A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance

RAP1 is one of the components of shelterin, the capping complex at chromosome ends or telomeres, although its role in telomere length maintenance and protection has remained elusive. RAP1 also binds subtelomeric repeats and along chromosome arms, where it regulates gene expression and has been shown to function in metabolism control. Telomerase is the enzyme that elongates telomeres, and its deficiency causes a premature aging in humans and mice. We describe an unanticipated genetic interaction between RAP1 and telomerase. While RAP1 deficiency alone does not impact on mouse survival, mice lacking both RAP1 and telomerase show a progressively decreased survival with increasing mouse generations compared to telomerase single mutants. Telomere shortening is more pronounced in Rap1^?/? Terc^?/? doubly deficient mice than in the single‐mutant Terc^?/? counterparts, leading to an earlier onset of telomere‐induced DNA damage and degenerative pathologies. Telomerase deficiency abolishes obesity and liver steatohepatitis provoked by RAP1 deficiency. Using genomewide ChIP sequencing, we find that progressive telomere shortening owing to telomerase deficiency leads to re‐localization of RAP1 from telomeres and subtelomeric regions to extratelomeric sites in a genomewide manner. These findings suggest that although in the presence of sufficient telomere reserve RAP1 is not a key factor for telomere maintenance and protection, it plays a crucial role in the context of telomerase deficiency, thus in agreement with its evolutionary conservation as a telomere component from yeast to humans.     

Validation of a universal set of primers to study animal-associated microeukaryotic communities

The application of metabarcoding to study animal-associated microeukaryotes has been restricted because the universal barcode used to study microeukaryotic ecology and distribution in the environment, the Small Subunit of the Ribosomal RNA gene (18S rRNA), is also present in the host. As a result, when host-associated microbial eukaryotes are analysed by metabarcoding, the reads tend to be dominated by host sequences. We have done an in silico validation against the SILVA 18S rRNA database of a non-metazoan primer set (primers that are biased against the metazoan 18S rRNA) that recovers only 2.6% of all the metazoan sequences, while recovering most of the other eukaryotes (80.4%). Among metazoans, the non-metazoan primers are predicted to amplify 74% of Porifera sequences, 4% of Ctenophora, and 15% of Cnidaria, while amplifying almost no sequences within Bilateria. In vivo, these non-metazoan primers reduce significantly the animal signal from coral and human samples, and when compared against universal primers provide at worst a 2-fold decrease in the number of metazoan reads and at best a 2800-fold decrease. This easy, inexpensive, and near-universal method for the study of animal-associated microeukaryotes diversity will contribute to a better understanding of the microbiome.     

Putative local adaptations modulate the interactions of the carnivorous plant Drosera uniflora Willd (1809) (Droseraceae) with cushion and shrub nurse plants

Cushion and shrub plants are typical high mountain nurse plants. In Magellanic tundras of the Cordillera del Sarao on the coast of Chile, the carnivorous plant Drosera uniflora grows in association with cushions of Donatia fascicularis and the shrubs Chusquea montana var. nigricans and Lepidothamnus fonkii. The different microhabitats for recruitment, in addition to the limited gene flow of D. uniflora, enable us to hypothesise that this plant manifests putative local adaptations, expressed in seed germination and population abundance. Our aim was to evaluate the local adaptation of D. uniflora to cushion and shrub microhabitats by estimating germination and abundance. Local adaptation of seed germination was determined by means of reciprocal transplant experiments. Abundance was determined in small plots located inside, on the edge and outside the nurse plants. Seed origin and growth substrate play a decisive role in D. uniflora germination. Seeds that originate from cushions plant habitat and germinate in the same substrate do so in greater numbers than when those which originate from shrubby habitats. Conversely, seeds that originate from shrubby habitat and germinate in the same substrate do not exhibit significant differences. Abundance was always greater inside than outside of the nurse plants. We concluded that availability and quality of different microhabitats for seed germination and recruitment, together with a limited gene flow, would trigger putative local adaptations in D. uniflora, modulating a long history of interactions with the plants that act as nurse plants.     

The Netrin-4/Laminin γ1/Neogenin-1 complex mediates migration in SK-N-SH neuroblastoma cells

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Netrin-4 (NTN4), a laminin-related protein, has been proposed as a key factor to target NB metastasis, although there is controversy about its function. Here, we show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Furthermore, NTN4 was shown to act as a cell adhesion molecule required for the migration induced by Neogenin-1 (NEO1) in SK-N-SH neuroblastoma cells. Therefore, we propose that NTN4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, acts as an essential extracellular matrix component, which induces the migration of SK-N-SH cells.     

The use of in vitro culture to improve the propagation of Rhododendron ponticum subsp. baeticum (Boiss. & Reuter)

Rhododendron ponticum subsp. baeticum is endemic in the southern region of the Iberian Peninsula. The relict populations of this species are vulnerable, due mainly to difficult conditions for the establishment of seedlings, resulting in a virtual lack of sexual recruitment. In order to preserve the surviving populations, in vitro culture methods have been applied for both the sexual and the agamic propagation of the species. The in vitro germination of seeds was high when conducted with Anderson’s medium without plant growth regulators. The self-rooted seedlings obtained were easily transplanted to outside conditions. The presence of growth regulators in the medium interfered with the development of the seedlings, causing heavy callus formation. The in vitro growth of explants took place readily in Anderson’s medium plus 0.072 mg L⁻¹ of BA and 0.036 mg L⁻¹ of NAA although the explants did not form roots. Rooting was achieved by the basal dipping of the explants in hydroalcoholic solutions of 500 mg L⁻¹ IAA during the outside transplanting process. Therefore, the combination of in vitro grown explants together with ex vitro rooting, results in a good method for the agamic propagation of Rhododendron ponticum subsp. baeticum.     

Thermal niche variation among individuals of the poison frog,Oophaga pumilio,in forest and converted habitats

The conversion of natural habitats to human land uses often increases local temperatures, creating novel thermal environments for species. The variable responses of ectotherms to habitat conversion, where some species decline while others persist, can partly be explained by variation among species in their thermal niches. However, few studies have examined thermal niche variation within species and across forest‐land use ecotones, information that could provide clues about the capacity of species to adapt to changing temperatures. Here, we quantify individual‐level variation in thermal traits of the tropical poison frog, Oophaga pumilio, in thermally contrasting habitats. Specifically, we examined local environmental temperatures, field body temperatures (T_b), preferred body temperatures (T_pref), critical thermal maxima (CT_max), and thermal safety margins (TSM) of individuals from warm, converted habitats and cool forests. We found that frogs from converted habitats exhibited greater mean T_b and T_pref than those from forests. In contrast, CT_max and TSM did not differ significantly between habitats. However, CT_max did increase moderately with increasing T_b, suggesting that changes in CT_max may be driven by microscale temperature exposure within habitats rather than by mean habitat conditions. Although O. pumilio exhibited moderate divergence in T_pref, CT_max appears to be less labile between habitats, possibly due to the ability of frogs in converted habitats to maintain their T_b below air temperatures that reach or exceed CT_max. Selective pressures on thermal tolerances may increase, however, with the loss of buffering microhabitats and increased frequency of extreme temperatures expected under future habitat degradation and climate warming. Abstract in Spanish is available with online material.     

Phlebotominae and Trichomyiinae (Diptera: Psychodidae) diversity in a tropical dry forest of central Mexico: a comparison of conserved and anthropized habitats

We recorded and analyzed the diversity of Phlebotominae and Trichomyiinae in two types of landscape (conserved and anthropized) over the course of three seasons (rainy, cold and dry) in southwestern Puebla, Mexico. A total of 578 specimens belonging to five genera and 10 species were obtained. Phlebotominae was represented by eight species, six of them new records for the state of Puebla, and for Trichomyiinae two species were recorded in the state of Puebla for the first time. There were eight species in the anthropized area and 10 species were recorded in the conservation area. The abundance of both subfamilies was high in the conservation area, representing 83.6% of the total sample, whereas in the anthropized area only 16.4% of the total sample was recorded. Eight species were found during the rainy and dry seasons, and seven species during the cold season. Abundance was highest in the dry season (76.30%), and lowest in the rainy season (5.9%). This kind of information is useful for understanding the transmission patterns of visceral leishmaniasis – endemic to central Mexico – by phlebotomine sand flies.     

Sperm protein (sp50) binds to acromosome and plasma membranes in a Ca2+-dependent manner: Possible role in acrosome reaction

Annexins are a family of Ca²⁺-binding proteins involved in the exocytotic process. The presence and the role of annexins in mammalian spermatozoa have not been well established. Two annexin-like proteins were obtained from guinea pig testis, a doublet of Mr 31–33 kD (p31/33) and a protein of Mr 50 kD (p50). Both proteins were able to bind to erythrocyte ghosts in a Ca²⁺-dependent fashion. Polyclonal antibodies against p31/33 reacted with two major proteins, Mrs 50 kD (sp50) and 42 kD (sp42), from mature and immature guinea pig spermatozoa. p50 and sp50 are likely the native proteins from testis and spermatozoa, respectively, and they are seemingly related. By immunofluorescence, sp50 was only found in the apical acrosome region of immature and capacitated and noncapacitated spermatozoa, and its location was intracellular. In spermatozoa undergoing acrosome reaction, sp50 was detected in the whole acrosome, while in spermatozoa that had undergone acrosome reaction sp50 was not detected. However, in the protein pattern of acrosome reaction vesicles, anti-p31/33 antibody revealed diffuse bands of Mr 35–38 kD. sp50 was able to bind to plasma membrane fragments and acrosome outer membrane from demembranated sperm in a Ca²⁺-dependent fashion. The presence of sp50 in the acrosome region, its distribution throughout the acrosome membrane just before the acrosome reaction, and its ability to bind both plasma and outer acrosome membranes in a Ca²⁺-dependent manner suggest that sp50 may participate in the acrosome reaction mechanism in guinea pig spermatozoa. © 1996 Wiley-Liss, Inc.