Showing papers by "Daniel Choquet published in 2007"

NMDA Receptor Surface Trafficking and Synaptic Subunit Composition Are Developmentally Regulated by the Extracellular Matrix Protein Reelin

Abstract: During postnatal development, changes in the subunit composition of glutamate receptors of the NMDA subtype (NMDARs) are key to the refinement of excitatory synapses. Hypotheses for maturation of synaptic NMDARs include regulation of their expression levels, membrane targeting, and surface movements. In addition, several members of extracellular matrix (ECM) proteins such as Reelin are involved in synaptic plasticity. However, it is not known whether and how ECM proteins regulate synaptic NMDAR maturation. To probe the participation of NMDARs to synaptic currents and NMDARs surface dynamics, we used electrophysiological recordings and single-particle tracking in cultured hippocampal neurons. Our results show that, during maturation, Reelin orchestrates the regulation of subunit composition of synaptic NMDARs and controls the surface mobility of NR2B subunits. During postnatal maturation, we observed a marked decrease of NR1/NR2B receptor participation to NMDAR-mediated synaptic currents concomitant with the accumulation of Reelin at active synapses. Blockade of the function of Reelin prevented the maturation-dependent reduction in NR1/NR2B-mediated synaptic currents. The reduction of NR1/NR2B receptors was not inhibited by blocking synaptic activity but required beta1-containing integrin receptors. Single-particle tracking showed that inhibition of Reelin decreased the surface mobility of native NR2B-containing NMDARs, whereas their synaptic dwell time increased. Conversely, recombinant Reelin dramatically reduced NR2B-mediated synaptic currents and the time spent by NR2B subunits within synapses. Our data reveal a new mode of control of synaptic NMDAR assembly at postnatal hippocampal synapses and an unprecedented role of ECM proteins in regulating glutamate receptor surface diffusion.

Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies.

Abstract: The cellular traffic of neurotransmitter receptors has captured a lot of attention over the last decade, mostly because synaptic receptor number is adjusted during synaptic development and plasticity. Although each neurotransmitter receptor family has its own trafficking characteristics, two main

Fast turnover of L1 adhesions in neuronal growth cones involving both surface diffusion and exo/endocytosis of L1 molecules.

Abstract: We investigated the interplay between surface trafficking and binding dynamics of the immunoglobulin cell adhesion molecule L1 at neuronal growth cones. Primary neurons were transfected with L1 constructs bearing thrombin-cleavable green fluorescent protein (GFP), allowing visualization of newly exocytosed L1 or labeling of membrane L1 molecules by Quantum dots. Intracellular L1–GFP vesicles showed preferential centrifugal motion, whereas surface L1–GFP diffused randomly, revealing two pathways to address L1 to adhesive sites. We triggered L1 adhesions using microspheres coated with L1–Fc protein or anti-L1 antibodies, manipulated by optical tweezers. Microspheres coupled to the actin retrograde flow at the growth cone periphery while recruiting L1–GFP molecules, of which 50% relied on exocytosis. Fluorescence recovery after photobleaching experiments revealed a rapid recycling of L1–GFP molecules at L1–Fc (but not anti-L1) bead contacts, attributed to a high lability of L1–L1 bonds at equilibrium. L1–GFP molecules truncated in the intracellular tail as well as neuronal cell adhesion molecules (NrCAMs) missing the clathrin adaptor binding sequence showed both little internalization and reduced turnover rates, indicating a role of endocytosis in the recycling of mature L1 contacts at the base of the growth cone. Thus, unlike for other molecules such as NrCAM or N-cadherin, diffusion/trapping and exo/endocytosis events cooperate to allow the fast renewal of L1 adhesions.

Neuroscience: wrestling with SUMO.

Abstract: The process of SUMOylation affects various cellular events by modifying the proteins involved. In neurons, it controls receptor numbers on the cell surface, thereby regulating neuronal communication.

Fluorescence microscopy of single autofluorescent proteins for cellular biology

Abstract: In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells.

Quand la lumière contrôle l’activité neuronale et le comportement

Abstract: 585 NO UV EL LE S M AG AZ IN E processus d’induction mécanique d’expression de gènes du développement par les mouvements morphogénétiques pose la question de l’existence d’un contrôlequalité de l’état de l’élaboration de la morphologie physique de l’embryon par le génome, maître d’œuvre du développement embryonnaire [9]. Il se peut même que les signaux mécaniques facilitent une communication rapide et de longue portée entre domaines de différenciation distants. Enfin, l’importance grandissante des signaux mécaniques dans la régulation de processus embryogénétiques ou organogénétiques pose nécessairement la question de leur rôle potentiel dans la dérégulation des processus morphogénétiques, conduisant à des développements pathologiques. ◊ In search of the molecular mechano-transductors of the cell

Multiple Routes for Glutamate Receptor Trafficking: Surface Diffusion and Membrane Traffic Cooperate to Bring Receptors to Synapses

Abstract: Trafficking of glutamate receptors into and out of synapses is critically involved in the plasticity of excitatory synaptic transmission. Endocytosis and exocytosis of receptors have initially been thought to account alone for this trafficking. However, membrane proteins also traffic through surface lateral diffusion in the plasma membrane. We describe developments in electrophysiological and optical approaches that have allowed for the real time measurement of glutamate receptor surface trafficking in live neurons. These include (i) specific imaging of surface receptors using a pH sensitive fluorescent protein, (ii) design of a photoactivable drug to inactivate locally surface receptors and monitor electrophysiologically their recovery, and (iii)application of single molecule fluorescence microscopy to directly track the movement of individual surface receptors with nanometer resolution inside and outside synapses. Altogether, these approaches have demonstrated that glutamate receptors diffuse at high rates in the neuronal membrane and suggest a key role for surface diffusion in the regulation of receptor numbers at synapses.

Fiber coupling of an infrared femtosecond laser to a multiphoton microscope

Abstract: We report on a new fiber delivery system for coupling a compact, turn-key infrared femtosecond laser into a multiphoton microscope. Using a hollow core photonics crystal fiber with minimized dispersion, we achieve beam propagation on distances up to 4 meters. We measured a pulse duration close to 300 fs at the output of the microscope, for a femtosecond laser pulse duration of 200 fs. We present experimental parameters and system optimisation, and compare images obtained with fiber coupling versus direct coupling of the femtosecond laser.