TY - JOUR
T1 - Stoichiometry of Scaffold Complexes in Living Neurons - DLC2 as a Dimerization Engine for GKAP
AU - Moutin, Enora
AU - Compan, Vincent
AU - Raynaud, Fabrice
AU - Clerté, Caroline
AU - Bouquier, Nathalie
AU - Labesse, Gilles
AU - Ferguson, Matthew L.
AU - Fagni, Laurent
AU - Royer, Catherine A.
AU - Perroy, Julie
PY - 2014/8/15
Y1 - 2014/8/15
N2 - 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 NMDA receptor-associated scaffolding complex, GKAP and DLC2, 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 dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of 2 DLC2 and 2 GKAP monomers, while in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP. 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 via ordering of GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2–GKAP interactions in glutamergic signalling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.
AB - 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 NMDA receptor-associated scaffolding complex, GKAP and DLC2, 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 dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of 2 DLC2 and 2 GKAP monomers, while in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP. 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 via ordering of GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2–GKAP interactions in glutamergic signalling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.
KW - Bioluminescence resonance energy transfer
KW - Dynein light chain
KW - Guanylate kinase-associated protein
KW - Oligomerization
KW - Scaffold
KW - Scanning number and brightness
UR - https://scholarworks.boisestate.edu/physics_facpubs/152
UR - http://dx.doi.org/10.1242/jcs.145748
UR - http://www.scopus.com/inward/record.url?scp=84906070886&partnerID=8YFLogxK
U2 - 10.1242/jcs.145748
DO - 10.1242/jcs.145748
M3 - Article
C2 - 24938595
VL - 127
SP - 3451
EP - 3462
JO - Physics Faculty Publications and Presentations
JF - Physics Faculty Publications and Presentations
IS - 16
ER -