RESUMEN
The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate in the dynamic equilibrium between synaptic and extrasynaptic GlyR in the neuronal membrane. Here we investigated the properties of this interaction in cells cotransfected with YFP-tagged gephyrin and GlyR subunits possessing an extracellular myc-tag. In HeLa cells and young neurons, single particle tracking was used to follow in real time individual GlyR, labeled with quantum dots, traveling into and out of gephyrin clusters. Analysis of the diffusion properties of two GlyR subunit types--able or unable to bind gephyrin--gave access to the association states of GlyR with its scaffolding protein. Our results indicated that an important portion of GlyR could be linked to a few molecules of gephyrin outside gephyrin clusters. This emphasizes the role of scaffolding proteins in the extrasynaptic membrane and supports the implication of gephyrin-gephyrin interactions in the stabilization of GlyR at synapses. The kinetic parameters controlling the equilibrium between GlyR inside and outside clusters were also characterized. Within clusters, we identified two subpopulations of GlyR with distinct degrees of stabilization between receptors and scaffolding proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Citometría de Flujo/métodos , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente/métodos , Neuronas/metabolismo , Receptores de Glicina/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Células HeLa , Humanos , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual biomolecules in living systems. Due to their remarkable photophysical properties and relatively small size, quantum dots can be used as ultrasensitive detection probes. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. This chapter presents methods used for tracking single biomolecules coupled to quantum dots in living cells from labeling procedures to the analysis of the quantum dot motion.
Asunto(s)
Microscopía Fluorescente/métodos , Puntos Cuánticos , Absorción , Biotinilación , Citoplasma/metabolismo , Colorantes Fluorescentes/farmacología , Células HeLa , Humanos , Modelos Estadísticos , Nanopartículas , Nanotecnología/métodos , Semiconductores , Estreptavidina/química , Factores de TiempoRESUMEN
Lateral diffusion of neurotransmitter receptors in and out of synapses has been postulated as a core mechanism for rapid changes in receptor number at synapses during plastic processes. In this study, we have used single particle tracking to investigate how changes in glycine receptor (GlyR) lateral diffusion properties might account for changes in receptor number at synapses after disruption of the cytoskeleton in dissociated spinal cord neurons. We found that pharmacological disruption of F-actin and microtubules decreased the amount of GlyR and gephyrin, the backbone of the inhibitory postsynaptic scaffold, at synapses. F-actin and microtubule disruption increased GlyR exchanges between the synaptic and extrasynaptic membranes and decreased receptor dwell time at synapses. GlyR lateral diffusion was predominantly controlled by microtubules in the extrasynaptic membrane and by actin at synapses. Both diffusion coefficients and confinement at synapses were affected after F-actin disruption. Our results indicate that receptor exchanges between the synaptic and extrasynaptic compartments depend on the properties of both the postsynaptic differentiation and the extrasynaptic membrane. Consequently, GlyR number at synapses may be rapidly modulated by the cytoskeleton through the regulation of lateral diffusion in the plasma membrane and of receptor stabilization at synapses.
Asunto(s)
Membrana Celular/metabolismo , Citoesqueleto/fisiología , Neuronas/metabolismo , Receptores de Glicina/metabolismo , Médula Espinal/metabolismo , Sinapsis/metabolismo , Actinas/fisiología , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas de la Membrana/metabolismo , Microtúbulos/fisiología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Factores de Tiempo , Distribución TisularRESUMEN
Dendritic spines show an activity-dependent cytoskeleton-based remodeling coupled with variations in receptor number and the functional properties of excitatory synapses. In this study, we analyzed the dynamics of gephyrin containing inhibitory postsynaptic scaffolds imaging a Venus::gephyrin (VeGe) chimera in dissociated spinal cord neurons. We provide evidence that the postsynaptic scaffolds at mature synapses display a submicrometric rapid lateral motion and are continuously moving on the dendritic shaft. This dynamic behavior is calcium dependent and is controlled by the cytoskeleton. Minute rearrangement within the gephyrin scaffold as well as the scaffold lateral displacements are F-actin dependent. The lateral movements are counteracted by microtubules. Moreover, the action of the potassium channel blocker 4-aminopyridine and receptor antagonists indicate that the dynamics of postsynaptic gephyrin scaffolds are controlled by synaptic activity.