Extended Data Fig. 10: Dendritically polarized GluA1 receptors are endocytosed in the AIS and targeted to Rab7 positive late endosomes.
From: Endocytosis in the axon initial segment maintains neuronal polarity
(a) Confocal images of mouse neurons fixed and stained at DIV14 for the indicated endogenous proteins. Bottom shows AIS zoom (brightness is increased for better visibility). (b) AP-2 labelled vesicle containing GluA1 in the AIS from the neuron shown in a (arrow designates cropped region). Brightness is increased for better visibility. (c) Confocal images of mouse neurons fixed and stained at DIV14 for the indicated endogenous proteins. Bottom shows AIS zoom. (d) Rab7 labelled vesicle containing GluA1 in the AIS from the neuron shown in c (arrow designates cropped region). (e) Confocal images of 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 proximal axon. (f) Proximal axonal region of neurons described in e (arrows show location of cropped region). (g) Average GluA1 fluorescence in the dendrite or proximal axon. (h) Confocal images of human neurons fixed and stained at DIV26 for the indicated proteins. Bottom shows AIS zoom (brightness is increased for better visibility). (i) AP-2 labelled vesicle containing Flag-GluA1 in the AIS from the neuron shown in h (arrow designates cropped region). Brightness is increased for better visibility. (j) Confocal images of human neurons fixed and stained at DIV26 for the indicated proteins. Bottom shows AIS zoom (brightness is increased for better visibility). (k) Rab7 labelled vesicle containing Flag-GluA1 in the AIS from the neuron shown in j (arrow designates cropped region). Brightness is increased for better visibility and extra-neuronal Rab7 fluorescence signal is from co-cultured glial cells. (l) Confocal images of human neurons pretreated with vehicle control (top, DMSO) or endocytic inhibitor (bottom, Dyngo 4a) for 18 h prior to being fixed and stained at DIV26. Arrows designate the axon. (m) Axonal region of neuron in l (arrows designate cropped region). (n) Average Flag-GluA1 fluorescence in the dendrite or axon. Data are shown as mean ± s.e.m. N-values are indicated on bar graphs and represent the number of cells. P values in g and n were calculated using a two-way ANOVA with Šidák multiple comparison test. Scale bars, 20 µm (a top, c top, e, h top, j top, l), 5 µm (f, m), 2 µm (a bottom, c bottom, d, h bottom, j bottom), 1 µm (b, i, k).
