Extended Data Fig. 1: Itsn1 and Syn1 form reversible condensates.
From: Intersectin and endophilin condensates prime synaptic vesicles for release site replenishment
a, A bar graph representing the number of condensates formed by co-expression of Syn1 with either a concatemer of two (Itsn1 AB, purple) or five (Itsn1 A–E, red) Itsn1 Src-homology 3 (SH3) domains, overlaid with the line graph representing the frequency distribution. Inset, representative images of HEK293 cells co-transfected with GFP–Syn1 and mCherry–Itsn1 AB or mCherry–Itsn1 A–E, respectively. Scale bar, 5 µm. b, A bar graph representing the number of Itsn1 A–E condensates with Syn1 (co-expression of mCherry–Itsn1 A–E and GFP–Syn1) at the indicated time after transfection, overlaid with the line graph representing the frequency distribution. Note that the effective concentration increases with time after transfection. c, A bar graph representing the number of Itsn1 FL condensates with Syn1 (co-expression of GFP–Itsn1 FL and mCherry–Syn1) at the indicated time after transfection, overlaid with the line graph representing the frequency distribution. Note that the effective concentration increases with time after transfection. d, Left, plot showing the condensate diameter of Itsn1 FL (red)/A–E (dark red)-Syn1 condensates with or without co-expression of untagged Syph. Itsn1 FL/Syn1 are tagged as in c, Itsn1 A–E/Syn1 are tagged with mCherry and GFP, respectively. Bars are the mean; error bars are s.e.m. Kruskal–Wallis test, with Dunn’s multiple comparisons test. The condensates are analyzed from three independent transfections; ****p < 0.0001 was calculated when comparing Itsn1 FL–Syn1–Syph co-expressing cells to Itsn1 A–E-Syn1–Syph co-expressing cells, and ****p < 0.0001 was calculated when comparing Itsn1 FL–Syn1 co-expressing cells to Itsn1 A–E–Syn1 co-expressing cells. Right, plot showing the number of Itsn1 FL/A–E–Syn1 condensates with or without co-expression of untagged Syph. Bars are the mean; error bars are s.e.m. Kruskal–Wallis test, with Dunn’s multiple comparisons test. The condensates are analyzed from three independent transfections; ****p < 0.0001 was calculated when comparing Itsn1 FL–Syn1–Syph co-expressing cells to Itsn1 A–E-Syn1–Syph co-expressing cells, and ****p < 0.0001 was calculated when comparing Itsn1 FL–Syn1 co-expressing cells to Itsn1 A–E–Syn1 co-expressing cells. e, Top, representative images of HEK293 cells co-expressing GFP–Syn1 and mCherry–Itsn1 AB domains before and after application of 3% 1,6-hexanediol (1,6-HD). Bottom, plots showing fluorescence intensity of Syn1 (blue) and Itsn1 AB (purple) at the indicated line on the images before (solid line) and after (dotted line) the application of 1,6-hexanediol. Scale bar, 5 µm. f, Top, representative images of HEK293 cells co-expressing GFP–Syn1 and mCherry–Itsn1 A–E domains before and after application of 3% 1,6-hexanediol (1,6-HD). Bottom, plots showing fluorescence intensity of Syn1 (blue) and Itsn1 A–E (purple) at the indicated line on the images before (solid line) and after (dotted line) the application of 1,6-hexanediol. Scale bar, 5 µm. g, Top, representative images of HEK293 cells co-expressing GFP–Syn1 and HaloTag–JF549–Itsn1 FL domains before and after application of 3% 1,6-hexanediol (1,6-HD). Bottom, plots showing fluorescence intensity of Syn1 (blue) and Itsn1 FL (purple) at the indicated line on the images before (solid line) and after (dotted line) the application of 1,6-hexanediol. Scale bar, 5 µm. See Supplementary Table 1 for additional information.
