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Spatial Distribution of DARPP-32 in Dendritic Spines
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-5584-9170
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
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2013 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 8, no 9, p. e75155-Article in journal (Refereed) Published
Abstract [en]

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, p. e75155-
Keywords [en]
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
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-133390DOI: 10.1371/journal.pone.0075155ISI: 000327538600129PubMedID: 24058659Scopus ID: 2-s2.0-84883766578OAI: oai:DiVA.org:kth-133390DiVA, id: diva2:661449
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council, VR-2006-3197 VR-2007-4582 VR-2010-4270
Note

QC 20131104

Available from: 2013-11-04 Created: 2013-10-31 Last updated: 2024-03-18Bibliographically approved
In thesis
1. Super resolution optical imaging – image analysis, multicolor development and biological applications
Open this publication in new window or tab >>Super resolution optical imaging – image analysis, multicolor development and biological applications
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on super resolution STED optical imaging. STED provides a wealth of new informational content to the acquired images by using stimulated emission to surpass the diffraction limit in optical fluorescence microscopy. To further increase the informational content, a new method to perform multicolor STED imaging by exploiting differences in the photostability and excitation spectra of dyes is presented. In order to extract information from the images, computational algorithms which handle the new type of high resolution informational content are developed.

We propose that multicolor super resolution imaging in combination with image analysis can reduce the amount of clinical samples required to perform accurate cancer diagnosis. To date, such diagnosis is based mainly on significant amounts of tissue samples extracted from the suspected tumor site. The sample extraction often requires anesthetics and can lead to complications such as hematoma, infections and even cancer cell ceding along the needle track. We show that by applying multicolor STED and image analysis, the information gained from single cells is greatly increased. We therefore propose that accurate diagnosis can be based on significantly less extracted tissue material, allowing for a more patient friendly sampling. This approach can also be applied when studying blood platelets, where we show how the high informational content can be used to identify platelet specific activational states. Since platelets are involved in many different types of diseases, such analysis could provide means of performing truly minimally invasive diagnostics based on a simple blood test.

In addition, our data makes it possible to understand in finer detail the underlying mechanisms rendering cells metastasis competent. We combine the high resolution spatial information provided by STED with information regarding the adhesive forces of cells measured by TFM (Traction Force Microscopy) and the cell stiffness measured by AFM (Atomic Force Microscopy). Such comparisons provide a link between the specific highly resolved protein distributions and different cellular mechanics and functions.

This thesis also includes STED imaging and analysis on the spatial organization of neuronal synaptic regulating proteins, implicating the speed with which neuronal signaling can be regulated.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. p. x, 86
Series
TRITA-FYS, ISSN 0280-316X ; 2014:04
Keywords
Stimulated emission depletion (STED) microscopy, nanoscopy, multicolor, image analysis, diagnostics, cancer, metastasis
National Category
Physical Sciences
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-141011 (URN)978-91-7595-001-3 (ISBN)
Public defence
2014-02-28, FB42, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:51 (English)
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Supervisors
Note

QC 20140207

Available from: 2014-02-07 Created: 2014-02-05 Last updated: 2022-06-23Bibliographically approved

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Blom, HansRönnlund, DanielWidengren, JerkerBrismar, Hjalmar

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