how the breeding system affects the relatedness structure of polygynous colonies. By combining the model and our empirical data, it was estimated that about 99.8% of the reproducing queens and males originated from within the nest, or from a nearby nest. This high rate of local mating and the rarity of long-distance dispersal maintain significant relatedness among nestmates, and contrast with the common view that unicoloniality is coupled with unrestricted gene flow among nests.