Extended Data Fig. 5: Point mutations block Itsn1 and EndoA1 binding in neurons.

a, Top, schematic showing Itsn1 and EndoA1 domain structures with the Itsn1–EndoA1 binding site denoted between participating domains by a dotted line; bottom, binding prevented by W949E, Y965E mutations in Itsn1. b, Left, western blot showing the reduction of EndoA1 binding to Itsn1^WEYE by comparing EndoA1 pull down in either Itsn1 KO⁺ ITSN1 WT or Itsn1 KO⁺ ITSN1^WEYE lysates; right, quantification of IP enrichment relative to Itsn1 KO⁺ ITSN1 WT. c, Example 2D STED images of different mouse hippocampal presynapses expressing GFP–Itsn1 showing its localization relative to the active zone, marked by Bassoon (cyan) or synaptic vesicles, marked by Syb2 (gray). GFP–Itsn1 was stained by an anti-GFP antibody and a secondary antibody conjugated to Atto647N. Endogenous Bassoon was used as a proxy for the active zone and was stained by an anti-Bassoon antibody and a secondary antibody conjugated to Alexa594. Endogenous Syb2 was stained by an anti-Syb2 antibody and a secondary antibody conjugated to Alexa594. Scale bar, 300 nm. d, Example 2D STED images of different mouse hippocampal presynapses expressing EndoA1–GFP showing its localization relative to the active zone, marked by Bassoon (cyan) or synaptic vesicles, marked by Syb2 (gray). EndoA1–GFP was stained by an anti-GFP antibody and a secondary antibody conjugated to Atto647N. Endogenous Bassoon was used as a proxy for the active zone and was stained by an anti-Bassoon antibody and a secondary antibody conjugated to Alexa594. Endogenous Syb2 was stained by an anti-Syb2 antibody and a secondary antibody conjugated to Alexa594. Scale bar, 300 nm. e, Cumulative plots showing the distribution of GFP–Itsn1 and GFP–EndoA1 puncta by 2D STED relative to the active zone boundary (dotted line), defined by Bassoon signal (blue fill). f, Cumulative plots showing the distribution of GFP–Itsn1 and GFP–EndoA1 puncta by 2D STED relative to synaptic vesicle localization boundary (dotted line), defined by Syb2 signal (gray). g, Plot showing distances between either GFP–Itsn1 or EndoA1–GFP puncta and either the active zone boundary or synaptic vesicle localization boundary. Bars are the mean; error bars are s.e.m. Kruskal–Wallis test, with Dunn’s multiple comparisons test. ****p < 0.0001. P values are shown for comparisons between both GFP–Itsn1 and EndoA1–GFP distances to Syb2 versus distances to Bassoon. See Supplementary Table 1 for additional information.

Source data