Design of plasmonic probes for the identification, localization and quantification of cell membrane receptors: evaluating the role of PKD1 in the distribution of neuronal glutamate receptors

FRAIRE, JC; MASSERONI, MARÍA LUJÁN; JAUSORO, IGNACIO; PERASSI, E; DIAZ AÑEL, A. M.; CORONADO, EDUARDO

ACS NANO

AMER CHEMICAL SOC

Lugar: Washington; Año: 2014 vol. 8 p. 8942 - 8958

1936-0851

Detecting, imaging, and being able to localize the distribution of several cell membrane receptor distribution on a single neuron is a very important topic in neuroscience research, as deficiencies and missorting of certain membrane proteins can be associated to several neurological and neurodegenerative diseases. In particular the metabotropic Glutamate Receptor 1a (mGluR1a) is associated to ischemia, brain trauma, epilepsy, multiple sclerosis, amyothropic lateral sclerosis, and Huntinton and Parkinson diseases. In the present work, the distribution of mGluR1a density on neuron cells on subcellular length scales (dendrites and axons) is determinate by evaluating the role played by the protein kinase D1 (PKD1) in the trafficking of membrane proteins comparing the distribution of mGluR1a for endogenous PKD1 expression with experiments performed in the presence of kinase-inactive protein kinase D1 (PKD1-kd). The localization, distribution and density of cell surface mGluR1a was evaluated using 90 nm diameter Au nanoparticles (NPs) probes specifically functionalized with high affinity and multivalent labeling function which allow not only to image NPs where this receptor is present but also to quantify by optical means the NP density. This is so because the NP generates a density ()-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  values were found to be significantly higher on dendrites than on axons for endogenous PKD1, and were observed a significant increase of mGluR1a on axons when PKD1 is altered. A detailed characterization of the spatial distribution of the NP immunolabels through scanning electron microscopy (SEM) confirmed the findings of the all-optical studies (fluorescence bright analysis and dark-field spectroscopy) and provided additional structural details. In addition, it is shown 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 demonstrated to be almost linear dependent on NP density using electrodynamics simulations, and therefore gives, indirectly, information about the subcellular distribution of a determinate membrane protein. This result is important since the calibration of the -dependent near field enhancement of the Au immunolabels through correlation of SERS and SEM, pave 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 alter the intracellular trafficking and membrane delivery of mGluR1a containing vesicles. After PKD1 suppression, dendritic membrane proteins are mispackaged into vesicles that are distributed to both axons and dendrites. These results reinforces the idea that in neurons PKD1 regulates the sorting of dendritic proteins and hence has a role in neuronal polarity.