Extended Data Fig. 4: Endocytosis of dendritic receptors in the AIS of human neurons.
From: Endocytosis in the axon initial segment maintains neuronal polarity
(a) Dendritically polarized TfR and axonal L1CAM are polarized to their appropriate domains in induced human neurons that were fixed and stained at DIV26. (b) Induced human neurons have a single ankyrinG-labelled AIS when co-cultured with glial cells. (c, d) Confocal images of human neurons fixed and stained at DIV26 for endocytic proteins. Arrows designate the AIS. (e) AIS of induced human neurons that were fixed and stained at DIV26 for ankyrinG, AP-2, and clathrin heavy chain. (f) Confocal images of human neurons fixed and stained at DIV26 for the indicated endogenous proteins. Bottom images show the AIS (brightness is increased for better visibility). Note that extra-neuronal fluorescence represents endogenous staining in co-cultured glial cells. (g) Zoomed in region from f of an endocytic vesicle in the AIS. Brightness is increased for better visibility. (h) Histogram analysis of axonal branches in the 50 microns distal to the AIS in the indicated animals (n = 15 animals/genotype). (i) Confocal images of cultured mouse neurons pretreated with vehicle control (top, DMSO) or endocytic inhibitor (bottom, Dyngo 4a) for 5 h prior to being fixed and stained at DIV14. Arrows designate the axon. (j) Endogenous TfR fluorescence in the axon from neurons described in i. Arrows designate the cropped region. (k) Average TfR fluorescence in the axon compared to the dendrite. All data are shown as mean ± s.e.m. N-values are indicated on bar graphs and represent the number of individual cells scored for each condition. P values in k were calculated using a two-way ANOVA with Šidák multiple comparison test. Scale bars, 20 µm (a, b, c, d, f top, i), 5 µm (f bottom, j), 2 µm (e), 1 µm (g).
