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. 2019 Dec 12;8:e50712. doi: 10.7554/eLife.50712

Figure 1. InSyn1 localization to the iPSD is DGC dependent.

(A) Depletion of Gephyrin or αDG by CRISPR in neurons. Cas9 knock-in hippocampal neurons were transduced with AAV:Cre/(control)gRNA [control], AAV:Cre/(Gphn)gRNA [gephyrin] or AAV:Cre/(Dag1)gRNA [αDG] at DIV1 and stained with gephyrin or αDG at DIV13 (left panel). GFP fluorescence of the Cas9-2A-GFP (right panel). Graphs to the right show the normalized puncta density. Gphn vs control (two-tailed t-test, gephyrin n = 19, control n = 17, p<0.0001), Dag1 vs control (two-tailed t-test, αDG n = 16, control n = 20, p<0.0001). (B, C) InSyn1-HA localization after αDG or gephyrin CRISPR depletion. Neurons were depleted of αDG or gephyrin, followed by AAV:InSyn1-HA transduction 3 days before fixation. Exogenously expressed InSyn1-HA is shown in red. Endogenous gephyrin or αDG are shown in green. Bar graph showing the distribution index of InSyn1-HA as arbitrary units. Control (n = 36), Gphn (n = 36), Dag1 (n = 36). One-way ANOVA followed by Tukey's multiple comparisons test, F (2, 105)=25.49, ***p<0.001. Scale bars, 20 µm. (D). HITI labeling of endogenous InSyn1 with smFP-HA in Cas9 KI neurons. InSyn1 is shown in red and αDG is shown in green. Of note, InSyn1:smFP showed clear puncta staining colocalized with αDG. (E) InSyn1 puncta-positive cells were quantified in either Control, Gphn, or Dag1 depleted neurons (n = 3).

Figure 1.

Figure 1—figure supplement 1. Schematic illustration of HITI labeling.

Figure 1—figure supplement 1.

To epitope tag the C-terminus of InSyn1, a guide targeting the end of the coding region was chosen. After CRISPR-dependent cutting, smFP-HA that contains 12 HA epitopes was directly inserted in-frame to label endogenous InSyn1 in neurons.