Brigitte David-Watine

The regulation and expression of MHC class I genes

The expression of mouse MHC class I genes and their products in vivo reveals complex patterns of regulation. Different promoter elements, which are required for gene activation or modulation in response to various external stimuli, have now been characterized as well as the proteins that bind to them. As described here by Brigitte David-Watine and colleagues, the picture that has gradually emerged from these in vitro studies is of an intricate interplay of transacting factors that ultimately lead to the fine tuning of MHC class I expression in vivo.

Activation of human α1 and α2 homomeric glycine receptors by taurine and GABA

1 Two ligand binding α subunits, α1 and α2, of the human (H) glycine receptor (GlyR) are involved at inhibitory synapses in the adult and neonatal spinal cord, respectively. The ability of homomeric αH1 and αH2 GlyRs to be activated by glycine, taurine and GABA was studied in Xenopus oocytes or in the human embryonic kidney HEK‐293 cell line. 2 In outside‐out patches from HEK cells, glycine, taurine and GABA activated both GlyRs with the same main unitary conductance, i.e. 85 ± 3 pS (n= 6) for αH1, and 95 ± 5 pS (n = 4) for αH2. 3 The sensitivity of both αH1 and αH2 GlyRs to glycine was highly variable. In Xenopus oocytes the EC50 for glycine (EC50gly) was between 25 and 280 μm for αH1 (n= 44) and between 46 and 541 μm for αH2 (n= 52). For both receptors, the highest EC50gly values were found on cells with low maximal glycine responses. 4 The actions of taurine and GABA were dependent on the EC50gly: (i) their EC50 values were linearly correlated to EC50gly, with EC50tau≈ 10 EC50gly and EC50GABA≈ 500‐800 EC50gly; (ii) they could act either as full or weak agonists depending on the EC50gly. 5 The Hill coefficient (nH) of glycine remained stable regardless of the EC50gly whereas nH for taurine decreased with increasing EC50tau. 6 The degree of desensitization, evaluated by fast application of saturating concentrations of agonist on outside‐out patches from Xenopus oocytes, was similar for glycine and taurine on both GlyRs and did not exceed 50 %. 7 Our data concerning the variations of EC50gly and the subsequent behaviour of taurine and GABA could be qualitatively described by the simple del Castillo‐Katz scheme, assuming that the agonist gating constant varies whereas the binding constants are stable. However, the stability of the Hill coefficient for glycine was not explained by this model, suggesting that other mechanisms are involved in the modulation of EC50.

Comparison of glycine and GABA actions on the zebrafish homomeric glycine receptor

1 Glycine and GABA can be co‐released from the same presynaptic terminals and in lower vertebrates they can activate the same glycine receptors (GlyRs). Thus we examined the effects of these two inhibitory transmitters on the homomeric GlyRs formed by the αZ1 subunit, of the zebrafish using two expression systems: Xenopus oocytes and the human BOSC 23 cell line. 2 The apparent affinity (EC50) of αZ1 for these neurotransmitters was highly variable. In Xenopus oocytes the EC50 ranged from 37 to 360 μm (mean ± s.d. EC50 116 ± 75 μm, n= 83) for glycine and from 8 to 120 mM (mean EC50 40 ± 30 mM, n= 37) for GABA. 3 In BOSC cells the EC50 varied from 9 to 92 μm (mean EC50 33 ± 17 μm, n= 19) and from 0.7 to 19.1 mM (mean EC50 4.9 ± 4.7 mM, n= 29) for glycine and GABA, respectively. 4 GABA activated αZ1 GlyRs either as a weak or full agonist: its efficacy (defined as Imax, GABA/Imax, Gly) was related to EC50 by an exponential relationship. A linear relationship was observed between EC50 values for GABA and glycine. 5 In outside‐out patches, GABA and glycine activated αZ1 with identical single‐channel conductances (85‐100 pS), but with different kinetics and marked effect of concentration on burst duration for glycine only. 6 In outside‐out patches deactivation time constants were concentration dependent for glycine, but not for GABA. 7 Our data demonstrate that the kinetics of glycine and GABA interactions with αZ1 are different and that they determine the properties of these neurotransmitter actions on the GlyR.

Characterization of the two zebrafish orthologues of the KAL-1 gene underlying X chromosome-linked Kallmann syndrome

The gene underlying X chromosome-linked Kallmann syndrome, KAL-1, has been identified for several years, yet its role in development is still poorly understood. In order to take advantage of the zebrafish as a model in developmental genetics, we isolated the two KAL-1 orthologues, kal1.1 and kal1.2, in this species. Comparison of deduced protein sequences with the human one shows 75.5 and 66.5% overall homology, respectively. The most conserved domains are the whey acidic protein-like domain and the first of four fibronectin-like type III repeats. However, kal1.2 putative protein lacks the basic C-terminal domain (20 residues) found in kal1.1 and KAL-1. The expressions of kal1.1 and kal1.2 were studied in the embryo between 6 and 96 hours post fertilization using whole-mount in situ hybridization. Although a few structures express both genes, kal1.1 and kal1.2 expression patterns are largely non-overlapping. Taken together, these patterns match fairly well those previously reported for human KAL-1 and chicken kal1. As regards the olfactory system, kal1.1 is expressed, from 37 h.p.f. onward, in the presumptive olfactory bulbs, whereas kal1.2 transcript is only detected, from 48 h.p.f., in the epithelium of the nasal cavity. The relevance of the zebrafish as an animal model for studying both the function of KAL-1 in normal development and the developmental failure leading to the olfactory defect in Kallmann syndrome, is discussed.

Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

BackgroundThere exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE) membrane system. However, this interaction has yet to be characterised in living interphase cells.ResultsUsing a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY) we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies).ConclusionWe conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

Functional integrity of green fluorescent protein conjugated glycine receptor channels

The alpha subunit (alphaZ1) of the zebrafish glycine receptor (GlyR) has been N-terminus fused with green fluorescent protein (GFP). We found that both pharmacological and electrophysiological properties of this chimeric alphaZ1-GFP are indistinguishable from those of the wild-type receptor when expressed in Xenopus oocytes and cell lines. The apparent affinities of this receptor for agonists (glycine, taurine and GABA), and the antagonist (strychnine) are unchanged, and single channel kinetics are not altered. In the same expression systems, alphaZ1-GFP was visualized using fluorescence microscopy. Fluorescence was distributed anisotropically across cellular membranes. In addition to the Golgi apparatus and endoplasmic reticulum, its presence was also detected on the plasmalemma, localized at discrete hot-spots which were identified as sites of high membrane turnover. Overall, the preservation in alphaZ1-GFPs of the wild type receptor functional properties makes it a promising new tool for further in situ investigations of GlyR expression, distribution and function.

Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells

Background Tpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human. Methodology/Principal Findings Here we show that depletion of Tpr by RNA interference triggers G0–G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell. Conclusions Our data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.

The human gephyrin (GPHN) gene: structure, chromosome localization and expression in non-neuronal cells.

Gephyrin was first described as a peripheral membrane protein of 93 kDa anchoring the glycine receptor (GlyR) to subsynaptic microtubules and cytoskeleton. Analysis of knock-out mice demonstrated that gephyrin has additional functions in GABA(A) receptor localization at the synapse and in the biosynthetic pathway of the molybdenum cofactor (Moco). Here we describe a human non-neuronal gephyrin cDNA and the exon/intron organization of the human gephyrin gene. We found the coding region to consist of 27 exons and to span approximately 800 kb on the long arm of chromosome 14. This structure is almost identical to that of the mouse gephyrin gene except that sequences corresponding to three exons described in rat and mouse could not be identified in human. Mutations of the GlyR subunits and of gephyrin lead to severe neuromotor phenotypes in human and mouse. Hyperekplexia involves most frequently a mutation in the GlyR alpha1 subunit in humans. However, inactivation of the Moco biosynthesis pathway results in very similar symptomatology. The recent characterization of a deletion of two exons of the gephyrin gene in a patient with symptoms typical of Moco deficiency confirmed that the involvement of gephyrin in these pathologies cannot be excluded. The precise localization of the gephyrin gene allowed us to exclude it from being a candidate for the autosomal dominant spastic paraplegia, the locus of which maps to 14q between markers D14S259 and D14S1018. A description of its structure and exon boundaries should lay the groundwork for further analysis of its expression in humans.

Tissue-specific expression of the mouse Q10 H-2 class-I gene during embryogenesis

We have studied the pattern of expression of the Q10 gene, a H-2 class-I gene located in the major histocompatibility complex which encodes a soluble class-I molecule, in the mid-gestation mouse embryo, and compared it to those of two other class-I genes, namely Kd and 37, the latter gene located in the thymus leukemia region. We found that the steady-state amount of these different mRNAs gradually increased from day 13 to day 18. By comparison with the level of expression of these genes in adult liver, the increase during gestation was fairly more marked for Q10 mRNA than for the others. Furthermore, we found that the Q10 gene is transiently expressed in the endoderm layer of the visceral yolk sac and in the fetal heart. Expression in the latter tissue decreases abruptly while increasing in the liver. It has been proposed that the Q10 protein is involved in immune tolerance. However, the time course of expression of Q10 mRNA and its tissue distribution during embryogenesis suggest that the Q10 protein could play a role in the differentiation of hematopoietic stem cells.

Real-time label-free detection of dividing cells by means of lensfree video-microscopy

Abstract. Quantification of cell proliferation and monitoring its kinetics are essential in fields of research such as developmental biology, oncology, etc. Although several proliferation assays exist, monitoring cell proliferation kinetics remains challenging. We present a novel cell proliferation assay based on real-time monitoring of cell culture inside a standard incubator using a lensfree video-microscope, combined with automated detection of single cell divisions over a population of several thousand cells. Since the method is based on direct visualization of dividing cells, it is label-free, continuous, and not sample destructive. Kinetics of cell proliferation can be monitored from a few hours to several days. We compare our method to a standard assay, the EdU proliferation assay, and as proof of principle, we demonstrate concentration-dependent and time-dependent effect of actinomycin D—a cell proliferation inhibitor.