Stoichiometry of scaffold complexes in living neurons - DLC2 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 selfassociation. In the dendritic shaft, the DLC2-GKAP heterooligomeric 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.

RGS2 expression predicts amyloid-β sensitivity, MCI and Alzheimer’s disease: genome-wide transcriptomic profiling and bioinformatics data mining

“… 79 Interference of the DLGAP1 – DLC2 interaction inhibits NMDA receptor activity in dendritic spines. 80 In turn, synaptic activity-induced DLGAP1 – DLC2 interaction in dendritic spines stabilizes the scaffolding complex and enhances the NMDA currents. 81 , 82 …”

Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability

“…Ty1 Gag-GFP expressed from a chromosomal Ty1 element was imaged in live yeast cells using two photon scanning N&B. This powerful technique extends fluorescence correlation spectroscopy (FCS) to provide 2D spatial variance analysis of fluorescent particles diffusing in live cells [35, 47, 48]. N&B analysis uses the variance in the fluorescence intensity at each pixel of a stack of rapid raster scans to calculate the absolute average number (N) of fluorescent molecules in the two photon excitation volume at each pixel, as well as their molecular brightness (B, in counts per pixel dwell-time per molecule).…”

The timing of Start is determined primarily by increased synthesis of the Cln3 activator rather than dilution of the Whi5 inhibitor

“…In their Letter, Schmoller et al (2022) stated that they are not familiar with 2psN&B and thus cannot judge the approach. 2psN&B is a well-established and widely used fluorescence fluctuation-based method for quantitative single-cell imaging (e.g., Nagy et al, 2010;Hellriegel et al, 2011;Moutin et al, 2014;Bourges et al, 2017;Cutrale et al, 2019; Volume 33 May 1, 2022 Cln3 synthesis activates Start | 5 Zamai et al, 2019). 2psN&B can provide absolute protein concentrations, protein complex stoichiometry, and/or dynamic information on diffusion rates and has several advantages over wide-field fluorescence imaging.…”