RESUMO
Homeostatic synaptic plasticity is a process by which neurons adjust their synaptic strength to compensate for perturbations in neuronal activity. Whether the highly diverse synapses on a neuron respond uniformly to the same perturbation remains unclear. Moreover, the molecular determinants that underlie synapse-specific homeostatic synaptic plasticity are unknown. Here, we report a synaptic tagging mechanism in which the ability of individual synapses to increase their strength in response to activity deprivation depends on the local expression of the spine-apparatus protein synaptopodin under the regulation of miR-124. Using genetic manipulations to alter synaptopodin expression or regulation by miR-124, we show that synaptopodin behaves as a "postsynaptic tag" whose translation is derepressed in a subpopulation of synapses and allows for nonuniform homeostatic strengthening and synaptic AMPA receptor stabilization. By genetically silencing individual connections in pairs of neurons, we demonstrate that this process operates in an input-specific manner. Overall, our study shifts the current view that homeostatic synaptic plasticity affects all synapses uniformly to a more complex paradigm where the ability of individual synapses to undergo homeostatic changes depends on their own functional and biochemical state.
Assuntos
MicroRNAs , Receptores de AMPA , Homeostase/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Plasticidade Neuronal/genética , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/metabolismoRESUMO
Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Neurons receive a large number of active synaptic inputs from their many presynaptic partners across their dendritic tree. However, little is known about how the strengths of individual synapses are controlled in balance with other synapses to effectively encode information while maintaining network homeostasis. This is in part due to the difficulty in assessing the activity of individual synapses with identified afferent and efferent connections for a synapse population in the brain. Here, to gain insights into the basic cellular rules that drive the activity-dependent spatial distribution of pre- and postsynaptic strengths across incoming axons and dendrites, we combine patch-clamp recordings with live-cell imaging of hippocampal pyramidal neurons in dissociated cultures and organotypic slices. Under basal conditions, both pre- and postsynaptic strengths cluster on single dendritic branches according to the identity of the presynaptic neurons, thus highlighting the ability of single dendritic branches to exhibit input specificity. Stimulating a single presynaptic neuron induces input-specific and dendritic branchwise spatial clustering of presynaptic strengths, which accompanies a widespread multiplicative scaling of postsynaptic strengths in dissociated cultures and heterosynaptic plasticity at distant synapses in organotypic slices. Our study provides evidence for a potential homeostatic mechanism by which the rapid changes in global or distant postsynaptic strengths compensate for input-specific presynaptic plasticity.
Assuntos
Dendritos/fisiologia , Terminações Pré-Sinápticas/fisiologia , Potenciais Sinápticos/fisiologia , Animais , Axônios , Região CA3 Hipocampal/fisiologia , Dendritos/metabolismo , Potenciais Pós-Sinápticos Excitadores , Hipocampo/fisiologia , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Células Piramidais/fisiologia , Sinapses/fisiologiaRESUMO
Leucine Rich Repeat Transmembrane proteins (LRRTMs) are neuronal cell adhesion molecules involved in synapse development and plasticity. LRRTM2 is the most synaptogenic isoform of the family, and its expression is strongly restricted to excitatory synapses in mature neurons. However, the mechanisms by which LRRTM2 is trafficked and stabilized at synapses remain unknown. Here, we examine the role of LRRTM2 intracellular domain on its membrane expression and stabilization at excitatory synapses, using a knock-down strategy combined to single molecule tracking and super-resolution dSTORM microscopy. We show that LRRTM2 operates an important shift in mobility after synaptogenesis in hippocampal neurons. Knock-down of LRRTM2 during synapse formation reduced excitatory synapse density in mature neurons. Deletion of LRRTM2 C-terminal domain abolished the compartmentalization of LRRTM2 in dendrites and disrupted its synaptic enrichment. Furtheremore, we show that LRRTM2 diffusion is increased in the absence of its intracellular domain, and that the protein is more dispersed at synapses. Surprisingly, LRRTM2 confinement at synapses was strongly dependent on a YxxC motif in the C-terminal domain, but was independent of the PDZ-like binding motif ECEV. Finally, the nanoscale organization of LRRTM2 at excitatory synapses depended on its C-terminal domain, with involvement of both the PDZ-binding and YxxC motifs. Altogether, these results demonstrate that LRRTM2 trafficking and enrichment at excitatory synapses are dependent on its intracellular domain.
Assuntos
Proteínas do Tecido Nervoso , Moléculas de Adesão de Célula Nervosa , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , SinapsesRESUMO
Synaptic adhesion molecules not only provide a physical link between pre- and post-synaptic membranes, but also contribute to synaptic differentiation and plasticity by organizing functional elements, in particular neurotransmitter receptors. The wealth of existing adhesive protein families including many isoforms and splice variants, calls for systematic identification of the levels and exchange rates of each of those protein members at specific synapse types. Complementary to electron microscopy to identify individual synaptic contacts and biochemistry to reveal protein-protein interactions, recent super-resolution light microscopy methods combined with appropriate fluorescent labeling provide a way to measure the dynamics and sub-micron organization of selective molecular components, and their inter-relations at the synapse. In this review, we summarize current knowledge on the dynamics, nanoscale localization, and function of key synaptic adhesion complexes.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Imagem Óptica/métodos , Imagem Individual de Molécula/métodos , Sinapses/metabolismo , Animais , Humanos , Plasticidade Neuronal , Transporte Proteico , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Espinhas Dendríticas/fisiologia , Modelos Biológicos , Densidade Pós-Sináptica/metabolismo , Transmissão Sináptica/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Forminas , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase , Proteínas , Ratos , Ratos Sprague-Dawley , Estatísticas não ParamétricasRESUMO
Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration.
Assuntos
Actinas/metabolismo , Caderinas/metabolismo , Cateninas/metabolismo , Movimento Celular/fisiologia , Cones de Crescimento/metabolismo , Hipocampo/citologia , Complexos Multiproteicos/metabolismo , Animais , Fenômenos Biomecânicos , Primers do DNA/genética , Embrião de Mamíferos/citologia , Fluorescência , Recuperação de Fluorescência Após Fotodegradação , Imuno-Histoquímica , Simulação de Dinâmica Molecular , Reação em Cadeia da Polimerase , Ratos , Fatores de TempoRESUMO
Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.
Assuntos
Canais de Cálcio Tipo L/biossíntese , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Dor , Biossíntese de Proteínas , Animais , RatosRESUMO
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.
Assuntos
Simulação por Computador , Modelos Neurológicos , Transporte Proteico , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Ligação Competitiva , Difusão , Endocitose , Potenciais Pós-Sinápticos Excitadores/fisiologia , Recuperação de Fluorescência Após Fotodegradação , Hipocampo/citologia , Membranas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Neurônios/ultraestrutura , Densidade Pós-Sináptica/metabolismo , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Super-resolution imaging provides unprecedented visualization of sub-cellular structures, but the two main techniques used, single-molecule localization microscopy (SMLM) and stimulated emission depletion (STED), are not easily reconciled. We present a protocol to super-impose nanoscale protein distribution reconstructed with SMLM to sub-cellular morphology obtained in STED. We describe steps for tracking cells on etched coverslips and registering images from two different microscopes with 30-nm accuracy. In this protocol, synaptic proteins are mapped in the dendritic spines of primary neurons. For complete details on the use and execution of this protocol, please refer to Inavalli et al.1.
RESUMO
Introduction: The synaptic adhesion molecule neuroligin-1 (NLGN1) is involved in the differentiation of excitatory synapses, but the precise underlying molecular mechanisms are still debated. Here, we explored the role of NLGN1 tyrosine phosphorylation in this process, focusing on a subset of receptor tyrosine kinases (RTKs), namely FGFR1 and Trks, that were previously described to phosphorylate NLGN1 at a unique intracellular residue (Y782). Methods: We used pharmacological inhibitors and genetic manipulation of those RTKs in dissociated hippocampal neurons, followed by biochemical measurement of NLGN1 phosphorylation and immunocytochemical staining of excitatory synaptic scaffolds. Results: This study shows that: (i) the accumulation of PSD-95 at de novo NLGN1 clusters induced by neurexin crosslinking is reduced by FGFR and Trk inhibitors; (ii) the increase in PSD-95 puncta caused by NLGN1 over-expression is impaired by FGFR and Trk inhibitors; (iii) TrkB activation by BDNF increases NLGN1 phosphorylation; and (iv) TrkB knock-down impairs the increase of PSD-95 puncta caused by NLGN1 over-expression, an effect which is not seen with the NLGN1 Y782A mutant. Discussion: Together, our data identify TrkB as one of the major RTKs responsible for NLGN1 tyrosine phosphorylation, and reveal that TrkB activity is necessary for the synaptogenic effects of NLGN1.
RESUMO
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-1ß 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.
Assuntos
Moléculas de Adesão Celular Neuronais/biossíntese , Guanilato Quinases/metabolismo , Hipocampo/fisiologia , Proteínas de Membrana/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Receptores de AMPA/metabolismo , Transmissão Sináptica/fisiologia , Animais , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/genética , Proteína 4 Homóloga a Disks-Large , Feminino , Guanilato Quinases/antagonistas & inibidores , Guanilato Quinases/genética , Hipocampo/metabolismo , Masculino , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , Técnicas de Patch-Clamp/métodos , Cultura Primária de Células , Ratos , Receptores de AMPA/fisiologia , Transmissão Sináptica/genética , Transfecção/métodosRESUMO
Neuroligins (NLGNs) form a family of cell adhesion molecules implicated in synapse development, but the mechanisms that retain these proteins at synapses are still incompletely understood. Recent studies indicate that surface-associated NLGN1 is diffusionally trapped at synapses, where it interacts with quasi-static scaffolding elements of the post-synaptic density. Whereas single molecule tracking reveals rapid diffusion and transient immobilization of NLGN1 at synapses within seconds, fluorescence recovery after photobleaching experiments indicate instead a long-term turnover of NLGN1 at synapse, in the hour time range. To gain insight into the mechanisms supporting NLGN1 anchorage at post-synapses and try to reconcile those experimental paradigms, we quantitatively analyzed here live-cell and super-resolution imaging experiments performed on NLGN1 using a newly released simulator of membrane protein dynamics for fluorescence microscopy, FluoSim. Based on a small set of parameters including diffusion coefficients, binding constants, and photophysical rates, the framework describes fairly well the dynamic behavior of extra-synaptic and synaptic NLGN1 over both short and long time ranges, and provides an estimate of NLGN1 copy numbers in post-synaptic densities at steady-state (around 50 dimers). One striking result is that the residence time of NLGN1 at synapses is much longer than what can be expected from extracellular interactions with pre-synaptic neurexins only, suggesting that NLGN1 is stabilized at synapses through multivalent interactions with intracellular post-synaptic scaffolding proteins.
RESUMO
MDGA molecules can bind neuroligins and interfere with trans-synaptic interactions to neurexins, thereby impairing synapse development. However, the subcellular localization and dynamics of MDGAs, or their specific action mode in neurons remain unclear. Here, surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons reveal that MDGAs are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation. Knocking-down/out MDGAs using shRNAs and CRISPR/Cas9 strategies increases the density of excitatory synapses, the membrane confinement of neuroligin-1, and the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, MDGA silencing reduces the mobility of AMPA receptors, increases the frequency of miniature EPSCs (but not IPSCs), and selectively enhances evoked AMPA-receptor-mediated EPSCs in CA1 pyramidal neurons. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors.
Assuntos
Proteínas do Tecido Nervoso , Receptores de AMPA , Moléculas de Adesão Celular Neuronais/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/fisiologiaRESUMO
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.
Assuntos
Caderinas/metabolismo , Espaço Extracelular/metabolismo , Receptores de AMPA/metabolismo , Coluna Vertebral/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Mutação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Técnicas de Patch-Clamp/métodos , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Interferência de RNA/fisiologia , Ratos , Transfecção/métodosRESUMO
A combination of cell culture and animal studies has recently shown that adhesion between neurexins and neuroligins played important roles in synapse initiation, maturation, and function. Binding of neurexin-1beta to neuroligin-1 triggers the postsynaptic clustering of the scaffold postsynaptic density protein 95, but the composition and timing of accumulation of glutamate receptors at those nascent contacts remain unclear. Using glutamate iontophoresis and patch-clamp recordings, we identified functional AMPA receptors (AMPARs) and NMDA receptors at postsynaptic density protein 95 clusters induced by neurexin-1beta coated microspheres on primary hippocampal neurons. The recruitment of AMPARs occurred as early as 2 h after initial contact, and was not blocked by TTX/2-amino-5-phosphovaleric acid (APV) treatment. The differential recruitment of recombinant subunits GluR1 and GluR2, as well as the absence of rectification in voltage/current curves, further indicate that neurexin/neuroligin contacts primarily recruit GluR2-containing AMPARs. Finally, by using glutamate un-caging and calcium imaging, we show that AMPARs participate in calcium entry at neurexin-1beta induced post-synapses, most likely through the activation of voltage-gated calcium channels. Such rapid and activity-independent accumulation of functional AMPARs at neurexin-1beta-induced postsynapses points to a new role of AMPARs in synaptogenesis.
Assuntos
Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Animais , Moléculas de Adesão Celular Neuronais , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Iontoforese/métodos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Técnicas de Patch-Clamp/métodos , Ligação Proteica/fisiologia , Ratos , Receptores de AMPA/genética , Fatores de Tempo , Valina/análogos & derivados , Valina/farmacologiaRESUMO
During development, neural circuit formation requires the stabilization of active γ-aminobutyric acidmediated (GABAergic) synapses and the elimination of inactive ones. Here, we demonstrate that, although the activation of postsynaptic GABA type A receptors (GABAARs) and adenosine A2A receptors (A2ARs) stabilizes GABAergic synapses, only A2AR activation is sufficient. Both GABAAR- and A2AR-dependent signaling pathways act synergistically to produce adenosine 3',5'-monophosphate through the recruitment of the calciumcalmodulinadenylyl cyclase pathway. Protein kinase A, thus activated, phosphorylates gephyrin on serine residue 303, which is required for GABAAR stabilization. Finally, the stabilization of pre- and postsynaptic GABAergic elements involves the interaction between gephyrin and the synaptogenic membrane protein Slitrk3. We propose that A2ARs act as detectors of active GABAergic synapses releasing GABA, adenosine triphosphate, and adenosine to regulate their fate toward stabilization or elimination.
Assuntos
Adenosina/metabolismo , Hipocampo/crescimento & desenvolvimento , Neurônios/fisiologia , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo , Antagonistas do Receptor A2 de Adenosina , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cognição , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hipocampo/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso , Fosforilação , Receptor A2A de Adenosina/genética , Receptores de GABA-A/metabolismoRESUMO
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.
Assuntos
Endocitose , Exocitose , Cones de Crescimento/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Adesividade , Animais , Células COS , Polaridade Celular , Chlorocebus aethiops , Difusão , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Microesferas , Modelos Biológicos , Ligação Proteica , Transporte Proteico , Ratos , Receptores Fc/metabolismo , Propriedades de SuperfícieRESUMO
Adhesion proteins play crucial roles at synapses, not only by providing a physical trans-synaptic linkage between axonal and dendritic membranes, but also by connecting to functional elements including the pre-synaptic neurotransmitter release machinery and post-synaptic receptors. To mediate these functions, adhesion proteins must be organized on the neuronal surface in a precise and controlled manner. Recent studies have started to describe the mobility, nanoscale organization, and turnover rate of key synaptic adhesion molecules including cadherins, neurexins, neuroligins, SynCAMs, and LRRTMs, and show that some of these proteins are highly mobile in the plasma membrane while others are confined at sub-synaptic compartments, providing evidence for different regulatory pathways. In this review article, we provide a biophysical view of the diffusional trapping of adhesion molecules at synapses, involving both extracellular and intracellular protein interactions. We review the methodology underlying these measurements, including biomimetic systems with purified adhesion proteins, means to perturb protein expression or function, single molecule imaging in cultured neurons, and analytical models to interpret the data. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.
Assuntos
Fenômenos Biofísicos/fisiologia , Moléculas de Adesão Celular Neuronais/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/análise , Moléculas de Adesão Celular Neuronais/genética , Humanos , Neurônios/química , Neurônios/metabolismo , Transporte Proteico/fisiologia , Sinapses/química , Sinapses/genéticaRESUMO
Fluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these issues, we present here a robust computer program, called FluoSim, which is an interactive simulator of membrane protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. FluoSim integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the localization and intensity of thousands of independent molecules in 2D cellular geometries, providing simulated data directly comparable to actual experiments. FluoSim was thoroughly validated against experimental data obtained on the canonical neurexin-neuroligin adhesion complex at cell-cell contacts. This unified software allows one to model and predict membrane protein dynamics and localization at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments.