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J Cell Sci. 2014 Aug 15;127(Pt 16):3451-62. doi: 10.1242/jcs.145748. Epub 2014 Jun 17.

The stoichiometry of scaffold complexes in living neurons - DLC2 functions as a dimerization engine for GKAP.

Author information

1
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France INSERM, U661, Montpellier, F-34094, France Universités de Montpellier 1 & 2, UMR-5203, Montpellier, F-34094, France.
2
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
3
Centre de Biochimie Structurale, INSERM U1054, Montpellier, F-34090, France CNRS UMR5048, Universités Montpellier 1 and 2, Montpellier F-34090, France.
4
Centre de Biochimie Structurale, INSERM U1054, Montpellier, F-34090, France CNRS UMR5048, Universités Montpellier 1 and 2, Montpellier F-34090, France royerc@rpi.edu julie.perroy@igf.cnrs.fr.
5
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France INSERM, U661, Montpellier, F-34094, France Universités de Montpellier 1 & 2, UMR-5203, Montpellier, F-34094, France royerc@rpi.edu julie.perroy@igf.cnrs.fr.

Abstract

Quantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the N-methyl-D-aspartate (NMDA)-receptor-associated scaffolding complex, GKAP (also known as DLGAP1) and DLC2 (also known as DYNLL2), using a novel variation of fluorescence fluctuation microscopy called two-photon scanning number and brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which, in turn, potentiated GKAP self-association. In the dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of two DLC2 and two GKAP monomers, whereas, in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP monomers. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission by ordering GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2-GKAP interactions in glutamatergic signaling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.

KEYWORDS:

Bioluminescence resonance energy transfer; Dynein light chain; Guanylate kinase-associated protein; Oligomerization; Scaffold; Scanning number and brightness

PMID:
24938595
DOI:
10.1242/jcs.145748
[Indexed for MEDLINE]
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