Structural basis for innate immune sensing by M-ficolin and its control by a pH-dependent conformational switch

Ficolins are soluble oligomeric proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. They act as innate immune sensors by recognizing conserved molecular markers exposed on microbial surfaces and thereby triggering effector mechanisms such as enhanced phagocytosis and inflammation. In humans, L- and H-ficolins have been characterized in plasma, whereas a third species, M-ficolin, is secreted by monocytes and macrophages. To decipher the molecular mechanisms underlying their recognition properties, we previously solved the structures of the recognition domains of L- and H-ficolins, in complex with various model ligands (Garlatti, V., Belloy, N., Martin, L., Lacroix, M., Matsushita, M., Endo, Y., Fujita, T., Fontecilla-Camps, J. C., Arlaud, G. J., Thielens, N. M., and Gaboriaud, C. (2007) EMBO J. 24, 623-633). We now report the ligand-bound crystal structures of the recognition domain of M-ficolin, determined at high resolution (1.75-1.8 A), which provides the first structural insights into its binding properties. Interaction with acetylated carbohydrates differs from the one previously described for L-ficolin. This study also reveals the structural determinants for binding to sialylated compounds, a property restricted to human M-ficolin and its mouse counterpart, ficolin B. Finally, comparison between the ligand-bound structures obtained at neutral pH and nonbinding conformations observed at pH 5.6 reveals how the ligand binding site is dislocated at acidic pH. This means that the binding function of M-ficolin is subject to a pH-sensitive conformational switch. Considering that the homologous ficolin B is found in the lysosomes of activated macrophages (Runza, V. L., Hehlgans, T., Echtenacher, B., Zahringer, U., Schwaeble, W. J., and Mannel, D. N. (2006) J. Endotoxin Res. 12, 120-126), we propose that this switch could play a physiological role in such acidic compartments.     

Structural and thermodynamic bases for the design of pure prolactin receptor antagonists: X-ray structure of Del1-9-G129R-hPRL

Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly(129) mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly(129) mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL.     

The human Nup107-160 nuclear pore subcomplex contributes to proper kinetochore functions

We previously demonstrated that a fraction of the human Nup107-160 nuclear pore subcomplex is recruited to kinetochores at the onset of mitosis. However, the molecular determinants for its kinetochore targeting and the functional significance of this localization were not investigated. Here, we show that the Nup107-160 complex interacts with CENP-F, but that CENP-F only moderately contributes to its targeting to kinetochores. In addition, we show that the recruitment of the Nup107-160 complex to kinetochores mainly depends on the Ndc80 complex. We further demonstrate that efficient depletion of the Nup107-160 complex from kinetochores, achieved either by combining siRNAs targeting several of its subunits excluding Seh1, or by depleting Seh1 alone, induces a mitotic delay. Further analysis of Seh1-depleted cells revealed impaired chromosome congression, reduced kinetochore tension and kinetochore-microtubule attachment defects. Finally, we show that the presence of the Nup107-160 complex at kinetochores is required for the recruitment of Crm1 and RanGAP1-RanBP2 to these structures. Together, our data thus provide the first molecular clues underlying the function of the human Nup107-160 complex at kinetochores.     

A new case of fish-eating in Japanese macaques: implications for social constraints on the diffusion of feeding innovation

This is the first detailed report of social factors affecting fish-eating in Japanese macaques under natural circumstances. We video-recorded a complete event of fish eating, involving a new fish food species for the monkeys on Koshima island. Following the discovery of a large beached sea bass by a peripheral male, we observed a total of 16 individuals feeding on the fish in turns, and interacting around it. The rank order of access to the fish was mainly explained by the spatial position of group members, whereas dominance determined how long the fish was monopolized. Although limited, the tolerated presence of close-bystanders while feeding was affected by kinship and affiliation. Genealogic data suggested that fish-eating behavior was well maintained in terms of maternal lineages. This report should contribute to a better understanding of how social features may constrain the long-term diffusion of feeding innovations in free-ranging primate groups.     

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, ‘mediator’ proteins are in charge of recruiting ‘signal transducers’ to molecules ‘sensing’ the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated.     

Influence of 6 or 8-substitution on the antiviral activity of 3-phenethylthiomethylimidazo[1,2-a]pyridine against human cytomegalovirus (HCMV) and varicella-zoster virus (VZV)

The synthesis of original imidazo[1,2-a]pyridines bearing a phenethylthiomethyl side chain at the 3 position and a (hetero)aryl substituent on the 6 or 8 position, and their antiviral activities are reported. From the synthesized compounds, the 6-halogeno and 6-phenylimidazo[1,2-a]pyridine derivatives 4c-d and 5b were the most potent against human cytomegalovirus (CMV) and/or varicella-zoster virus (VZV), whereas several other congeners (i.e., 5e, 5g, 5i, 5l, 5n, 5p, 5q, and 5t), while less potent, were equally or more selective in their inhibitory activity against both VZV and CMV. These compounds showed similar activity against thymidine kinase competent (TK(+)) and deficient (TK(-)) VZV strains, demonstrating a mechanism of action independent of the viral thymidine kinase.     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.     

Anterior regions of monkey parietal cortex process visual 3D shape

The intraparietal cortex is involved in the control of visually guided actions, like reach-to-grasp movements, which require extracting the 3D shape and position of objects from 2D retinal images. Using fMRI in behaving monkeys, we investigated the role of the intraparietal cortex in processing stereoscopic information for recovering the depth structure and the position in depth of objects. We found that while several areas (CIP, LIP, and AIP on the lateral bank; PIP and MIP on the medial bank) are activated by stereoscopic stimuli, AIP and an adjoining portion of LIP are sensitive only to depth structure. Furthermore, only these two regions are sensitive to both the depth structure and the 2D shape of small objects. These results indicate that extracting 3D spatial information from stereo involves several intraparietal areas, among which AIP and anterior LIP are more specifically engaged in extracting the 3D shape of objects.     

Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis

The Drosophila melanogaster body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. Here, we investigate the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. We show that exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. We show that, in vivo, Rab6 is found in a complex with Bicaudal-D, and that Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila.