RESUMO
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
Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.
Assuntos
Caenorhabditis elegans , Proteína Neuronal da Síndrome de Wiskott-Aldrich , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Animais , Humanos , Caenorhabditis elegans/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Sinapsinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
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
Background: Although several approaches have revealed much about individual factors that regulate pancreatic development, we have yet to fully understand their complicated interplay during pancreas morphogenesis. Gfi1 is transcription factor specifically expressed in pancreatic acinar cells, whose role in pancreas cells fate identity and specification is still elusive. Methods: In order to gain further insight into the function of this factor in the pancreas, we generated animals deficient for Gfi1 specifically in the pancreas. Gfi1 conditional knockout animals were phenotypically characterized by immunohistochemistry, RT-qPCR, and RNA scope. To assess the role of Gfi1 in the pathogenesis of diabetes, we challenged Gfi1-deficient mice with two models of induced hyperglycemia: long-term high-fat/high-sugar feeding and streptozotocin injections. Results: Interestingly, mutant mice did not show any obvious deleterious phenotype. However, in depth analyses demonstrated a significant decrease in pancreatic amylase expression, leading to a diminution in intestinal carbohydrates processing and thus glucose absorption. In fact, Gfi1-deficient mice were found resistant to diet-induced hyperglycemia, appearing normoglycemic even after long-term high-fat/high-sugar diet. Another feature observed in mutant acinar cells was the misexpression of ghrelin, a hormone previously suggested to exhibit anti-apoptotic effects on ß-cells in vitro. Impressively, Gfi1 mutant mice were found to be resistant to the cytotoxic and diabetogenic effects of high-dose streptozotocin administrations, displaying a negligible loss of ß-cells and an imperturbable normoglycemia. Conclusions: Together, these results demonstrate that Gfi1 could turn to be extremely valuable for the development of new therapies and could thus open new research avenues in the context of diabetes research.
Assuntos
Proteínas de Ligação a DNA/deficiência , Diabetes Mellitus/metabolismo , Diabetes Mellitus/prevenção & controle , Fatores de Transcrição/deficiência , Células Acinares/citologia , Células Acinares/metabolismo , Amilases/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Grelina/metabolismo , Proteínas de Homeodomínio/metabolismo , Hiperglicemia/complicações , Hiperglicemia/genética , Integrases/metabolismo , Camundongos Transgênicos , Mutação/genética , Pâncreas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Presenilina-1/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Fibroblastos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microscopia , Presenilina-1/metabolismoRESUMO
R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/ß-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Oócitos , Ovário , Trombospondinas/fisiologia , Via de Sinalização Wnt , Animais , Comunicação Celular , Proliferação de Células , Embrião de Mamíferos , Feminino , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , Oócitos/metabolismo , Ovário/citologia , Ovário/metabolismoRESUMO
Basal autophagy is as a compressive catabolic mechanism engaged in the breakdown of damaged macromolecules and organelles leading to the recycling of elementary nutrients. Thought essential to cellular refreshing, little is known about the origin of a constitutional rate of basal autophagy. Here, we found that loss of Drosophila vacuolar peduncle (vap), a presumed GAP enzyme, is associated with enhanced basal autophagy rate and physiological alterations resulting in a wasteful cell energy balance, a hallmark of overactive autophagy. By contrast, starvation-induced autophagy was disrupted in vap mutant conditions, leading to a block of maturation into autolysosomes. This phenotype stem for exacerbated biogenesis of PI(3)P-dependent endomembranes, including autophagosome membranes and ectopic fusions of vesicles. These findings shed new light on the neurodegenerative phenotype found associated to mutant vap adult brains in a former study. A partner of Vap, Sprint (Spri), acting as an endocytic GEF for Rab5, had the converse effect of leading to a reduction in PI(3)P-dependent endomembrane formation in mutants. Spri was conditional to normal basal autophagy and instrumental to the starvation-sensitivity phenotype specific of vap. Rab5 activity itself was essential for PI(3)P and for pre-autophagosome structures formation. We propose that Vap/Spri complexes promote a cell surface-derived flow of endocytic Rab5-containing vesicles, the traffic of which is crucial for the implementation of a basal autophagy rate.
Assuntos
Autofagia , Drosophila/citologia , Animais , Autofagossomos/metabolismo , Proteínas de Transporte/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Deleção de Genes , Fatores de Troca do Nucleotídeo Guanina , Masculino , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico , Interferência de RNA , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Finely tuned regulation of epithelial cell death maintains tissue integrity and homeostasis. At the cellular level, life and death decisions are controlled by environmental stimuli such as the activation of death receptors. We show that cell polarity and adherens junction formation prevent proapoptotic signals emanating from the Fas death receptor. Fas is sequestered in E-cadherin actin-based adhesion structures that are less able to induce downstream apoptosis signaling. Using a proteomic-based approach, we find that the polarity molecule Dlg1 interacts with the C-terminal PDZ-binding site in Fas and that this interaction decreases formation of the death-inducing complex upon engagement with Fas ligand (FasL), thus acting as an additional cell death protection mechanism. We propose that E-cadherin and Dlg1 inhibit FasL-induced cell death by two complementary but partially independent mechanisms that help to maintain epithelial homeostasis by protecting normal polarized epithelia from apoptosis. When polarity is lost, the Fas-cadherin-Dlg1 antiapoptotic complex is disrupted, and FasL can promote the elimination of compromised nonpolarized cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Células Epiteliais/metabolismo , Proteína Ligante Fas/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Receptor fas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Junções Aderentes/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Polaridade Celular , Proteína 1 Homóloga a Discs-Large , Células Epiteliais/citologia , Proteína Ligante Fas/genética , Humanos , Proteínas de Membrana/genética , Complexos Multiproteicos/genética , Domínios Proteicos , Proteômica , Receptor fas/genéticaRESUMO
Testis differentiation requires high levels of proliferation of progenitor cells that give rise to two cell lineages forming the testis, the Sertoli and the Leydig cells. Hence defective cell cycling leads to testicular dysgenesis that has profound effects on androgen production and fertility. The growth factor NRG1 has been implicated in adult Leydig cell proliferation, but a potential function in the fetal testis has not been analysed to date. Here we show that Nrg1 and its receptors ErbB2/3 are already expressed in early gonadal development. Using tissue-specific deletion, we further demonstrate that Nrg1 is required in a dose-dependent manner to induce proliferation of Sertoli progenitor cells and then differentiated Sertoli cells. As a result of reduced numbers of Sertoli cells, Nrg1 knockout mice display a delay in testis differentiation and defects in sex cord partitioning. Taken together Nrg1 signalling is essential for the establishment of the stock of Sertoli cells and thus required to prevent testicular hypoplasia.
Assuntos
Neuregulina-1/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Transdução de Sinais , Testículo/citologia , Animais , Contagem de Células , Diferenciação Celular , Proliferação de Células , Receptores ErbB/metabolismo , Feminino , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Processos de Determinação Sexual , Células-Tronco/citologia , Células-Tronco/metabolismo , Testículo/embriologia , Trombospondinas/metabolismoRESUMO
The recent demonstration that pancreatic α cells can be continuously regenerated and converted into ß-like cells upon ectopic expression of Pax4 opened new avenues of research in the endocrine cell differentiation and diabetes fields. To determine whether such plasticity was also shared by δ cells, we generated and characterized transgenic animals that express Pax4 specifically in somatostatin-expressing cells. We demonstrate that the ectopic expression of Pax4 in δ cells is sufficient to induce their conversion into functional ß-like cells. Importantly, this conversion induces compensatory mechanisms involving the reactivation of endocrine developmental processes that result in dramatic ß-like cell hyperplasia. Importantly, these ß-like cells are functional and can partly reverse the consequences of chemically induced diabetes.
Assuntos
Diabetes Mellitus Experimental/genética , Expressão Ectópica do Gene , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Box Pareados/genética , Células Secretoras de Somatostatina/metabolismo , Animais , Proliferação de Células , Transdiferenciação Celular/genética , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Terapia Genética/métodos , Glucagon/biossíntese , Glucagon/genética , Proteínas de Homeodomínio/metabolismo , Insulina/biossíntese , Insulina/genética , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição Box Pareados/metabolismo , Somatostatina/biossíntese , Somatostatina/genética , Células Secretoras de Somatostatina/citologia , EstreptozocinaRESUMO
The symbiotic interaction between cnidarians (e.g., corals and sea anemones) and photosynthetic dinoflagellates of the genus Symbiodinium is triggered by both host-symbiont recognition processes and metabolic exchange between the 2 partners. The molecular communication is crucial for homeostatic regulation of the symbiosis, both under normal conditions and during stresses that further lead to symbiosis collapse. It is therefore important to identify and fully characterise the key players of this intimate interaction at the symbiotic interface. In this study, we determined the cellular and subcellular localization and expression of the sterol-trafficking Niemann-Pick type C proteins (NPC1 and NPC2) in the symbiotic sea anemones Anemonia viridis and Aiptasia sp. We first established that NPC1 is localised within vesicles in host tissues and to the symbiosome membranes in several anthozoan species. We demonstrated that the canonical NPC2-a protein is mainly expressed in the epidermis, whereas the NPC2-d protein is closely associated with symbiosome membranes. Furthermore, we showed that the expression of the NPC2-d protein is correlated with symbiont presence in healthy symbiotic specimens. As npc2-d is a cnidarian-specific duplicated gene, we hypothesised that it probably arose from a subfunctionalisation process that might result in a gain of function and symbiosis adaptation in anthozoans. Niemann-Pick type C proteins may be key players in a functional symbiosis and be useful tools to study host-symbiont interactions in the anthozoan-dinoflagellate association.
Assuntos
Dinoflagellida/metabolismo , Dinoflagellida/fisiologia , Doença de Niemann-Pick Tipo C/metabolismo , Anêmonas-do-Mar/metabolismo , Anêmonas-do-Mar/fisiologia , Simbiose/fisiologia , Animais , Perfilação da Expressão Gênica/métodos , Doença de Niemann-Pick Tipo C/genética , Simbiose/genéticaRESUMO
We investigated whether microRNAs could regulate AMPA receptor expression during activity blockade. miR-92a strongly repressed the translation of GluA1 receptors by binding the 3' untranslated region of rat GluA1 (also known as Gria1) mRNA and was downregulated in rat hippocampal neurons after treatment with tetrodotoxin and AP5. Deleting the seed region in GluA1 or overexpressing miR-92a blocked homeostatic scaling, indicating that miR-92a regulates the translation and synaptic incorporation of new GluA1-containing AMPA receptors.
Assuntos
Regulação para Baixo/genética , Homeostase/genética , MicroRNAs/genética , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/genética , Sinapses/genética , Animais , Sequência de Bases , Células HEK293 , Hipocampo/metabolismo , Hipocampo/fisiologia , Humanos , MicroRNAs/antagonistas & inibidores , Dados de Sequência Molecular , Neurônios/metabolismo , Neurônios/fisiologia , Ligação Proteica/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Regiões não Traduzidas/genéticaRESUMO
The role of adhesion molecules in the assembly of synapses in the nervous system is an important issue. To characterize the role of neurexin/neuroligin adhesion complexes in synapse differentiation, various imaging assays can be performed in primary hippocampal cultures. First, to temporally control contact formation, biomimetic assays can be performed using microspheres coated with purified neurexin or with antibody clusters that aggregate neurexin. These models are combined with live fluorescence imaging to study the dynamics of accumulation of post-synaptic components, including scaffolding molecules and glutamate receptors. To demonstrate that AMPA receptors can be recruited to nascent neurexin/neuroligin contacts through lateral diffusion, the mobility of AMPA receptors in the neuronal membrane is monitored by tracking individual quantum dots (QDs) conjugated to antibodies against AMPA receptors. Experiments monitoring the attachment and detachment of Nrx-coated QDs to measure the rates of neurexin/neuroligin interaction can also be performed. Each of these assays is detailed in this unit.
Assuntos
Biomimética/métodos , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Células HEK293 , Humanos , Moléculas de Adesão de Célula Nervosa , Neurônios/citologia , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , RatosRESUMO
Adhesion between neurexin-1ß (Nrx1ß) 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 Nrx1ß or nonactivating HA antibodies. Time-lapse imaging, fluorescence recovery after photobleaching, and single-particle tracking demonstrated that in addition to aggregating Nlg1, Nrx1ß 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.
Assuntos
Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Moléculas de Adesão Celular Neuronais/genética , Proteína 4 Homóloga a Disks-Large , Humanos , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Fosforilação , Mutação Puntual , Ratos , Homologia de Sequência de Aminoácidos , Tirosina/farmacologiaRESUMO
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
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
The trafficking of ionotropic glutamate receptors to and from synaptic sites is regulated by proteins that interact with their cytoplasmic C-terminal domain. Profilin IIa (PfnIIa), an actin-binding protein expressed in the brain and recruited to synapses in an activity-dependent manner, was shown previously to interact with the C-terminal domain of the GluK2b subunit splice variant of kainate receptors (KARs). Here, we characterize this interaction and examine the role of PfnIIa in the regulation of KAR trafficking. PfnIIa directly and specifically binds to the C-terminal domain of GluK2b through a diproline motif. Expression of PfnIIa in transfected COS-7 cells and in cultured hippocampal neurons from PfnII-deficient mice decreases the level of extracellular of homomeric GluK2b as well as heteromeric GluK2a/GluK2b KARs. Our data suggest a novel mechanism by which PfnIIa exerts a dual role on the trafficking of KARs, by a generic inhibition of clathrin-mediated endocytosis through its interaction with dynamin-1, and by controlling KARs exocytosis through a direct and specific interaction with GluK2b.
Assuntos
Exocitose/fisiologia , Hipocampo/metabolismo , Neurônios/metabolismo , Profilinas/metabolismo , Receptores de Ácido Caínico/metabolismo , Sinapses/metabolismo , Animais , Células COS , Chlorocebus aethiops , Clatrina/genética , Clatrina/metabolismo , Dinamina I/genética , Dinamina I/metabolismo , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Profilinas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores de Ácido Caínico/genética , Sinapses/genética , Receptor de GluK2 CainatoRESUMO
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
In response to glomerular injury, mesangial cells are activated into myofibroblasts, which contribute to the physiopathology of glomerulosclerosis. We have previously shown that chronic treatment of cultured human mesangial cells with mycophenolic acid (MPA), a specific inhibitor of guanosine nucleotide synthesis, prevents their activation and alters cytoskeleton protein expression and associated functions, such as contractility and migratory capacity. The aim of the present study was to explore the mechanisms underlying MPA-induced mesangial cytoskeleton alterations. We therein show that coincubation with guanosine (100 microM) compensates for the effects of MPA on mesangial cell proliferation and migration, and prevents MPA-induced overexpression of alpha-smooth muscle actin (SMA) and basic calponin (b-calp), indicating that guanylates are involved in mesangial responses to MPA. MPA decreased the GTP-bound (active) form of both RhoA, Rac1 and Cdc42, and specifically altered the expression level of Rac1. Pharmacological inhibition of RhoA activity reduced expression of both SMA and calponin, whereas overexpression of a dominant-negative form of Rac1 increased SMA expression. Conversely, overexpression of constitutively active Rac1 resulted in SMA and b-calp down-regulation, and fully prevented their stimulation by MPA, indicating that Rac inactivation is responsible for MPA effects on mesangial cytoskeletal expression. These results show that in human mesangial cells, RhoA and Rac1 exert opposite effects on the expression of two major cytoskeletal proteins: SMA and basic calponin. Moreover, these data highlight for the first time an integrated mechanism whereby MPA regulates mesangial phenotype, which is mediated by loss of Rac activity.