Abstract
The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3´, 5´-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.
Highlights
Communication between nerve cells in the brain can simplistically be described as a biochemical concert of synaptic neurotransmitters, receptors, ion channels and effector molecules, coding and controlling signal transmission
The postsynaptic localization of DARPP-32 in dendritic spines was studied in cultured striatal neurons
Due to the diffraction limit of light, classical fluorescence microscopy cannot resolve the postsynaptic distribution of DARPP-32 within a single spine. To overcome this inherent problem, we applied superresolution stimulated emission depletion microscopy (STED) microscopy to dissect the nanoscale topology of immunofluorescently labeled DARPP-32
Summary
Communication between nerve cells in the brain can simplistically be described as a biochemical concert of synaptic neurotransmitters, receptors, ion channels and effector molecules, coding and controlling signal transmission. Regulation of signaling efficiency is basically controlled by down-stream (and up-stream) regulating molecular system that modulates synaptic transmission. An important effector molecule in the dopaminergic signaling pathway, mediating the action of dopamine, is the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) [3]. This phosphoprotein is expressed primarily in medium-sized spiny neurons of the neostriatum [4], which receive dopaminergic as well as glutamatergic stimulation of connecting neurons from the midbrain, cortex and thalamus
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