RESUMO
Detecting, imaging, and being able to localize the distribution of several cell membrane receptors on a single neuron are very important topics in neuroscience research. In the present work, the distribution of metabotropic glutamate receptor 1a (mGluR1a) density on neuron cells on subcellular length scales is determined by evaluating the role played by protein kinase D1 (PKD1) in the trafficking of membrane proteins, comparing the distribution of mGluR1a in experiments performed in endogenous PKD1 expression with those in the presence of kinase-inactive protein kinase D1 (PKD1-kd). The localization, distribution, and density of cell surface mGluR1a were evaluated using 90 nm diameter Au nanoparticle (NP) probes specifically functionalized with a high-affinity and multivalent labeling function, which allows not only imaging NPs where this receptor is present but also quantifying by optical means the NP density. This is so because the NP generates a density (ρ)-dependent SERS response that facilitated a spatial mapping of the mGluR1a density distribution on subcellular length scales (dendrites and axons) in an optical microscope. The measured ρ values were found to be significantly higher on dendrites than on axons for endogenous PKD1, while an increase of ρ on axons was observed when PKD1 is altered. The spatial distribution of the NP immunolabels through scanning electron microscopy (SEM) confirmed the results obtained by fluorescence bright-field analysis and dark-field spectroscopy and provided additional structural details. In addition, it is shown using electrodynamic simulations that SERS spectroscopy could be a very sensitive tool for the spatial mapping of cell membrane receptors on subcellular length scales, as SERS signals are almost linearly dependent on NP density and therefore give indirect information on the distribution of cell membrane proteins. This result is important since the calibration of the ρ-dependent near-field enhancement of the Au immunolabels through correlation of SERS and SEM paves the way toward quantitative immunolabeling studies of cell membrane proteins involved in neuron polarity. From the molecular biology point of view, this study shows that in cultured hippocampal pyramidal cells mGluR1a is predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive protein kinase D1 (PKD1-kd) dramatically and selectively alters the intracellular trafficking and membrane delivery of mGluR1a-containing vesicles.
Assuntos
Membrana Celular/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Neurônios/citologia , Proteína Quinase C/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Axônios/metabolismo , Regulação Enzimológica da Expressão Gênica , Ouro/química , Ouro/metabolismo , Nanopartículas Metálicas , Imagem Óptica , Transporte Proteico , RatosRESUMO
BACKGROUND INFORMATION: Heterotrimeric GTP-binding proteins play a key role in cell trafficking regulation. Above all, specific Gßγ subunits have been shown to be a major component of a signal transduction pathway, which also involves phospholipases C (PLC), protein kinases C (PKC) and D (PKD), whose main function is to regulate transport between Golgi and plasma membrane. It was the involvement of PLC which led us to study the role of the other member of this G protein family, the α subunits, in the regulation of membrane fission at the Golgi apparatus. RESULTS: Among constitutive active (QL) variants of different G protein α subunit sub-families, only GαqQL subunits were able to induce Golgi fragmentation, a phenotype that mainly reflects a membrane fission increase at this organelle. This phenotype was not observed with a GαqQL palmitoylation mutant, showing the need for a membrane-bounded subunit. Besides, GαqQL-dependent Golgi fission was blocked by specific PLC and PKC inhibitors, and in the presence of a PKD1-kinase dead variant. In addition, GαqQL was the only α subunit capable of inducing PKD1 phosphorylation. Finally, Vesicular Stomatitis Virus thermosensitive mutant glycoprotein (VSVG tsO45) transport assays have demonstrated that GαqQL acts directly on Golgi membranes to regulate trafficking between this organelle and plasma membrane. CONCLUSIONS: All these results indicate Gαq subunits for the first time as a regulator of PKD-mediated intracellular trafficking between Golgi apparatus and plasma membrane, opening new perspectives in the understanding of internal trafficking regulation by external signals through G protein-coupled receptors.
Assuntos
Membrana Celular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Proteína Quinase C/metabolismo , Membrana Celular/enzimologia , Citoesqueleto/metabolismo , Complexo de Golgi/enzimologia , Células HeLa , Humanos , Microtúbulos/metabolismo , Modelos Biológicos , Fosfoinositídeo Fosfolipase C/metabolismo , Fosforilação , Proteína Quinase C/química , Estrutura Terciária de Proteína , Transporte Proteico , Rede trans-Golgi/metabolismoRESUMO
In non-neuronal cells, inactivation of protein kinase D (PKD) blocks fission of trans-Golgi network (TGN) transport carriers, inducing the appearance of long tubules filled with cargo. We now report on the function of PKD1 in neuronal protein trafficking. In cultured hippocampal pyramidal cells, the transferrin receptor (TfR) and the low-density receptor-related protein (LRP) are predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive PKD1 or its depletion by RNA interference treatment dramatically and selectively alter the intracellular trafficking and membrane delivery of TfR- and LRP-containing vesicles, without inhibiting exit from the TGN or inducing Golgi tubulation. After PKD1 suppression, dendritic membrane proteins are mispackaged into carriers that transport VAMP2; these vesicles are distributed to both axons and dendrites, but are rapidly endocytosed from dendrites and preferentially delivered to the axonal membrane. A kinase-defective mutant of PKD1 lacking the ability to bind diacylglycerol and hence its Golgi localization does not cause missorting of TfR or LRP. These results suggest that in neurons PKD1 regulates TGN-derived sorting of dendritic proteins and hence has a role in neuronal polarity.