Fig. 4: R-mAbs can be genetically expressed in neurons as intrabodies.
From: Genetically encoded intrabody probes for labeling and manipulating AMPA-type glutamate receptors
a Schematic of i-mAb approach. b Representative images of live hippocampal neurons transfected with designated i-mAb (cyan) along with mCherry cell fill (red) and PSD95FingR-GFP (yellow). Arrowheads indicate colocalization of i-mAbs with PSD95FingR-GFP. Scale bars are 10 µm, insets are 5 µm. c Fraction of PSD95FingR-GFP puncta, averaged per cell, with detectable GluA1 or GluA2 i-mAb signal (A1 scFv-Halo n = 14 neurons, A2 Fab-Halo n = 8 neurons, A2 scFv-Halo n = 8 neurons, Tukey’s multiple comparisons test, A1 scFv-Halo vs. A2 Fab-Halo *p = 0.0164, A1 scFv-Halo vs. A2 scFv-Halo *p = 0.0110, A2 Fab-Halo vs. A2 scFv-Halo n.s. p = 0.9883). d Quantification of designated i-mAb signal stability, normalized to signal immediately following JF635i washout at t = 0 (A1 scFv-Halo n = 35 dendrite ROIs, A2 Fab-Halo n = 20 dendrite ROIs, A2 scFv-Halo n = 40 dendrite ROIs all from 2 cultures). e Representative mEPSC traces from neurons transfected with plasmids encoding control (antibody heavy-chain only) or designated i-mAb (scale bar is 15 pA/1 s). f mEPSC frequency (left) and amplitude (right) from neurons transfected with A1 scFv-Halo compared to control (control n = 8 neurons, A1 scFv-Halo n = 10 neurons, two-tailed Student’s t-tests: frequency n.s. p = 0.9369; amplitude n.s. p = 0.1044). g mEPSC frequency (left) and amplitude (right) from neurons transfected with control, A2 Fab-Halo, or A2 scFv-Halo i-mAbs (control n = 14 neurons, A2 Fab-Halo n = 20 neurons, A2 scFv-Halo n = 7 neurons, Dunnett’s multiple comparisons test: frequency control vs. A2 Fab-Halo ****p < 0.0001, control vs. A2 scFv-Halo ****p < 0.0001; amplitude control vs. A2 Fab-Halo n.s. p = 0.2139, control vs. A2 scFv-Halo n.s. p = 0.0695). h Image of a hippocampal neuron dendrite that was infected with A1 scFv-Halo AAV and labeled with JF635i (cyan), anti-Homer (yellow), and anti-GluA2 (red). Arrowheads indicate A1 scFv-Halo colocalization with anti-Homer and anti-GluA2. Scale bar is 5 µm. i Plot of synaptic anti-GluA2 intensity in control neurons compared to neurons expressing A1 scFv-Halo AAV (control n = 13 cells, A1 scFv-Halo AAV n = 28 cells, two-tailed Student’s t-test, n.s. p = 0.6835). Data are presented as mean values +/- SEM. n.s. not significant. Source data are provided as a Source Data file.
