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Accueil > Agenda > Les séminaires Jean Roche > Faire et défaire : un rôle pour la gephyrine dans les échafaudages (...)

Faire et défaire : un rôle pour la gephyrine dans les (...)

Lundi 17 octobre 2005,11h, salle Lissitsky.


N°1 Hanus, C ; Vannier, C ; Triller, A. 2004.

Intracellular association of glycine receptor with gephyrin increases its plasma membrane accumulation rate.

JOURNAL OF NEUROSCIENCE 24 (5) : 1119-1128.

Abstract : Gephyrin, a tubulin-binding protein, is the core of inhibitory postsynaptic scaffolds stabilizing glycine receptors (GlyRs) and/or GABA(A) receptors. Previous ultrastructural studies in vivo and in vitro have reported a localization of gephyrin to intracellular cisternas during development or after glycinergic denervation (Seitanidou et al., 1992 ; Colin et al., 1996, 1998). These data were compatible with a traffic of this cytoplasmic, but membrane-associated, protein together with membrane proteins such as GlyR after exocytosis and/or endocytosis pathways. We have now investigated the consequences of a GlyR-gephyrin interaction on the localization and the dynamics of these two molecules in African green monkey kidney cells (COS-7) cells and in neurons transfected with green fluorescent protein-tagged-gephyrin and myc-tagged GlyR alpha(1) subunits. In these experiments, myc-tagged GlyR alpha(1) contained, or did not contain, the gephyrin-binding sequence (betagb) of the GlyR beta subunit. We report here that GlyR-gephyrin interaction localizes gephyrin to GlyR-containing organelles. Videomicroscopy and nocodazole treatment indicate that the movements of these vesicles are microtubule dependent. Expressing GlyR alpha(1) with a thrombin cleavage site between the myc-tag and the N-terminal of the GlyR alpha(1) subunit (Rosenberg et al., 2001) allowed monitoring of newly inserted receptors in the cell surface. Using temperature changes to block GlyR in, and then release it from, the trans-Golgi network, we show that gephyrin accelerates the accumulation of GlyR at the cell surface. Therefore, our data strongly suggest that some GlyR clusters are associated with gephyrin on their way to the cell surface and that this association increases the accumulation of GlyR at the plasma membrane.

N°2 Dahan, M ; Humbert, M ; Hanus, C ; Levi, S ; Vannier, C ; Triller, A. 2003.

Dynamics of Glycine and GABA receptors in spinal cord neurons studied by single molecule fluorescence imaging..

BIOPHYSICAL JOURNAL 84 (2) : 123A-123A, Part 2 Suppl. S.

N°3 Bainier, C ; Vannier, C ; Courjon, D ; Rivoal, JC ; Ducourtieux, S ; De Wilde, Y ; Aigouy, L ; Formanek, F ; Belliard, L ; Siry, P ; Perrin, B. 2003.

Comparison of test images obtained from various configurations of scanning near-field optical microscopes.

APPLIED OPTICS 42 (4) : 691-700.

Abstract : The characteristics of a few experimental near-field optical microscopes, located in different laboratories, have been compared on the basis of their ability to image a well-defined submicrometer test, object. (C) 2003 Optical Society of America.

N°4 Legendre, P ; Muller, E ; Badiu, CI ; Meier, J ; Vannier, C ; Triller, A. 2002.

Desensitization of homomeric alpha 1 glycine receptor increases with receptor density.


Abstract : Variations in the number of receptors at glycinergic synapses are now established and are believed to contribute to inhibitory synaptic plasticity. However, the relation between glycine receptor (GlyR) kinetics and density is still unclear. We used outside-out patch-clamp recordings and fast-flow application techniques to resolve fast homomeric GlyRalpha1 kinetics and to determine how the functional properties of these receptors depend on their density and on the presence of the anchoring protein gephyrin. The expression of GlyRs in human embryonic kidney cells increased with time and was correlated with an increase in GlyR desensitization at 2 days after transfection. Cotransfection of homomeric GlyRalpha1 bearing the gephyrin-binding site with gephyrin also increased desensitization but at 1 day after transfection compared with transfections of homomeric GlyRalpha1 without gephyrin. This increase results from the occurrence of a fast desensitization component and short applications of a saturating concentration of glycine suffice to promote a rapidly entered desensitized closed state. The level of desensitization changed neither the EC50 value nor the Hill coefficient of the glycine dose-response curves because the amplitude of the current was measured at the peak of the responses. These results demonstrate that variations in GlyR density during cluster formation result from a change in GlyR efficiency due to modifications in their desensitization properties.

N°5 Rasmussen, H ; Rasmussen, T ; Triller, A ; Vannier, C. 2002.

Strychnine-blocked glycine receptor is removed from synapses by a shift in insertion/degradation equilibrium.


Abstract : The long-term inhibition by strychnine of glycine receptor activity in neurons provokes the receptor’s selective intracellular accumulation and disappearance from synapses. This could result either from a disruption of the postsynaptic anchoring of the receptor or from an arrest of its exocytic transport. In this study we combined biochemical and fluorescence microscopy analyses to determine on a short time scale the fate of the strychnine-inactivated glycine receptor. Quantification of the cellular content of receptor showed that the rapid accumulation depends on protein synthesis. Cell surface biotinylation of neurons demonstrated that strychnine did not accelerate the turnover rate of the receptor. Labeling of endosomes indicated that, in strychnine-treated cells, the accumulated receptor is not blocked in the endosomal transport pathway. Taken together, these results indicate that strychnine does not destabilize the postsynaptic receptor but triggers its disappearance from synapses by a nondegradative sequestration of newly synthesized molecules in a nonendocytic compartment.

N°6 Humbert, M ; Vannier, C ; Triller, A ; Dahan, M. 2002.

Dynamics of post-synaptic membrane receptors studied by single molecule fluorescence imaging.

BIOPHYSICAL JOURNAL 82 (1) : 48A-48A, Part 2.

N°7 Rosenberg, M ; Meier, J ; Triller, A ; Vannier, C. 2001.

Dynamics of glycine receptor insertion in the neuronal plasma membrane.

JOURNAL OF NEUROSCIENCE 21 (14) : 5036-5044.

Abstract : The exocytosis site of newly synthesized glycine receptor was defined by means of a morphological assay to characterize its export from the trans-Golgi Network to the plasma membrane. This was achieved by expressing in transfected neurons an alpha1 subunit bearing an N-terminal tag selectively cleavable from outside the cell by thrombin. This was combined with a transient temperature-induced block of exocytic transport that creates a synchronized exocytic wave. Immunofluorescence microscopy analysis of the cell surface appearance of newly synthesized receptor revealed that exocytosis mainly occurred at nonsynaptic sites in the cell body and the initial portion of dendrites. At the time of cell surface insertion, the receptors existed as discrete clusters. Quantitative analysis showed that glycine receptor clusters are stable in size and subsequently appeared in more distal dendritic regions. This localization resulted from diffusion in the plasma membrane and not from exocytosis of transport vesicles directed to dendrites. Kinetic analysis established a direct substrate-product relationship between pools of somatic and dendritic receptors. This indicated that clusters represent intermediates between newly synthesized and synaptic receptors. These results support a diffusion-retention model for the formation of receptor-enriched postsynaptic domains and not that of a vectorial intracellular targeting to synapses.

N°8 Meier, J ; Vannier, C ; Serge, A ; Triller, A ; Choquet, D. 2001.

Fast and reversible trapping of surface glycine receptors by gephyrin.

NATURE NEUROSCIENCE 4 (3) : 253-260.

Abstract : Variations in receptor number at a given synapse are known to contribute to synaptic plasticity, but methods used to establish this idea usually do not allow for the determination of the dynamics of these phenomena. We used single-particle tracking to follow in real time, on the cell surface, movements of the glycine receptor (GlyR) with or without the GlyR stabilizing protein gephyrin. GlyR alternated within seconds between diffusive and confined states. In the absence of gephyrin, GlyR were mostly freely diffusing. Gephyrin induced long confinement periods spatially associated with submembranous clusters of gephyrin. However, even when most receptors were stabilized, they still frequently made transitions through the diffusive state. These data show that receptor number in a cluster results from a dynamic equilibrium between the pools of stabilized and freely mobile receptors. Modification of this equilibrium could be involved in regulation of the number of receptors at synapses.

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