<big>The PDZ-Binding Site of GluR2 Controls Its Surface Expression but Not Its Lateral Mobility</big>
<big>The PDZ-Binding Site of GluR2 Controls Its Surface Expression but Not Its Lateral Mobility</big>
* Given the striking role of Stargazin C terminus in controlling AMPAR surface diffusion, we wondered if AMPAR subunits C termini had any role in controlling surface movements. The direct interaction of '''GluR2''' C terminus with the PDZ-containing proteins ABP/GRIP and PICK1 has been shown to play an important role in the regulation of AMPARs expression at synaptic sites.
* Given the striking role of Stargazin C terminus in controlling AMPAR surface diffusion, we wondered if AMPAR subunits C termini had any role in controlling surface movements. The direct interaction of '''GluR2''' C terminus with the PDZ-containing proteins ABP/GRIP and PICK1 has been shown to play an important role in the regulation of AMPARs expression at synaptic sites. Whether these proteins are involved solely in modulating the surface expression of the AMPARs or whether they also anchor surface AMPARs at synapse, however, remains unclear.
* Whether these proteins are involved solely in modulating the surface expression of the AMPARs or whether they also anchor surface AMPARs at synapse, however, remains unclear.
* We first used a mutant '''GluR2''', GluR2-DC, in which the last C-terminal four amino acids corresponding to the PDZ binding site were removed.
* We first used a mutant '''GluR2''', GluR2-DC, in which the last C-terminal four amino acids corresponding to the PDZ binding site were removed.
* We compared the surface expression of '''GluR2''' DC::GFP and wild-type '''GluR2''' in cultured hippocampal neurons. Since the GFP tag is coupled to the extracellular N terminus of '''GluR2''', the surface receptors could be specifically immunolabeled with an anti-GFP. The signal coming from this surface staining was normalized to that of the signal of the GFP, which corresponds to the total intracellular and surface expression of the recombinant protein. <br>'''''Note: Interesting! Never heard of this methodology for determining the proportion of receptors expressed at the membrane vs intracellular'''''
* We compared the surface expression of '''GluR2''' DC::GFP and wild-type '''GluR2''' in cultured hippocampal neurons. Since the GFP tag is coupled to the extracellular N terminus of '''GluR2''', the surface receptors could be specifically immunolabeled with an anti-GFP. The signal coming from this surface staining was normalized to that of the signal of the GFP, which corresponds to the total intracellular and surface expression of the recombinant protein. <br>'''''Note: Interesting! Never heard of this methodology for determining the proportion of receptors expressed at the membrane vs intracellular'''''
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* GluR2 DC still colocalized with Homer1c so, while GluR2 DC is less expressed at the neuronal membrane, it's still clustered at excitatory synapses.
* GluR2 DC still colocalized with Homer1c so, while GluR2 DC is less expressed at the neuronal membrane, it's still clustered at excitatory synapses.
* To investigate the role of GluR2 PDZ interactors in controlling GluR2 lateral mobility, we tracked in real time the movement of GluR2:WT:GFP or GluR2:DC:GFP at the neuronal surface using QDots coupled to anti-GFP. <br> '''''Note: This thing is a monster... <br>GluR2:DC{{Nc}}GFP{{Nc}}anti-GFP{{Nc}}Qdot <br>Remember from their 2004 paper, they were already reporting a decrease in diffusion rate, specifically at the synapse, when they compared Qdots and at the synapse compared to a fluorescent marker'''''
* To investigate the role of GluR2 PDZ interactors in controlling GluR2 lateral mobility, we tracked in real time the movement of GluR2:WT:GFP or GluR2:DC:GFP at the neuronal surface using QDots coupled to anti-GFP. <br> '''''Note: This thing is a monster... <br>GluR2:DC{{Nc}}GFP{{Nc}}anti-GFP{{Nc}}Qdot <br>Remember from their 2004 paper, they were already reporting a decrease in diffusion rate, specifically at the synapse, when they compared Qdots and at the synapse compared to a fluorescent marker'''''
* The diffusing properties of GluR2 were not significantly changed by the deletion of the PDZ binding site. Indeed, the fraction of immobile receptors and the median diffusion coefficients of mobile receptors were similar for GluR2:WT:GFP and GluR2:DC:GFP
* '''diffusion''' of '''GluR2''' were '''not changed''' by the '''deletion''' of the '''PDZ binding site'''. Indeed, the fraction of immobile receptors, percentage time in confined sites, and the median diffusion coefficients of mobile receptors were similar for GluR2:WT:GFP and GluR2:DC:GFP
In this study they tested the effects of glutamate application and calcium influx on AMPAR diffusion. Being one of their earlier studies aimed at measuring diffusion, they used antibodies instead of Qdots.
Glutamate Effect
Bath application of 100 uM Glutamate
increased the diffusion rate of GluR2-containting AMPAR
Glutamate causes endocytosis of AMPARs, and internal AMPARs are immobile. Therefore it seems like glutamate may be causing a general endocytotic episode at non-synaptic AMPARs, perhaps not even at the synapse that received the glutamate application.
Newly inserted receptors were found to be initially diffusive and then stabilized at synaptic sites.
Summary
they found that bath application of glutamate induces rapid depletion of AMPARs from PSDs increases synaptic diffusion rate, decreases % of completely immobile receptors, increases proportion of receptors in the area surrounding the synapse (juxtasynaptic region). Activation of NMDARs results in increased surface expression of AMPARs -- in the first few minutes there is mainly a decrease in the proportion of immobile synaptic receptors, but after 40 min, both diffusion rates and percentages of immobile synaptic receptors are back to control values and the proportion of juxtasynaptic receptors is decreased. This observation relates to the fate of newly exocytosed AMPARs: using cleavable extracellular tags, it was observed that at early times after exocytosis, new GluR1 containing AMPARs are diffusively distributed along dendrites. This is followed by their lateral translocation and accumulation into synapses (Passafaro et al., 2001). GluR2 subunits were addressed directly at synapses. In our experiments, we followed the movement of native GluR2 containing AMPARs, where the data suggests that at the level of synapses themselves, newly added receptors are initially diffusive and then stabilize over time.
Methods
Anti-GluR2 antibodies were labeled with Cy5 or Alexa-647 molecules at low labeling ratio (mean labeling ratio of 0.4 dye per antibody) so that individual antibodies were labeled at most with one fluorophore. A small proportion of surface expressed AMPA receptors containing the GluR2 subunit were selectively labeled in live neurons through short incubations with these antibodies. We could thus image and resolve discrete fluorescence spots with an epifluorescence imaging setup
2004
Groc L, Heine M, Cognet L, Brickley K, Stephenson FA, Lounis B, Choquet D.
• 2004 • Nature Neuroscience - - PDF
In this study they tried out Qdots for the first time, but didn't use the data. They simply made the claim that the results from Qdot-antibodies gave similar results as the Cy3-antibodies (but only in extrasynaptic space, Qdots were 5x slower in synaptic space). They found that AMPARs diffuse 4x faster than NMDARs when outside the synapse (10.0 vs 2.3 nm/s) and at similar speeds in the synapse (28.0 vs 21.0 nm/s). Note that diffusion was faster outside the synapse than inside the synapse (that doesn't make sense). KCl was used to stimulate neurons - this caused extrasynaptic AMPARs to diffuse 5x faster than baseline; whereas AMPAR mobility didn't change much in the synapse (but lower N). They didn't report NMDAR changes from KCl. They stimulated PKC activity using TPA, resulting in significant mobility increases for both NMDAR and AMPAR in synaptic and extrasynaptic space.
Error creating thumbnail: File missingDifferential lateral diffusion of AMPARs (grey) and NMDARs (black) at the surface of hippocampal neurons. Because AMPARs and NMDARs undergo exo-endocytosis cycling we carried out controls to show that the vast majority of labeled receptors were located at the surface of neurons during recording sessions. (a,b) Histograms of extrasynaptic AMPAR (a) and NMDAR diffusions (b). AMPAR diffusion was approximately four times higher than NMDAR. Insets, examples of GluR2R (a) and NR1R (b) trajectories (bars, 100 nm). Average trajectory length is 455, range 260–9750 ms. (c,d) Diffusion histograms of synaptic AMPARs and NMDARs. (e) Fractions of mobile AMPARs and NMDARs. Note the smaller fraction of mobile receptors in extrasynaptic membranes. (f) Median diffusion of mobile AMPAR and NMDAR
Abstract
The basis for differences in activity-dependent trafficking of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) remains unclear. Using single-molecule tracking, we found different lateral mobilities for AMPARs and NMDARs: changes in neuronal activity modified AMPAR but not NMDAR mobility, whereas protein kinase C activation modified both. Differences in mobility were mainly detected for extrasynaptic AMPARs, suggesting that receptor diffusion between synaptic and extrasynaptic domains is involved in plasticity processes.
Methods
Here, we directly compared the lateral mobilities of AMPARs and NMDARs. For this, we measured the diffusion of GluR2 subunit–containing AMPAR and NR1 subunit–containing NMDAR at the surface of cultured hippocampal neurons at 9–11 days in vitro by single-molecule fluorescence microscopy tracking of receptors labeled with appropriate Cy3-coupled antibodies. We comparedAMPAR diffusion obtained with Cy3-conjugated antibodies with quantum dot (QD)-coupled antibodies, because QDs are more photostable than organic dyes. In the extrasynaptic membrane, diffusion distributions measured using Cy3-conjugated and QD-coupled antibodies were similar, thus validating the diffusion estimates obtained with Cy3-conjugated antibodies. Distributions of synaptic receptor diffusions were significantly different, however, with QD-coupled receptor diffusion being five times slower. This effect could be due to limitation of receptor diffusion within the glutamatergic synaptic cleft by the bound QD (QD diameter 10–15 nm). Therefore, in the present study, AMPAR and NMDAR diffusions were estimated only from Cy3-conjugated antibodies.
Results
We treated neurons with either potassium chloride increase neural activity, or tetrodotoxin to decrease neuronal activity. After 2 min of 40 mM KCl treatment, extrasynaptic AMPAR diffusion greatly increased (560% of control), owing to less immobile receptors (from 47% to 32%). Notably, the AMPAR diffusion measured after KCl treatment is comparable to the previously published ‘control’ value (median 0.11 um2/s) obtained after similar KCl treatment needed to stain synapses with FM1-43. When the spontaneous neuronal activity was blocked with TTX for only 10 min, no changes were observed, consistent with the low spontaneous basal activity of our cultured neurons. However, blocking neuronal activity for 48 h greatly decreased extrasynaptic AMPAR diffusion (12% of control), owing to more immobile receptors (from 47% to 67%).
Activation of protein kinase C (PKC) induces rapid dispersal of NMDARs from a clustered to a uniform membrane distribution as well as endocytosis and redistribution of GluR2-containing AMPARs to synaptic sites. We thus investigated whether NMDAR and AMPAR mobilities were affected by the PKC agonist TPA. After TPA treatment, NMDAR diffusion was increased in both extrasynaptic (12-fold) and synaptic membranes (5-fold). This is consistent with the reported TPA-induced dispersion of NMDARs. Extrasynaptic and synaptic AMPAR diffusions were also significantly affected by TPA treatment (extrasynaptic +34%) (synaptic +333%).
Concentration of neurotransmitter receptors at synapses is thought to result from stable binding to subsynaptic scaffold proteins. Recent data on synaptic plasticity have shown that changes in synaptic strength derive partly from modification of postsynaptic receptor numbers. This has led to the notion of receptor trafficking into and out of synapses. The proposed underlying mechanisms have under-evaluated the role of extrasynaptic receptors. Recent technological advances have allowed imaging of receptor movements at the single-molecule level, and these experiments demonstrate that receptors switch at unexpected rates between extrasynaptic and synaptic localizations by lateral diffusion. Variation in receptor numbers at postsynaptic sites is therefore likely to depend on regulation of diffusion by modification of the structure of the membrane and/or by transient interactions with scaffolding proteins. This review is part of the TINS Synaptic Connectivity series.
To assess if membrane diffusion could affect the kinetics of receptor recruitment at adhesive contacts, we transfected neurons with green fluorescent protein-tagged immunoglobin cell adhesion molecules of varying length (25-180 kD), and measured the lateral mobility of single quantum dots bound to those receptors at the cell surface. The diffusion coefficient varied within a physiological range (0.1-0.5 microm(2)/s), and was inversely proportional to the size of the receptor. We then triggered adhesive contact formation by placing anti-green fluorescent protein-coated microspheres on growth cones using optical tweezers, and measured surface receptor recruitment around microspheres by time-lapse fluorescence imaging. The accumulation rate was rather insensitive to the type of receptor, suggesting that the long-range membrane diffusion of immunoglobin cell adhesion molecules is not a limiting step in the initiation of neuronal contacts.
2006
Thoumine, Lambert, Mège, Choquet
• 2006 • Journal - PDF
N-cadherin plays a key role in axonal outgrowth and synaptogenesis, but how neurons initiate and remodel N-cadherin-based adhesions remains unclear. We addressed this issue with a semiartificial system consisting of N-cadherin coated microspheres adhering to cultured neurons transfected for N-cadherin-GFP. Using optical tweezers, we show that growth cones are particularly reactive to N-cadherin coated microspheres, which they capture in a few seconds and drag rearward. Such strong coupling requires an intact connection between N-cadherin receptors and catenins. As they move to the basis of growth cones, microspheres slow down while gradually accumulating N-cadherin-GFP, demonstrating a clear delay between bead coupling to the actin flow and receptor recruitment. Using FRAP and photoactivation, N-cadherin receptors at bead-to-cell contacts were found to continuously recycle, consistently with a model of ligand-receptor reaction not limited by membrane diffusion. The use of N-cadherin-GFP receptors truncated or mutated in specific cytoplasmic regions show that N-cadherin turnover is exquisitely regulated by catenin partners. Turnover rates are considerably lower than those obtained previously in single molecule studies, demonstrating an active regulation of cadherin bond kinetics in intact cells. Finally, spontaneous neuronal contacts enriched in N-cadherin exhibited similar turnover rates, suggesting that such dynamics of N-cadherin may represent an intrinsic mechanism underlying the plasticity of neuronal adhesions.
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 locally inactivate 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. Together, 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.
Tracking individual nano-objects in live cells during arbitrary long times is a ubiquitous need in modern biology. We present here a method for tracking individual 5-nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against single fluorescent molecule tracking in live COS7 cells on subsecond timescales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. Single Nanoparticle Photothermal Tracking has the unique potential to record arbitrary long trajectory of membrane proteins using nonfluorescent nanometer-sized labels.
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 modes of receptor delivery to the synapse have emerged: endo-exocytotic cycling and surface diffusion [e.g., for glutamatergic receptors, see Bredt and Nicoll (2003) and Groc and Choquet (2006)]. Receptor cycling through endo-exocytotic processes can be measured by several experimental means, from biochemical to imaging assays. The use of fluorescent protein (XFP)-tag imaging provides a powerful approach to investigate the trafficking of receptor clusters between neuronal compartments (e.g., soma, dendrite, spine) (Kennedy and Ehlers, 2006). A disadvantage of the XFP-tag approach in live experiment is extreme difficulty in detecting XFP fluorescence signals from small nonclustered receptor pool (Cognet et al., 2002; Lippincott-Schwartz and Patterson, 2003). XFP-tagged neurotransmitter receptors are often present in several cellular compartments from the endoplasmic reticulum to the plasma membrane with various relative contents. For instance, surface XFP-tagged neurotransmitter receptors represent only a minor fraction of the total receptor population, precluding their specific detection. Alternative live-cell imaging approaches were thus required to specifically isolate surface receptors. Interestingly, a variant of the green fluorescent protein (GFP), ecliptic pHluorin, shows a reversible excitation ratio change between pH 7.5 and 5.5, and its absorbance decreases as the pH is lowered. Most neurotransmitter receptors, including the ionotropic glutamate ones, display an extracellular N-terminal region, implying that the N terminus will always be in an acidic environment inside the cell, whereas it will be exposed to a neutral pH after insertion into the plasma membrane. By this means, surface receptors can be specifically detected and tracked with live-imaging approaches (Ashby et al., 2004). Alternatively, surface receptors can be labeled and detected by immunocytochemical approaches using antibodies directed against receptor extracellular epitopes. The purpose of this Toolbox is to outline currently available approaches to measure the surface trafficking of receptor in neurons, with a special emphasis on single-molecule (organic dye) and quantum dot (QDot) detection for neurotransmitter receptor tracking.
Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.
Accumulation of AMPA receptors at synapses is a fundamental feature of glutamatergic synaptic transmission. Stargazin, a member of the TARP family, is an AMPAR auxiliary subunit allowing interaction of the receptor with scaffold proteins of the postsynaptic density, such as PSD-95. How PSD-95 and Stargazin regulate AMPAR number in synaptic membranes remains elusive. We show, using single quantum dot and FRAP imaging in live hippocampal neurons, that exchange of AMPAR by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 AMPAR subunit C terminus. Disruption of interactions between Stargazin and PSD-95 strongly increases AMPAR surface diffusion, preventing AMPAR accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin-PSD-95 interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density.
Notes
Quantum Dot
FRAP
Live hippocampal neurons
exchange of AMPAR by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 AMPAR subunit C terminus.
Disruption of interactions between Stargazin and PSD-95 strongly increases AMPAR surface diffusion, preventing AMPAR accumulation at postsynaptic sites.
AMPARs and Stargazin diffuse as complexes in and out synapses.
This article describes imaging techniques using single optical labels, ranging from fluorescent dyes to scattering particles, for the study of the movement of individual or small assemblies of membrane proteins. These techniques have been used to track the movements of different types of plasma membrane proteins, such as neurotransmitter receptors and adhesion proteins. They can be used to probe the degree of interaction between membrane proteins and cytoplasmic stabilizing elements in live cells.
AMPA glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission. Upon fast consecutive synaptic stimulation, transmission can be depressed. Recuperation from fast synaptic depression has been attributed solely to recovery of transmitter release and/or AMPAR desensitization. We show that AMPAR lateral diffusion, observed in both intact hippocampi and cultured neurons, allows fast exchange of desensitized receptors with naïve functional ones within or near the postsynaptic density. Recovery from depression in the tens of millisecond time range can be explained in part by this fast receptor exchange. Preventing AMPAR surface movements through cross-linking, endogenous clustering, or calcium rise all slow recovery from depression. Physiological regulation of postsynaptic receptor mobility affects the fidelity of synaptic transmission by shaping the frequency dependence of synaptic responses.
Neurotransmitter receptor trafficking in and out synapses has emerged as a key process to regulate synaptic transmission during synaptic development and plasticity both at excitatory and inhibitory synapses. Lateral diffusion of surface neurotransmitter receptors has recently emerged as a key pathway to regulate receptor trafficking to and from synapses. Receptors enter and exit synapses mainly by lateral diffusion within the plane of the membrane while their retrieval and addition from and to the plasma membrane by endo and exocytotic processes occur largely at extrasynaptic sites. As a consequence, regulation of receptor surface trafficking is likely to be a major process to regulate receptor numbers at synapses. Measurement of receptor surface diffusion has required the development of new experimental approaches to specifically label and track surface receptor with appropriate time- and space-resolutions. In this review, we first discuss the approaches that have been used to measure receptor surface diffusion, such as the ensemble approach that measure average diffusion of a defined surface receptor population and the single molecule/particle approaches that measure the surface diffusion of isolated receptors. To date, surface diffusion has been described for a variety of neurotransmitter receptors that exhibit common as well as specific features. These points are discussed in a comparative manner and emerging rules of surface trafficking as well as potential interplay between receptor classes are further commented. Because our knowledge on neurotransmitter receptor surface diffusion is fairly recent, open questions and experimental challenges facing the field are highlighted throughout the review.
Single-molecule approaches give access to the full distribution of molecule behaviors and overcome the averaging intrinsic to bulk measurement methods. They allow access to complex processes where a given molecule can have heterogeneous properties over time. Recent developments in single-molecule imaging technologies have been followed by their wide application in cellular biology and are leading to the unraveling of new mechanisms related to molecular movements. They are shaping new concepts in the dynamic equilibria of complex biological macromolecular assemblies such as synapses. These advances were made possible thanks to improvements in visualization approaches combined with new strategies to label proteins with nanoprobes. In this primer, we will review the different approaches used to track single molecules in live neurons, compare them to bulk measurements, and discuss the different concepts that have emerged from their application to synaptic biology.
2009
Renner, Cognet L, Lounis B, Triller A, Choquet D.
• 2009 • Neuropsychopharm - PDF
Receptors are concentrated in the postsynaptic membrane but can enter and exit synapses rapidly during both basal turnover and processes of synaptic plasticity. How the exchange of receptors by lateral diffusion between synaptic and extrasynaptic areas is regulated remains largely unknown. We investigated the structural properties of the postsynaptic membrane that allow these movements by addressing the diffusion behaviors of AMPA receptors (AMPARs) and different lipids. Using single molecule tracking we found that not only AMPARs but also lipids, which are not synaptically enriched, display confined diffusion at synapses. Each molecule type displays a different average confinement area, smaller molecules being confined to smaller areas. Glutamate application increases the mobility of all molecules. The structure of the synaptic membrane is thus probably organized as a size exclusion matrix and this controls the rate of exchange of molecules with the extrasynaptic membrane.
The physical properties of the postsynaptic membrane (PSM), including its viscosity, determine its capacity to regulate the net flux of synaptic membrane proteins such as neurotransmitter receptors. To address these properties, we studied the lateral diffusion of glycophosphatidylinositol-anchored green fluorescent protein and cholera toxin bound to the external leaflet of the plasma membrane. Relative to extrasynaptic regions, their mobility was reduced at synapses and even more at inhibitory than at excitatory ones. This indicates a higher density of obstacles and/or higher membrane viscosity at inhibitory contacts. Actin depolymerization reduced the confinement and accelerated a population of fast, mobile molecules. The compaction of obstacles thus depends on actin cytoskeleton integrity. Cholesterol depletion increased the mobility of the slow diffusing molecules, allowing them to diffuse more rapidly through the crowded PSM. Thus, the PSM has lipid-raft properties, and the density of obstacles to diffusion depends on filamentous actin. Therefore, lipid composition and actin-dependent protein compaction regulate viscosity of the PSM and, consequently, the molecular flow in and out of synapses.
Many synapses in the mature CNS are wrapped by a dense extracellular matrix (ECM). Using single-particle tracking and fluorescence recovery after photobleaching, we found that this net-like ECM formed surface compartments on rat primary neurons that acted as lateral diffusion barriers for AMPA-type glutamate receptors. Enzymatic removal of the ECM increased extrasynaptic receptor diffusion and the exchange of synaptic AMPA receptors. Whole-cell patch-clamp recording revealed an increased paired-pulse ratio as a functional consequence of ECM removal. These results suggest that the surface compartments formed by the ECM hinder lateral diffusion of AMPA receptors and may therefore modulate short-term synaptic plasticity.
Petrini EM, Lu J, Cognet L, Lounis B, Ehlers MD, Choquet D.
• 2009 • Neuron - PDF
At excitatory glutamatergic synapses, postsynaptic endocytic zones (EZs), which are adjacent to the postsynaptic density (PSD), mediate clathrin-dependent endocytosis of surface AMPA receptors (AMPAR) as a first step to receptor recycling or degradation. However, it remains unknown whether receptor recycling influences AMPAR lateral diffusion and whether EZs are important for the expression of synaptic potentiation. Here, we demonstrate that the presence of both EZs and AMPAR recycling maintain a large pool of mobile AMPARs at synapses. In addition, we find that synaptic potentiation is accompanied by an accumulation and immobilization of AMPARs at synapses resulting from both their exocytosis and stabilization at the PSD. Displacement of EZs from the postsynaptic region impairs the expression of synaptic potentiation by blocking AMPAR recycling. Thus, receptor recycling is crucial for maintaining a mobile population of surface AMPARs that can be delivered to synapses for increases in synaptic strength.
Saint-Michel E, Giannone G, Choquet D, Thoumine O.
• 2009 • Biophys J - PDF
We report what to our knowledge is a new method to characterize kinetic rates between cell-surface-attached adhesion molecules. Cells expressing specific membrane receptors are surface-labeled with quantum dots coated with their respective ligands. The progressive diminution in the total number of surface-diffusing quantum dots tracked over time collectively reflects intrinsic ligand/receptor interaction kinetics. The probability of quantum dot detachment is modeled using a stochastic analysis of bond formation and dissociation, with a small number of ligand/receptor pairs, resulting in a set of coupled differential equations that are solved numerically. Comparison with the experimental data provides an estimation of the kinetic rates, together with the mean number of ligands per quantum dot, as three adjustable parameters. We validate this approach by studying the calcium-dependent neurexin/neuroligin interaction, which plays an important role in synapse formation. Using primary neurons expressing neuroligin-1 and quantum dots coated with purified neurexin-1beta, we determine the kinetic rates between these two binding partners and compare them with data obtained using other techniques. Using specific molecular constructs, we also provide interesting information about the effects of neurexin and neuroligin dimerization on the kinetic rates. As it stands, this simple technique should be applicable to many types of biological ligand/receptor pairs.
2010
Opazo P, Labrecque S, Tigaret CM, Frouin A, Wiseman PW, De Koninck P, Choquet D.
• 2010 • Neuron - PDF
The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is critically required for the synaptic recruitment of AMPA-type glutamate receptors (AMPARs) during both development and plasticity. However, the underlying mechanism is unknown. Using single-particle tracking of AMPARs, we show that CaMKII activation and postsynaptic translocation induce the synaptic trapping of AMPARs diffusing in the membrane. AMPAR immobilization requires both phosphorylation of the auxiliary subunit Stargazin and its binding to PDZ domain scaffolds. It does not depend on the PDZ binding domain of GluA1 AMPAR subunit nor its phosphorylation at Ser831. Finally, CaMKII-dependent AMPAR immobilization regulates short-term plasticity. Thus, NMDA-dependent Ca(2+) influx in the post-synapse triggers a CaMKII- and Stargazin-dependent decrease in AMPAR diffusional exchange at synapses that controls synaptic function.
Giannone G, Hosy E, Levet F, Constals A, Schulze K, Sobolevsky AI, Rosconi MP, Gouaux E, Tampé R, Choquet D, Cognet L.
• 2010 • Biophys - PDF
Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.
Brachet A, Leterrier C, Irondelle M, Fache MP, Racine V, Sibarita JB, Choquet D, Dargent B.
• 2010 • J Cell Bio - PDF
In mammalian neurons, the precise accumulation of sodium channels at the axonal initial segment (AIS) ensures action potential initiation. This accumulation precedes the immobilization of membrane proteins and lipids by a diffusion barrier at the AIS. Using single-particle tracking, we measured the mobility of a chimeric ion channel bearing the ankyrin-binding motif of the Nav1.2 sodium channel. We found that ankyrin G (ankG) limits membrane diffusion of ion channels when coexpressed in neuroblastoma cells. Site-directed mutants with decreased affinity for ankG exhibit increased diffusion speeds. In immature hippocampal neurons, we demonstrated that ion channel immobilization by ankG is regulated by protein kinase CK2 and occurs as soon as ankG accumulates at the AIS of elongating axons. Once the diffusion barrier is formed, ankG is still required to stabilize ion channels. In conclusion, our findings indicate that specific binding to ankG constitutes the initial step for Nav channel immobilization at the AIS membrane and precedes the establishment of the diffusion barrier.
Bard L, Sainlos M, Bouchet D, Cousins S, Mikasova L, Breillat C, Stephenson FA, Imperiali B, Choquet D, Groc L.
• 2010 • PNAS - PDF
The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using high-resolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions.
2011
Opazo P, Choquet D.
• 2011 • Molec Cell Neuro - PDF
The amount of AMPARs at synapses is not a fixed number but varies according to different factors including synaptic development, activity and disease. Because the number of AMPARs sets the strength of synaptic transmission, their trafficking is subject to fine and tight regulation. In this review, we will describe the different steps taken by AMPARs in order to reach the synapse. We propose a three-step mechanism involving exocytosis at extra/perisynaptic sites, lateral diffusion to synapses and a subsequent rate-limiting diffusional trapping step. We will describe how the different trafficking steps are regulated during synaptic plasticity or altered during neurodegenerative diseases such as Alzheimer's.
Sainlos M, Tigaret C, Poujol C, Olivier NB, Bard L, Breillat C, Thiolon K, Choquet D, Imperiali B.
• 2011 • Nature ChemBio - PDF
The interactions of the AMPA receptor (AMPAR) auxiliary subunit Stargazin with PDZ domain-containing scaffold proteins such as PSD-95 are critical for the synaptic stabilization of AMPARs. To investigate these interactions, we have developed biomimetic competing ligands that are assembled from two Stargazin-derived PSD-95/DLG/ZO-1 (PDZ) domain-binding motifs using 'click' chemistry. Characterization of the ligands in vitro and in a cellular FRET-based model revealed an enhanced affinity for the multiple PDZ domains of PSD-95 compared to monovalent peptides. In cultured neurons, the divalent ligands competed with transmembrane AMPAR regulatory protein (TARP) for the intracellular membrane-associated guanylate kinase resulting in increased lateral diffusion and endocytosis of surface AMPARs, while showing strong inhibition of synaptic AMPAR currents. This provides evidence for a model in which the TARP-containing AMPARs are stabilized at the synapse by engaging in multivalent interactions. In light of the prevalence of PDZ domain clusters, these new biomimetic chemical tools could find broad application for acutely perturbing multivalent complexes.
Grunwald C, Schulze K, Giannone G, Cognet L, Lounis B, Choquet D, Tampé R.
• 2011 • J Am Chem - PDF
Single-molecule applications, saturated pattern excitation microscopy, and stimulated emission depletion (STED) microscopy demand bright as well as highly stable fluorescent dyes. Here we describe the synthesis of quantum-yield-optimized fluorophores for reversible, site-specific labeling of proteins or macromolecular complexes. We used polyproline-II (PPII) helices as sufficiently rigid spacers with various lengths to improve the fluorescence signals of a set of different trisNTA-fluorophores. The improved quantum yields were demonstrated by steady-state and fluorescence lifetime analyses. As a proof of principle, we characterized the trisNTA-PPII-fluorophores with respect to in vivo protein labeling and super-resolution imaging at synapses of living neurons. The distribution of His-tagged AMPA receptors (GluA1) in spatially restricted synaptic clefts was imaged by confocal and STED microscopy. The comparison of fluorescence intensity profiles revealed the superior resolution of STED microscopy. These results highlight the advantages of biocompatible and, in particular, small and photostable trisNTA-PPII-fluorophores in super-resolution microscopy.
Mondin M, Labrousse V, Hosy E, Heine M, Tessier B, Levet F, Poujol C, Blanchet C, Choquet D, Thoumine O.
• 2011 • J Neuro - PDF
The mechanisms governing the recruitment of functional glutamate receptors at nascent excitatory postsynapses following initial axon-dendrite contact remain unclear. We examined here the ability of neurexin/neuroligin adhesions to mobilize AMPA-type glutamate receptors (AMPARs) at postsynapses through a diffusion/trap process involving the scaffold molecule PSD-95. Using single nanoparticle tracking in primary rat and mouse hippocampal neurons overexpressing or lacking neuroligin-1 (Nlg1), a striking inverse correlation was found between AMPAR diffusion and Nlg1 expression level. The use of Nlg1 mutants and inhibitory RNAs against PSD-95 demonstrated that this effect depended on intact Nlg1/PSD-95 interactions. Furthermore, functional AMPARs were recruited within 1 h at nascent Nlg1/PSD-95 clusters assembled by neurexin-1β multimers, a process requiring AMPAR membrane diffusion. Triggering novel neurexin/neuroligin adhesions also caused a depletion of PSD-95 from native synapses and a drop in AMPAR miniature EPSCs, indicating a competitive mechanism. Finally, both AMPAR level at synapses and AMPAR-dependent synaptic transmission were diminished in hippocampal slices from newborn Nlg1 knock-out mice, confirming an important role of Nlg1 in driving AMPARs to nascent synapses. Together, these data reveal a mechanism by which membrane-diffusing AMPARs can be rapidly trapped at PSD-95 scaffolds assembled at nascent neurexin/neuroligin adhesions, in competition with existing synapses.
Trafficking of AMPA receptors (AMPARs) plays a key role in synaptic transmission. However, a general framework integrating the two major mechanisms regulating AMPAR delivery at postsynapses (i.e., surface diffusion and internal recycling) is lacking. To this aim, we built a model based on numerical trajectories of individual AMPARs, including free diffusion in the extrasynaptic space, confinement in the synapse, and trapping at the postsynaptic density (PSD) through reversible interactions with scaffold proteins. The AMPAR/scaffold kinetic rates were adjusted by comparing computer simulations to single-particle tracking and fluorescence recovery after photobleaching experiments in primary neurons, in different conditions of synapse density and maturation. The model predicts that the steady-state AMPAR number at synapses is bidirectionally controlled by AMPAR/scaffold binding affinity and PSD size. To reveal the impact of recycling processes in basal conditions and upon synaptic potentiation or depression, spatially and temporally defined exocytic and endocytic events were introduced. The model predicts that local recycling of AMPARs close to the PSD, coupled to short-range surface diffusion, provides rapid control of AMPAR number at synapses. In contrast, because of long-range diffusion limitations, extrasynaptic recycling is intrinsically slower and less synapse-specific. Thus, by discriminating the relative contributions of AMPAR diffusion, trapping, and recycling events on spatial and temporal bases, this model provides unique insights on the dynamic regulation of synaptic strength.
Izeddin I, Boulanger J, Racine V, Specht CG, Kechkar A, Nair D, Triller A, Choquet D, Dahan M, Sibarita JB.
• 2012 • Opt Express - PDF
Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 10(5) single molecule detections to reconstruct a single image. We hereby present an algorithm based on image wavelet segmentation and single particle centroid determination, and compare its performance with the commonly used gaussian fitting of the point spread function. We performed realistic simulations at different signal-to-noise ratios and particle densities and show that the calculation time using the wavelet approach can be more than one order of magnitude faster than that of gaussian fitting without a significant degradation of the localization accuracy, from 1 nm to 4 nm in our range of study. We propose a simulation-based estimate of the resolution of an experimental single molecule acquisition.
Opazo P, Sainlos M, Choquet D.
• 2012 • Curr Opin Neurobio - PDF
Excitatory synaptic transmission is largely mediated by AMPA receptors (AMPARs) present at the postsynaptic density. Recent studies in single molecule tracking of AMPAR has revealed that extrasynaptic AMPARs are highly mobile and thus might serve as a readily available pool for their synaptic recruitment during synaptic plasticity events such as long-term potentiation (LTP). Because this hypothesis relies on the cell's ability to increase the number of diffusional traps or 'slots' at synapses during LTP, we will review a number of protein-protein interactions that might impact AMPARs lateral diffusion and thus potentially serve as slots. Recent studies have identified the interaction between the AMPAR-Stargazin complex and PSD-95 as the minimal components of the diffusional trapping slot. We will overview the molecular basis of this critical interaction, its activity-dependent regulation and its potential contribution to LTP.
Hoze N, Nair D, Hosy E, Sieben C, Manley S, Herrmann A, Sibarita JB, Choquet D, Holcman D.
• 2012 • PNAS - PDF
Simultaneous tracking of many thousands of individual particles in live cells is possible now with the advent of high-density superresolution imaging methods. We present an approach to extract local biophysical properties of cell-particle interaction from such newly acquired large collection of data. Because classical methods do not keep the spatial localization of individual trajectories, it is not possible to access localized biophysical parameters. In contrast, by combining the high-density superresolution imaging data with the present analysis, we determine the local properties of protein dynamics. We specifically focus on AMPA receptor (AMPAR) trafficking and estimate the strength of their molecular interaction at the subdiffraction level in hippocampal dendrites. These interactions correspond to attracting potential wells of large size, showing that the high density of AMPARs is generated by physical interactions with an ensemble of cooperative membrane surface binding sites, rather than molecular crowding or aggregation, which is the case for the membrane viral glycoprotein VSVG. We further show that AMPARs can either be pushed in or out of dendritic spines. Finally, we characterize the recurrent step of influenza trajectories. To conclude, the present analysis allows the identification of the molecular organization responsible for the heterogeneities of random trajectories in cells.
2013
Giannone G, Hosy E, Sibarita JB, Choquet D, Cognet L.
• 2013 • Methods Molec Bio - PDF
In this chapter, we present the uPAINT method (Universal Point Accumulation Imaging in Nanoscale Topography), a simple single-molecule super-resolution method which can be implemented on any wide field fluorescence microscope operating in oblique illumination. The key feature of uPAINT lies in recording high numbers of single molecules at the surface of a cell by constantly labeling while imaging. In addition to generating super-resolved images, uPAINT can provide dynamical information on a single live cell with large statistics revealing localization-specific diffusion properties of membrane biomolecules. Interestingly, any membrane biomolecule that can be labeled with a fluorescent ligand can be studied, making uPAINT an extremely versatile method.
Carta M, Opazo P, Veran J, Athané A, Choquet D, Coussen F, Mulle C.
• 2013 • EMBO - PDF
Calmodulin-dependent kinase II (CaMKII) is key for long-term potentiation of synaptic AMPA receptors. Whether CaMKII is involved in activity-dependent plasticity of other ionotropic glutamate receptors is unknown. We show that repeated pairing of pre- and postsynaptic stimulation at hippocampal mossy fibre synapses induces long-term depression of kainate receptor (KAR)-mediated responses, which depends on Ca(2+) influx, activation of CaMKII, and on the GluK5 subunit of KARs. CaMKII phosphorylation of three residues in the C-terminal domain of GluK5 subunit markedly increases lateral mobility of KARs, possibly by decreasing the binding of GluK5 to PSD-95. CaMKII activation also promotes surface expression of KARs at extrasynaptic sites, but concomitantly decreases its synaptic content. Using a molecular replacement strategy, we demonstrate that the direct phosphorylation of GluK5 by CaMKII is necessary for KAR-LTD. We propose that CaMKII-dependent phosphorylation of GluK5 is responsible for synaptic depression by untrapping of KARs from the PSD and increased diffusion away from synaptic sites.
Sainlos M, Iskenderian-Epps WS, Olivier NB, Choquet D, Imperiali B.
• 2013 • J Am Chem - PDF
We report a general method for light-assisted control of interactions of PDZ domain binding motifs with their cognate domains by the incorporation of a photolabile caging group onto the essential C-terminal carboxylate binding determinant of the motif. The strategy was implemented and validated for both simple monovalent and biomimetic divalent ligands, which have recently been established as powerful tools for acute perturbation of native PDZ domain-dependent interactions in live cells.
Kechkar A, Nair D, Heilemann M, Choquet D, Sibarita JB.
• 2013 • PLoS One - PDF
Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.
Giannone G, Mondin M, Grillo-Bosch D, Tessier B, Saint-Michel E, Czöndör K, Sainlos M, Choquet D, Thoumine O.
• 2013 • Cell Rep - PDF
Adhesion between neurexin-1β (Nrx1β) and neuroligin-1 (Nlg1) induces early recruitment of the postsynaptic density protein 95 (PSD-95) scaffold; however, the associated signaling mechanisms are unknown. To dissociate the effects of ligand binding and receptor multimerization, we compared conditions in which Nlg1 in neurons was bound to Nrx1β or nonactivating HA antibodies. Time-lapse imaging, fluorescence recovery after photobleaching, and single-particle tracking demonstrated that in addition to aggregating Nlg1, Nrx1β binding stimulates the interaction between Nlg1 and PSD-95. Phosphotyrosine immunoblots and pull-down of gephyrin by Nlg1 peptides in vitro showed that Nlg1 can be phosphorylated at a unique tyrosine (Y782), preventing gephyrin binding. Expression of Nlg1 point mutants in neurons indicated that Y782 phosphorylation controls the preferential binding of Nlg1 to PSD-95 versus gephyrin, and accordingly the formation of inhibitory and excitatory synapses. We propose that ligand-induced changes in the Nlg1 phosphotyrosine level control the balance between excitatory and inhibitory scaffold assembly during synapse formation and stabilization.
