title: The stoichiometry of scaffold complexes in living neurons - DLC2 functions as a dimerization engine for GKAP.
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.
authors.affiliation: 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.
authors.forename: Enora
authors.initials: E
authors.lastname: Moutin
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Vincent
authors.initials: V
authors.lastname: Compan
authors.affiliation: 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.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Raynaud
authors.affiliation: Centre de Biochimie Structurale, INSERM U1054, Montpellier, F-34090, France CNRS UMR5048, Universités Montpellier 1 and 2, Montpellier F-34090, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Clerté
authors.affiliation: 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.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bouquier
authors.affiliation: Centre de Biochimie Structurale, INSERM U1054, Montpellier, F-34090, France CNRS UMR5048, Universités Montpellier 1 and 2, Montpellier F-34090, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Labesse
authors.affiliation: Centre de Biochimie Structurale, INSERM U1054, Montpellier, F-34090, France CNRS UMR5048, Universités Montpellier 1 and 2, Montpellier F-34090, France.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Ferguson
authors.affiliation: 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.
authors.forename: Laurent
authors.initials: L
authors.lastname: Fagni
authors.affiliation: 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.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Royer
authors.affiliation: 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.
authors.forename: Julie
authors.initials: J
authors.lastname: Perroy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D049229:Dendritic Spines / Q000378:metabolism; D019281:Dimerization; D020690:GTPase-Activating Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D000074407:SAP90-PSD95 Associated Proteins; D016415:Sequence Alignment; D013569:Synapses / Q000737:chemistry / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C000614187:DLGAP1 protein, human; C000614190:Dlgap1 protein, mouse; D020690:GTPase-Activating Proteins; D009419:Nerve Tissue Proteins; D000074407:SAP90-PSD95 Associated Proteins; C473607:STARD13 protein, human; D025521:Tumor Suppressor Proteins
keywords: Bioluminescence resonance energy transfer; Dynein light chain; Guanylate kinase-associated protein; Oligomerization; Scaffold; Scanning number and brightness