In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons

Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of 'click chemistry'. After a brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single-particle tracking and quantum dots. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous

Sense and Senselessness; An Absurd Argument

Senior Project submitted to The Division of Social Studies of Bard College

Régulation de la diffusion latérale et du nombre de récepteurs GABA de type A aux synapses inhibitrices

Le nombre de récepteurs aux neurotransmetteurs aux synapses est un paramètre déterminant de la transmission synaptique. Les récepteurs entrent et sortent continuellement des synapses, mais peuvent aussi être stabilisés via leur interaction avec certaines molécules synaptiques. Dans le cerveau, la transmission inhibitrice rapide est majoritairement assurée par le récepteur du GABA (RGABAA). L accumulation de RGABAA aux synapses dépend de la géphyrine, principale protéine de l échafaudage post-synaptique inhibiteur. Au cours de ma thèse je me suis intéressée aux mécanismes qui régulent la diffusion latérale et le nombre de RGABAA aux synapses. J ai d abord contribué au développement de méthodes de marquage pour la technique de suivi de molécules uniques (SPT), qui permet d étudier les paramètres de diffusion des récepteurs membranaires. En utilisant cette technique, j ai montré que la présence d un site d interaction avec la géphyrine sur la sous-unité alpha1 du RGABAA modifiait la diffusion des RGABAA aux synapses, ce qui apporte une nouvelle preuve de l interaction dans les neurones vivants. J ai aussi étudié l influence de l activité du RGABAA sur son accumulation synaptique dans les neurones d hippocampe. J ai montré que l agoniste et l antagoniste du RGABAA avaient des actions opposées sur la diffusion ainsi que sur l accumulation du récepteur aux synapses. Le diazépam, un modulateur positif du RGABAA, a un effet stabilisateur inattendu qui va à l encontre des effets induits par l agoniste. Cet effet sur le trafic de surface du RGABAA pourrait constituer un nouveau mécanisme d action des benzodiazépines et expliquerait leur efficacité lors de leur utilisation cliniquePARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

High-Affinity Labeling and Tracking of Individual Histidine-Tagged Proteins in Live Cells Using Ni 2+ Tris-nitrilotriacetic Acid Quantum Dot Conjugates

International audienceInvestigation of many cellular processes using fluorescent quantum dots (QDs) is hindered by the nontrivial requirements for QD surface functionalization and targeting. To address these challenges, we designed, characterized and applied QD−trisNTA, which integrates tris-nitrilotriacetic acid, a small and high-affinity recognition unit for the ubiquitous polyhistidine protein tag. Using QD−trisNTA, we demonstrate two-color QD tracking of the type-1 interferon receptor subunits in live cells, potentially enabling direct visualization of protein−protein interactions at the single molecule level

The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive, α1 subunit-containing GABA(A)Rs; however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation we demonstrate that GABA(A)Rs and gephyrin are intimately associated at inhibitory synapses in cultured rat hippocampal neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ~20 μM, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phospho-mimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, receptor accumulation at synapses and the amplitude of mIPSCs. Finally, single particle tracking reveals that gephyrin reduces the diffusion of α1 subunit-containing GABA(A)Rs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABA(A)Rs at synaptic sites, thereby modulating the strength of synaptic inhibition