Fig. 1: Itsn1 forms condensates containing presynaptic proteins and structures.
From: Intersectin and endophilin condensates prime synaptic vesicles for release site replenishment
a, The protein domain structure of Itsn1. b, Two HEK293T cells expressing GFP–Itsn1 FL. Scale bar, 10 µm. c, Example live-HEK293T cell images showing GFP–Itsn1 FL condensates undergoing a fusion event. Times after the initiation of image acquisitions are indicated. Scale bar, 2 µm. d, Example live-cell fluorescence micrographs showing GFP–Itsn1 signals over 8 s with the addition of 4% 1,6-HD after 2 s (green line). Scale bar, 10 µm. e, Fluorescence recovery of GFP–Itsn1 signals when signals within the condensate were photobleached (green line) with a diameter of 800 nm. Each frame represents 0.6 s. Scale bar, 2 µm. f, Plot showing FRAP with a bleaching spot of the indicated diameters in HEK293T cells expressing GFP–Itsn1. g, Plot showing time constant Tau of fluorescence recovery in f. Bars are the mean; error bars are s.e.m. Student’s t-test. **P = 0.0013. h, Top, HEK293 cells co-expressing mCherry–Syn1, GFP–Itsn1 FL and synaptophysin-emiRFP (red fluorescent protein) 670 (Syph). Bottom, cells in top after 3% 1,6-HD treatment. Scale bar, 10 µm. i, Top, HEK293 cells co-expressing BFP–Syn1, mCerulean–EndoA1 and HaloTag–JF549-Itsn1 FL. Bottom, cells after 3% 1,6-HD treatment. Scale bars, 10 µm. j, Top, HEK293 cells co-expressing BFP–Syn1, mCerulean–EndoA1, HALO (JF549)–Itsn1 FL and Syph–emiRFP670. Each individual channel is separated and then merged. Bottom, cells in top after 3% 1,6-HD treatment. Scale bars, 10 µm. k–m, HEK293 cells co-expressing mutant mCherry–Itsn1 A–E (Itsn1 W949E and Y965E; Itsn1 A–EWEYE) and mCerulean–EndoA1 (k), mCherry–Itsn1 A–EWEYE and BFP–Syn1 (l) and mCherry–Itsn1 A–EWEYE, mCerulean–EndoA1 and BFP–Syn1 (m). Scale bar, 3 µm. See Supplementary Table 1 for additional information. HD, hexanediol.
