Workshop for French Young Scientists 2009 - San Diego - Abstracts
Understanding agonist differentiation of mucosal CD8AA intraepithelial lymphocyte through TCR analysis
Intraepithelial lymphocytes (IELs) from the small intestine constitute a heterogenous population of T cells that form the first line of adaptive immune defense at the main entry port for pathogens. Although all IELs characteristically display an antigen-experienced phenotype, they differ in the antigen-major histocompatibility complex (MHC) restriction and antigen specificity of their TCRs (T cell receptor, TCR). In addition, the mechanisms and pathways they employed to accumulate at this mucosal interface and to adapt to this effector memory phenotype are different. Two main groups can be distinguished based on their ontogeny and the nature of their cognate antigens (Ag). The first one consists of T cells characterized by expression of a TCRαβ with either CD4 or CD8αβ co-receptors, that followed a conventional thymic selection pathway and that mostly adapted to the antigen-experienced phenotype upon recognition of their non-self cognate antigens post thymically in the periphery. The second subgroup comprises lymphocytes bearing either TCRγδ or TCRαβ together with CD8αα homodimer. In contrast to the conventional subset, the CD8αα IELs have adapted their activated phenotype during a unique "self-agonist Ag based" selection process in the thymus. Nevertheless, although generated in diverse compartments and under different conditions, all IELs have in common that an agonist-driven TCR activation process lays at the basis of their functional and phenotypic differentiation. Our research is focusing on two IEL populations: TCRαβ CD8αα and TCRαβ CD4 CD8αα cells. We thoroughly characterized their TCR repertoire using two novel approaches: (1) using fluorescence-activated cell sorting (FACS) screening and (2) characterization of entire TCRα and TCRβ chain pairs on a single cell level. The data obtained form this latter analysis will lead to the first complete TCR repertoire detail data set of the α- and β-chain of the IEL sub-populations at a single cell level. The goal of this approach is to unravel the ways in which IEL see their cognate antigens setting the stage for further quest to identify the antigens and their restricting MHC molecules. The results of this study might provide us with a tremendous amount of new and important information regarding the selective processes that drive their unique differentiation and ultimately also with new insights regarding the protective and regulatory functions of these mucosal T cells.
The Arabidopsis NRT1.1 transporter acts as a nitrate sensor and governs root colonization of nitrate-rich patches via modification of local auxin concentration
Nitrate is both the main nitrogen source for nutrition of higher plants, and a signal molecule regulating their metabolism and development. The roots sense the nitrate concentration in the soil solution, and trigger signalling pathways allowing plant adaptation to changes in the external availability of this key nutrient. Localized proliferation of lateral roots in NO3—rich patches is a striking example of the nutrient-induced plasticity of root development. In Arabidopsis, NO3- stimulation of lateral root elongation is apparently under the control of a NO3- signaling pathway involving the ANR1 transcription factor that is thought to transduce the NO3- signal internally, but the upstream NO3- sensing system is unknown. On the basis of a fine analysis of root development, we show that mutants of the NRT1.1 nitrate transporter display a strongly decreased root colonization of NO3—rich patches, resulting from reduced lateral root elongation and delayed lateral root emergence. Our results show that NRT1.1 promotes localized root proliferation independently of any nutritional effect, and we concluded that NRT1.1 is a key component of the NO3—sensing system that enables the plant to detect and exploit NO3—rich soil patches. However, the signalling mechanism is unknown but studies based on DR5::GUS expression pattern suggest differential auxin accumulation in nrt1-1 lateral root primordia and in short lateral roots. Recently, using the xenopus oocyte heterologous expression system, we showed that NRT1.1 not only transports nitrate but also facilitates influx of the phytohormone auxin in a nitrate concentration dependant manner. Based on these results we propose that the role of NRT1.1 is to repress lateral root development at low nitrate availability by preventing auxin accumulation in these roots. This defines a new mechanism for sensing environmental stimuli, and for connecting nutrient and hormone signalling in the control of organ development.
Internal Waves and Mixing above the Southwest Indian Ridge
Global climate models generally do not have mixing parameterizations that can simulate
observations of elevated mixing over rough topography. Here we look at an area of heightened mixing in the Indian Ocean, just above the Southwest Indian Ridge (SWIR). Global models indicate significant barotropic tidal dissipation over the rough topography of the ridge though there have been few previous in situ measurements. Here we present 50 days of data collected over the SWIR by two moorings, each consisting of two McLane Moored Profilers, deployed on the north and south side of the ridge. Our analysis suggests elevated mixing is occurring in narrow tidal beams along the ridge. The magnitude, spatial structure, and variability of the internal tide will be presented, and the causes of elevated mixing in the Southwest Indian Ocean, as well as implications for turbulent dissipation and local and basin-wide energy budgets, will be discussed.
Successful treatment of the murine model of cystinosis using bone marrow cell transplantation
Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. Cystinosis results from a genetic defect in the gene CTNS encoding the lysosomal cystine transporter protein, cystinosin. Cystine accumulates in every organ in the body and leads to organ damage and dysfunction including renal defects. We performed syngeneic BMC, HSC or MSC transplantation in lethally irradiated 2 months old Ctns-/- mice. Cells were isolated from wildtype or GFP-transgenic mice, both expressing a functional Ctns gene. MSC did not integrate efficiently in any organ. In contrast, organ-specific cystine contentwas reduced by 57% to 94% in all organs tested in the BMC and HSC-treated mice. Confocal microscopy and quantitative-PCR (qPCR) revealed a large quantity of engrafted bone marrowderived cells in all organs tested, from 5% to 19% of the total cells. Most of these cells were not from the lymphoid lineage, but part of the intrinsic structure of the organ. The natural progression of renal dysfunction was prevented and deposition of corneal cystine crystals was significantly improved in BMC-treated mice. This work is a proof of concept for using BMC transplantation as a therapy for cystinosis. Moreover, the extensive re-population of tissue compartments highlights the efficiency of this strategy as therapy for a chronic, progressive degenerative disease.
Stoichiometry and subunit composition of the CRAC channel complex in the resting and active state
Recent genome-wide RNAi screens have revealed Stim and Orai as critical components of the Ca2+ release-activated Ca2+ (CRAC) channel required for T cell activation. Upon release of Ca2+ from the ER, Stim senses Ca2+ depletion, aggregates, relocalizes to ER-plasma membrane (PM) junctions, and interacts with Orai pore-forming subunits in the PM to open the CRAC channel. We analyzed the quaternary structure of the Drosophila Orai subunit and showed by cross-linking, and by non-denaturing gel electrophoresis that Orai is predominantly a dimer under resting conditions. Single-molecule imaging of GFP-tagged Orai expressed in Xenopus oocytes revealed predominantly two-step photo-bleaching, consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the C-terminus of Drosophila Stim as a cytosolic protein to activate the Orai channel without inducing Ca2+ store depletion or clustering of Orai into punctae yielded predominantly four-step photobleaching, consistent with a tetrameric Orai stoichiometry of the active CRAC channel. Single-molecule imaging of YFP-tagged C-Stim in presence of non-tagged Orai showed mainly two-step photobleaching suggesting that, in the active state, a dimer of Stim is bound to each Orai tetramer. Results that we recently further extended to the human STIM1 and ORAI1/ORAI3 proteins by single-molecule photobleaching on fixed Human HEK cells. Interaction of the Orai C-terminal coiled-coil domain (as shown by structure-disruptive mutations) with the C-terminus of Stim thus induces Orai dimers to dimerize, forming a tetramer that constitutes the Ca2+-selective pore. This represents a novel mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule and may reveal a new channel gating mechanism. Finally, we developed an extensive and sensitive proteomics approach to screen for additional protein interactors of Stim and Orai by tandem affinity purification and LC-MS/MS, providing a detailed proteomic profiling of the CRAC channel supramolecular complex and the dynamic protein interaction network in the CRAC channel pathway in resting and store-depleted conditions
Evidence of Little Ice Age cooling in West Antarctica from borehole temperature
We measured the temperature in a 300 m dry hole at the West Antarctica-Ice Sheet Divide site, adjacent to the deep ice core hole (79° 28’S-and 112° 07’W). WAIS Divide mean annual temperature of -31°C and accumulation rate of 22 cm-ice/year make it an ideal polar opposite to the Greenland Summit site. The record shows a clear cooling signal with a minimum at about 153 m, very similar in amplitude to the GISP2 record. Preliminary inversion results suggest an amplitude of 1°C for the Little Ice Age cooling, similar to that of Greenland Summit. This result differs from an unpublished Taylor Dome borehole record discussed by Broecker (PNAS, 2000). This inter-hemispheric synchroneity does not provide support for a bipolar see-saw mechanism but is consistent with a solar forcing cause for the Little Ice Age.
Transportin regulates major mitotic assembly events: from spindle assembly to nuclear pore assembly
Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures, the mitotic spindle and nucleus. For the latter, this further requires the precise formation of both nuclear membranes and nuclear pore complexes (NPC). Previously, importin alpha/beta and RanGTP were shown to act as dueling regulators to ensure that these processes, from spindle assembly to nuclear pore assembly, occur only in the vicinity of the mitotic chromosomes. We now find that the distantly related karyopherin, transportin, is a cell cycle regulator. Transportin negatively regulates both nuclear membrane fusion and nuclear pore assembly in Xenopus egg extracts. For pore assembly, we show that these karyopherins initiate their regulation as early as the first known step, recruitment of the critical pore-targeting nucleoporin ELYS/MEL-28 to chromatin. Indeed, both transportin and importin beta can interact directly with ELYS. We further define the nucleoporin subunit targets for transportin and importin beta and find them to be largely the same: ELYS, the Nup107/160 complex, Nup53, and the FG nucleoporins. Equally importantly, we find that transportin negatively regulates mitotic spindle assembly. All of the above negative regulatory events are counteracted by RanGTP. We conclude that the interplay of the two negative regulators, transportin and importin beta, along with that of the positive regulator RanGTP, allows a precise choreography of multiple major cell cycle assembly events.
Molecular determinants of CD8+ T Cell Memory
A number of studies have contributed to our understanding of how CD4+ T lymphocytes provide the ‘help’ necessary for optimal priming and memory establishment in CD8+ T lymphocytes. A central event in this process is believed to occur through the sequential interaction of CD4+ T cells with antigen-presenting cells (APC) leading to activation of the latter to a state in which they can autonomously provide the necessary signals for directly priming the CD8+ T cells in a subsequent interaction. Once “helped”, the progeny of these CD8+ T cells are endowed with the capacity for homeostatic survival and secondary expansion, while those primed in the absence of CD4+ T cells give rise to daughter cells which undergo TRAIL-mediated apoptosis upon restimulation. Although we, and others, have identified the CD40-L/CD40 pathway as being crucial for initiation of the “help” message from CD4+ T lymphocytes to APC, it has remained elusive how this message is delivered from the activated APC to the naïve CD8+ T cells. Using in vivo models of immunization by infectious pathogens and cross-priming, we now show using blocking antibodies and knockout mice that the “help” message can be transmitted from activated APC to CD8+ T cells via the CD70/CD27 costimulatory pathway.
Using acoustic methods to study sperm whale depredation of longline gear in the Gulf of Alaska
Sperm whales depredate black cod (Anoploma fimbria) from demersal longlines in the Gulf Alaska.
This causes an important loss for the fishermen as well as a change in the feeding behavior of the
whales. SEASWAP (South-East Alaska Sperm Whale Avoidance Project) was created in 2002 to
assess the severity of depredation. Over the past few years, many acoustic recorders have been
deployed (acoustic autonomous recorders, vertical array of hydrophones, underwater camera with
microphones, bioacoustics tags) to understand how sperm whales modify their natural behavior (in
terms of dive depth and dive duration for example) and how they use echolocation to catch fish from
the longlines. I will discuss the most recent results.
Non-anchored Peptide Binding to MHC Increases CD4+ T cell Repertoire
The first structure of I-A molecules revealed a new “non-anchored” mode of peptide binding to Major Histocompatibility Molecules (MHC) that occurred through interactions between peptide backbone and side chains of MHC residues located on the lips of the groove, and scarcely used MHC pocket/peptide side chain anchoring. The appearance of this new mode of peptide binding is coincidental with the apparition of I-A/HLA-DQ MHC class II molecules in evolution and has been conserved in a majority of species. Mechanistically, it is opposed to classical anchor-dependent binding of MHC class I molecules and more ancestral I-E/HLA-DR molecules, by being endowed with more promiscuous peptide binding and less MHC specificity than anchor binding that defined strong peptide binding motifs. The translation of this observation into the biology of the immune response remained obscure until we were able to produce and use MHC tetramers that could display short 9-mer peptides, or “individual register” peptides, within known MHC class II epitopes. These reagents demonstrated that long peptides, as they occur in MHC class II antigen processing, were indeed presented in multiple registers by the same MHC molecule during a normal immune response and recruited individual T cell populations. Thus, I-A/HLA-DQ peptide binding allows the expansion of the diversity of CD4+ T cells against single long peptides. It appears that during infections, depending on the compartmentalization of peptide processing, similar antigens can access different MHC class II presentation pathways and generate a different repertoire of responding T cells based on the number of registers present in the final processed antigen. The implications for vaccine design, especially subunit vaccines, are obvious.
Molecular physiology of sound perception and hearing loss
Our ability to perceive sound and maintain balance is crucially dependent on the process of mechanotransduction, that is, the conversion of mechanical stimuli that are evoked by sound waves and head movements into electrical signals that are processed by the nervous system. Hair cells in the inner ear are the specialized mechanosensory cells that carry out the conversion process. Hair cells carry at their apical surface a bundle of F-actin-rich stereocilia that form the mechanically sensitive organelle of the cell. Upon deflection, calcium and potassium are entering the tips of sterocilia trough unidentified mechanically channel. We previously identify the molecular component of the nanofilament which gates the channel. Here I will present the identification of a third genuine component of the mechanosensitive auditory machinery, the PDZ protein Harmonin, required for setting proper tension in the system.
Another aspect of my work is to identify genes causing hearing loss. By a mutagenesis screen performed in the mice, we previously obtain strains affected by hearing loss. I focus my work on the strain named Samba, and identified the induced mutation in an uncharacterized gene named Loxhd1. Loxhd1 encodes a protein of 2068 amino acids with an unusual structure. It consists of a 15 so-called PLAT (Polycystin/Lipoxygenase/Alpha-toxin) domains. While the function of PLAT domains is unknown, previous works suggested that PLAT domains are able to interact with the plasma membrane, and in some cases also with other proteins. Loxhd1 mRNA is expressed specifically in hair cells, and the corresponding protein is located at the inner face of the stereocilia membrane, likely providing stiffness and elasticity to the structure.
Based on our findings in mice, we searched for mutations in human families segregating autosomal recessive hearing loss. We linked a genomic region on human chromosome 18 that includes the LOXHD1 gene to hearing impairment in a consanguineous Iranian family. Subsequent analysis of the genomic DNA by sequencing revealed a missense mutation in LOXHD1 that is homozygous only in affected family members. The mutation is predicted to truncate LOXHD1 after the 5th PLAT domain. Affected members of the family show progressive hearing loss that can be detected already in childhood and is aggravated during ageing, leading finally to complete deafness.
In summary, our findings demonstrate that the mouse is a powerful instrument for auditory research, enabling the discovery of genes that cause hearing loss in humans and providing model systems to study disease mechanisms.
The autophagy machinery is required to initiate hepatitis C virus replication
In addition to its cellular homeostasis function, autophagy is emerging as a central component of antimicrobial host defense against diverse infections. To counteract this mechanism, many pathogens have evolved to evade, subvert, or exploit autophagy. Here, we report that autophagy proteins (i.e. Beclin-1, Atg4B, Atg5 and Atg12) are proviral factors for hepatitis C virus (HCV) productive infection. Moreover, we show that these autophagy proteins are needed at an early step in the virus life cycle downstream of membrane fusion and upstream or at the level of viral translation, presumably by contributing to the delivery of incoming HCV RNA to the translation apparatus and/or the recruitment of cellular factors required to initiate translation. Specifically, we demonstrate that autophagy proteins are required to initiate HCV RNA translation/replication of the incoming viral RNA, but not to maintain it once these processes are established. These results illustrate a previously unappreciated role for autophagy in the establishment of a viral infection and they suggest that different host factors regulate the translation of incoming viral genome and translation of progeny HCV RNA once replication is established.
Role of SKIP/SNW1 splicing protein in HIV-1 transcription regulation
HIV-1 Tat binds human CyclinT1 and recruits the CDK9/P-TEFb complex to the viral TAR RNA in a step that links RNA polymerase II (RNAPII) C-terminal domain (CTD) Ser 2 phosphorylation with transcription elongation. Previous studies have suggested a connection between Tat and pre-mRNA splicing factors. Our previous study revealed that the Ski-interacting protein, SKIP/SNW1, associates with the P-TEFb/CDK9 elongation factor and coactivates inducible genes, including HIV-1. We showed recently that SKIP also associates with c-Myc and Menin, a subunit of the MLL1 histone methyltransferase (H3K4me3) complex, and that HIV-1 Tat transactivation requires c-Myc and Menin, but not MLL1 or H3K4me3. RNAi-ChIP experiments reveal that SKIP acts downstream of Tat:P-TEFb to recruit c-Myc and its partner TRRAP, a scaffold for histone acetyltransferases, to the HIV-1 promoter. By contrast, SKIP is recruited by the RNF20 H2B ubiquitin ligase to the basal HIV-1 promoter, in a step that is bypassed by Tat and down-regulated by c-Myc. Interestingly, we find that SKIP and P-TEFb are dispensable for UV stress-induced HIV-1 transcription, which is strongly up-regulated by treating cells with the CDK9 inhibitor, flavopiridol. Thus SKIP acts with c-Myc and Menin to promote HIV-1 Tat:P-TEFb transcription at an elongation step that is bypassed under stress.