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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cells, Cultured
  • Dendritic Spines / metabolism
  • Dimerization
  • GTPase-Activating Proteins
  • Humans
  • Mice
  • Molecular Sequence Data
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Neurons / chemistry
  • Neurons / metabolism*
  • Protein Binding
  • SAP90-PSD95 Associated Proteins
  • Sequence Alignment
  • Synapses / chemistry
  • Synapses / metabolism
  • Tumor Suppressor Proteins / chemistry
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*


  • DLGAP1 protein, human
  • Dlgap1 protein, mouse
  • GTPase-Activating Proteins
  • Nerve Tissue Proteins
  • SAP90-PSD95 Associated Proteins
  • STARD13 protein, human
  • Tumor Suppressor Proteins