INFIQC   05475
INSTITUTO DE INVESTIGACIONES EN FISICO- QUIMICA DE CORDOBA
Unidad Ejecutora - UE
artículos
Título:
Identification, Localization, and Quantification of Neuronal Cell Membrane Receptors with Plasmonic Probes: Role of Protein Kinase D1 in Their Distribution
Autor/es:
JUAN C. FRAIRE; M. LUJAN MASSERONI; IGNACIO JAUSORO; EDUARDO M. PERASSI; ALBERTO M. DIAZ AÑEL; EDIARDO A. CORONADO
Revista:
ACS NANO
Editorial:
AMER CHEMICAL SOC
Referencias:
Lugar: Washington; Año: 2014 vol. 8 p. 8942 - 8958
ISSN:
1936-0851
Resumen:
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 (F)-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 F values were found to be significantly higher on dendrites than on axons for endogenous PKD1, while an increase of F 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 F-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 kinaseinactive
protein kinase D1 (PKD1-kd) dramatically and selectively alters the intracellular trafficking and membrane delivery of mGluR1a-containing vesicles.