Fig. 2: Itsn1 forms dynamic condensed structures that are active zone-adjacent and overlap EndoA1.
From: Intersectin and endophilin condensates prime synaptic vesicles for release site replenishment
a, Left, axon co-expressing GFP–Itsn1 and mCherry–Syn1 before the application of 7% 1,6-HD. Scale bar, 2 µm. Right, the same axon, but 30 s after the application of 7% 1,6-HD. b, CV of GFP–Itsn1 and mCherry–Syn1 in a. Bars are the mean; error bars are s.e.m. Two-sided Student’s t-test. *P = 0.0313 and **P = 0.0013. c, Left, axon co-expressing GFP–Itsn1 and mCherry–Syn1 before the application of 7% 1,4-BD. Scale bar, 2 µm. Right, the same axon, but 30 s after the application of 7% 1,6-BD. d, Same as b, CV of GFP–Itsn1 and mCherry–Syn1 in c. Bars are the mean; error bars are s.e.m. Two-sided Student’s t-test. ***P = 0.0001 and ****P < 0.0001. e, Three-color 2D STED representative images of side-view synapses. Each row is a different synapse stained for either Syn1, Itsn1 or EndoA1 along with the synaptic markers PSD-95 and/or Bassoon, with separate channels and merged channels displayed. Scale bar, 200 nm. f, Left, plot showing distances (nm) between either Itsn1, EndoA1 or Syn1 puncta to the Bassoon signal midline. Bars are the mean; error bars are s.e.m. Kruskal–Wallis test, with Dunn’s multiple comparisons test. ****P < 0.0001. Comparisons between Itsn1, EndoA1 and Syn1 to the Bassoon signal midline. Right, plot showing distances between either Itsn1, EndoA1 or Syn1 puncta to the midpoint of the Bassoon signal. Bars are the mean; error bars are s.e.m. g, Cumulative plot showing the distribution of Itsn1, EndoA1 and Syn1 puncta distances from the Bassoon midline. h, Plots showing GFP–Itsn1 fluorescence intensity at synaptic boutons or axonal processes. Traces shown are average values; error bars are s.e.m. i, Plots showing EndoA1–mRFP fluorescence intensity at synaptic boutons or axonal processes. Traces shown are average values; error bars are s.e.m. j, Different blots showing the indicated proteins on purified SV and CCV. Actual protein size marker or size indicator (in kDa) on the left-hand side. Fluorescence intensity in h and i was measured as depicted in Extended Data Fig. 3i and normalized to the baseline. See Supplementary Table 1 and source data for additional information. SV, synaptic vesicles; CCV, clathrin-coated vesicles; BD, butanediol.
