Extended Data Fig. 2: Itsn1 and EndoA1 can assemble into distinct condensates.

a, Three example images of live HEK293 cells expressing mCerulean–Endo A1. Scale bar, 10 µm. b, Left, HEK293 cells co-expressing mCherry–Syn1 and mCerulean–EndoA1 before (top) and after application of 3% 1,6-hexanediol (bottom). Right, plot showing fluorescence intensity of EndoA1 (green) at the indicated line on the images before (solid line) and after (dotted line) the application of 1,6-hexanediol. Scale bar, 5 µm. c, Same as b, but with GFP–Itsn1 FL and BFP–EndoA1. d, Plot showing the fraction of condensates with high circularity (≥0.89 ratio of x and y diameter, where x is the smaller axis) of either Itsn1–EndoA1, Syn1–EndoA1 or Itsn1–EndoA1–Syn1 condensates. The concatemer of Itsn1 was either AB or A–E. Itsn1 AB/A–E are mCherry, Itsn1 FL is fused to HaloTag–JF549, EndoA1 is mCerulean-tagged and Syn1 is BFP-tagged. 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 = 0.0332 was calculated when comparing Itsn1 A–E–EndoA1 to Syn1–EndoA1, ***p = 0.0003 was calculated when comparing Itsn1 FL–Syn1–EndoA1 to Itsn1 AB–EndoA1, and ***p=0.0001 was calculated when comparing Itsn1 FL–Syn1–EndoA1 to Itsn1 A–E–EndoA1 or Itsn1 A–E–EndoA1. e, Plot showing the diameter of condensates containing either Itsn1–EndoA1, Syn1–EndoA1, or Itsn1–EndoA1–Syn1. Fusion tags are the same as d. The concatemer of Itsn1 was either AB or A–E. 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 AB–EndoA1 to either Itsn1 AB–Syn1–EndoA1, Itsn1 A–E–Syn1–EndoA1 or Itsn1 FL–Syn1–EndoA1. ****p < 0.0001 was calculated when comparing Itsn1 A–E–EndoA1 to either Syn1–EndoA1, Itsn1 AB–Syn1–EndoA1, Itsn1 A–E–Syn1–EndoA1 or Itsn1 FL–Syn1–EndoA1. ***p = 0.0008 was calculated when comparing Syn1–EndoA1 to Itsn1 AB–Syn1–EndoA1 and ****p < 0.0001 was calculated when comparing Syn1–EndoA1 to Itsn1 FL–Syn1–EndoA1. ****p < 0.001 was calculated when comparing Itsn1 FL–Syn1–EndoA1 to Itsn1 AB–Syn1–EndoA1 or Itsn1 A–E-Syn1–EndoA1. f, Plot showing the fraction of cells containing either Itsn1–EndoA1, Syn1–EndoA1 or Itsn1–EndoA1–Syn1 condensates. The concatemer of Itsn1 was either AB or A–E. Fusion tags are the same as in d and e. Bars are the mean; error bars are s.e.m. g, Top, HEK293 cells expressing mCerulean–EndoA1, mCherry–Itsn1 AB and Syph–emiRFP670. Bottom, cells in the top after treatment with 3% 1,6-hexanediol. Scale bar, 10 µm. h, Top, HEK293 cells expressing BFP–EndoA1, GFP–Itsn1 FL and Syph (untagged). Bottom, cells in top after treatment with 3% 1,6-hexanediol. Scale bar, 10 µm. i, Same as Fig.1f, but with GFP–Itsn1^WEYE. j, Same as Fig. 1g, but with GFP–Itsn1^WEYE. Bars are the mean; error bars are s.e.m. Two-sided Student’s t test. **p = 0.0069. See Supplementary Table 1 for additional information.

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