Fig 2. Bin1 regulates recycling endosomes in spines and interacts with Arf6.
(A) Single plane SIM images of relative localization of Bin1 and major trafficking markers in the dendritic compartment. Regions in boxes are shown below each image set in high magnification where region of co-localization are highlighted. Scale bar = 5 μm.
(B) Quantification of Bin1 co-localization with major trafficking markers based on SIM images. One-way ANOVA with Bonferroni post-hoc tests * P<0.05, ** P<0.01
(C) Left: Representative flattened confocal images of Rab11 staining in GFP filled control (scr) and Bin1 knockdown (kd) neurons. The outline indicates the GFP-cell fill (Fig S2F). Scale bar = 10 μm.
Right: Quantification of area occupied by and number of Rab11 positive puncta in scr and kd neurons (n = 12 cells). Area occupied: Mann-Whitney test; Puncta number: unpaired t-test *** P<0.001, **** P<0.0001
(D) Single plane SIM images of the relative localization of Bin1 and Arf6 in dendrites and spines. Scale bar = 5 μm.
(E) Quantification of Bin1 co-localization with trafficking markers in SIM images. One-way ANOVA with Bonferroni post-hoc tests ** P<0.01, *** P<0.001
(F) Representative 3D reconstruction of a stack of SIM images showing the relative localization of Bin1 and Arf6 in spines. Inset enlargement; scale bar = 50 nm.
(G) Montage of spine depicted in (F) to illustrate Arf6 and Bin1 contact sites (white arrowheads).
(H) Results of co-immunoprecipitation experiment that demonstrates pulldown of Arf6 with Bin1 immunoprecipitation from rat cortex homogenates.
(I) Flattened confocal image showing proximity-ligation assay (PLA) reveals an interaction of Bin1 and Arf6 in rat cortical neuron cultures, reflected by green PLA puncta. Red outline represents mCherry cell-filled pyramidal neuron. Inset: Zoomed in view of PLA puncta in dendritic spines and shaft. Scale bar = 20 μm
(J) Quantification of PLA puncta normalized to number of cell bodies in the imaging frame (identified by DAPI nuclear staining). Bin1 Ab and Arf6 Ab conditions represent negative controls where each antibody was used singly in the PLA assay. Kruskal-Wallis with Dunn’s post-hoc tests * P<0.05, *** P<0.001