Fig. 5: Synaptic distribution of NTD-deleted receptors.
From: AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions
a Exogenous surface HA-tagged receptors in cultured hippocampal neurons were live-labelled with fluorescently conjugated single-chain Fv-Clasp (ScFv-Clasp) avoiding artificial protein cross-linking. b–c Representative confocal images of full-length (top) and ΔNTD (bottom) GluA1 b and GluA2 (c) (AMPAR – magenta, GFP – green, Scale bars: 1 µm). For n-numbers see legend to panel d. d ΔNTD receptors were slightly de-enriched at dendritic spines (spine/dendrite fluorescence, left, GluA1 full-length: 2.42 ± 2.11–2.63, n = 294 spines/16 cells; GluA1 ΔNTD: 2.18 ± 2.03–2.37, n = 267 spines/16 cells, five preparations; p = 0.0473, two-tailed Mann–Whitney test; right, GluA2 full-length: 2.14 ± 1.90–2.44, n = 200 spines/14 cells; GluA2 ΔNTD: 1.79 ± 1.65-1.93, n = 187 spines/12 cells, four preparations; p < 0.0001, two-tailed Mann–Whitney test). e–h Representative 3D STORM images of full-length (top) and ΔNTD (bottom) GluA1 e and GluA2 f receptors (magenta) co-labelled with presynaptic marker bassoon (green). Widefield (wf) GFP images overlayed in grey (Scale bars: 1 µm). g–h Magnified synaptic clusters within boxed areas in e–f (Scale bars: 100 nm). i ΔNTD GluA1 (areas (µm2), left, GluA1 full-length: 0.183 ± 0.009, GluA1 ΔNTD: 0.325 ± 0.015; p < 0.0001, two-tailed unpaired t test) and ΔNTD GluA2 receptors (areas (µm2), right, GluA2 full-length: 0.220 ± 0.015, GluA2 ΔNTD: 0.274 ± 0.019; p < 0.0193, two-tailed unpaired t test) occupied larger synaptic areas than respective full-length controls upon 3D STORM imaging. j NTD deletion in both GluA1 (densities (Localisations/µm2), left, GluA1 full-length: 4087 ± 248.8, GluA1 ΔNTD: 2648 ± 164.6; p < 0.0001, two-tailed unpaired t test) and GluA2 (densities (Localisations/µm2), right, GluA2 full-length: 3787 ± 276.4, GluA2 ΔNTD: 2590 ± 208.5; p < 0.0001, two-tailed unpaired t test) reduced receptor densities at the synapse. k–l Postsynaptic AMPAR areas correlated with presynaptic bassoon areas (k, slope ± S.E. GluA1 full-length: 0.5175 ± 0.049 (R2 0.5024), GluA1 ΔNTD: 0.6235 ± 0.049 (R2 = 0.6768), p = 0.1268, F test: F (DFn, DFd) = 2.353 (1, 189)) and GluA2 ΔNTD (l, slope ± S.E. GluA2 full-length: 0.7698 ± 0.1056 (R2 0.5202), GluA2 ΔNTD: 0.7155 ± 0.082 (R2 = 0.6435), p = 0.6857, F test: F (DFn, DFd) = 0.1648 (1, 91)). Data shown as median ± lower-upper 95% CI (panel d) or mean ± S.E.M (panels i–j), * indicates p < 0.05, and ***p < 0.001. STORM data (panels e–l) were obtained from three culture preparations for GluA1 (GluA1 full-length: 113 synapses/12 cells, GluA1 ΔNTD: 80 synapses/16 cells) and from four preparations for GluA2 (GluA2 full-length: 51 synapses/nine cells, GluA2 ΔNTD: 44 synapses/10 cells). Source data are provided as a source data file.
