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Venezia, Italy

Guescini M.,Urbino University | Leo G.,University of Modena and Reggio Emilia | Genedani S.,University of Modena and Reggio Emilia | Carone C.,University of Modena and Reggio Emilia | And 8 more authors.
Experimental Cell Research

Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A 2A and D 2 receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D 2R-CFP or A 2AR-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D 2R-CFP and A 2AR-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24h with A 2AR positive MVs were treated with the adenosine A 2A receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A 2ARs were functionally competent in target cells. These findings demonstrate that A 2A receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells. © 2012 Elsevier Inc. Source

Ciruela F.,Unitat de Farmacologia | Fernandez-Duenas V.,Unitat de Farmacologia | Llorente J.,Unitat de Farmacologia | Borroto-Escuela D.,Karolinska Institutet | And 6 more authors.
Brain Research

The control of glutamatergic corticostriatal transmission is essential for the induction and expression of plasticity mechanisms in the striatum, a phenomenon thickly regulated by G protein-coupled receptors (GPCRs). Interestingly, in addition to dopamine receptors, adenosine and metabotropic glutamate receptors also play a key role in striatal functioning. The existence of a supramolecular organization (i.e. oligomer) containing dopamine, adenosine and metabotropic glutamate receptors in the striatal neurons is now being widely accepted by the scientific community. Indeed, these oligomers may enhance the diversity and performance by which extracellular striatal signals are transferred to the G-proteins in the process of receptor transduction, and also may allow unpredictable receptor-receptor allosteric regulations. Overall, here we want to review how formations of adenosine, dopamine and metabotropic glutamate receptors-containing oligomers impinge into striatal functioning in both normal and pathological conditions. This article is part of a Special Issue entitled: Brain Integration. © 2012 Elsevier B.V. All rights reserved. Source

Fernandez-Duenas V.,University of Barcelona | Llorente J.,University of Barcelona | Gandia J.,University of Barcelona | Borroto-Escuela D.O.,Karolinska Institutet | And 4 more authors.

Understanding of the molecular mechanisms of protein-protein interactions (PPIs) at the cell surface of living cells is fundamental to comprehend the functional meaning of a large number of cellular processes. Here we discuss how new methodological strategies derived from non-invasive fluorescence-based approaches (i.e. fluorescence resonance energy transfer, FRET) have been successfully developed to characterize plasma membrane PPIs. Importantly, these technologies alone - or in concert with complementary methods (i.e. SNAP-tag/TR-FRET, TIRF/FRET) - can become extremely powerful approaches for visualizing cell surface PPIs, even between more than two proteins and also in native tissues. Interestingly, these methods would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Accordingly, herein we provide a thorough assessment on all biotechnological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied in the study of PPIs occurring at the cell surface of living cells. © 2012 Elsevier Inc. Source

Guescini M.,Urbino University | Guidolin D.,University of Padua | Vallorani L.,Urbino University | Casadei L.,Urbino University | And 8 more authors.
Experimental Cell Research

Micro-vesicles can be released by different cell types and operate as 'safe containers' mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway.In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases. © 2010 Elsevier Inc. Source

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