Fig. 5: Proper SDII folding and build-up of Gephyrin filaments are essential for synaptic cluster formation and LLPS.
From: Gephyrin filaments represent the molecular basis of inhibitory postsynaptic densities
a Representative SDS-PAGE of sedimentation experiment showing the amount of protein present in pellet (P) and supernatant (S) fractions of GephE variants (WT, ∆SDII, D422N, R379D) either without GlyT-LD (ctrl) or with (+GlyR-LD). b Quantification of band intensities in the pellet fraction; n = 3 presented as means ± SD. Data were analyzed by one-way ANOVA (F9,20 = 47.78, p = 1.42·10-11), Tukey’s post hoc test; p-values are indicated. c Representative images of gephyrinfloxed/floxed murine hippocampal neurons infected with moxBFP-Cre and either mScarlet-GephWT or mScarlet-GephΔSDII (pseudo-colored fire LUT, magenta) and immunostained against vesicular GABA transporter (vGAT, green) and GABAAR γ2 subunit (yellow). d Quantification of gephyrin clusters from c per cell area; n = 20 images per condition from four independent cultures. Each data point represents cluster density per acquired image, means ± SD are indicated. Data were analyzed by unpaired, two-tailed Welch’s two-sample t-test (t(19.3) = 13.8, p = 1.92·10-11). e Surface representation of GephEG375D dimer highlighting the positions of SDI (red), SDIII (yellow) and SDIV (blue) of one monomer. The second monomer is shown in gray. Scale bar 50 Å. f Surface representation of GephED422N dimer highlighting the positions of SDI (red), SDII (green) SDIII (yellow) and SDIV (blue) of one monomer. The second monomer is shown in gray. Scale similar to (f). g Close up depiction of the SDII interface highlighting D422 situated in SDII‘ and R379 in close proximity situated in SDII. h Representative images of gephyrinfloxed/floxed murine hippocampal neurons infected with moxBFP-Cre and either mScarlet-GephWT, mScarlet-GephG375D or mScarlet-GephD422N (pseudo-colored fire LUT, magenta) and immunostained against vesicular GABA transporter (vGAT, green) and GABAAR γ2 subunit (yellow). i Quantification of gephyrin clusters from h per cell area; n = 20 images per condition from four independent cultures. Each data point represents cluster density per acquired image; means ± SD are indicated. Data were analyzed by one-way ANOVA (F2,57 = 34.94, p = 1.25·10-10), Tukey’s post hoc test; p-values are indicated. Cartoon bar diagrams illustrate the Geph constructs used in this experiment (green: GephG, gray: GephC, light green: SDII, red: position of amino acid substitution).
