Spatial Distribution of DARPP-32 in Dendritic Spines
2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 9, e75155- p.Article in journal (Refereed) Published
The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3́, 5́-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an "aggregated" manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32's possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed.
Place, publisher, year, edition, pages
2013. Vol. 8, no 9, e75155- p.
dopamine 1 receptor, phosphoprotein DARPP 32, animal cell, article, cell aggregation, cellular distribution, corpus striatum, dendritic spine, embryo, microscopy, nonhuman, protein expression, protein localization, rat, signal transduction, stimulated emission depletion microscopy
IdentifiersURN: urn:nbn:se:kth:diva-133390DOI: 10.1371/journal.pone.0075155ISI: 000327538600129ScopusID: 2-s2.0-8488376657OAI: oai:DiVA.org:kth-133390DiVA: diva2:661449
FunderScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council, VR-2006-3197 VR-2007-4582 VR-2010-4270
QC 201311042013-11-042013-10-312014-02-07Bibliographically approved