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. 2019 Oct 23;104(2):305–321.e8. doi: 10.1016/j.neuron.2019.07.029

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© 2019 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

AnkG Allows for the Stable Accumulation of NF186 at the AIS by Inhibiting Its Endocytosis

(A and B) Temporal-color coded maximum projection (A) from live imaging of a DIV4 neuron co-transfected at DIV0 with indicated constructs and incubated with a fluorescently tagged anti-NF antibody (640-antiNF). (B) Number of internalized NF vesicles in indicated compartments.

(C and D) Temporal-color coded maximum projections (C) from live imaging of DIV4 neurons co-transfected at DIV0 with indicated constructs incubated with 640-antiNF. (D) Mean number of 640-antiNF vesicles during the first 5 min post incubation in the proximal axons. One-way ANOVA, ^∗∗∗p < 0.0001, n = 8 neurons, N = 2.

(E–G) DIV4 neurons (E) co-transfected at DIV0 with indicated constructs. (F) Fluorescence intensity ratio of internalized over surface NF186 in the proximal axon. (G) Mean number of internalized NF186 vesicles per 100 μm² of axon. Mann-Whitney test, p = 1.7^∗10^–7 n = 17 axons.

(H–J) DIV1 neurons (H) transfected at DIV0 with indicated constructs. (I) Fluorescence intensity ratio of internalized over surface NF186 in the first 10 μm of proximal neurites. Kruskal-Wallis test, Dunn’s multiple comparison test. ns, p > 0.99, ^∗∗∗p < 0.0001, n = 33 ROIs. (J) Number of internalized NF186 vesicles per 10 μm. Unpaired t test, ^∗∗∗p < 0.0001, n = 14 neurons, N = 2.

(K) Model of the molecular pathways involved in AIS formation. See text for details.

In (A), (C), and (D), scale bars are 10 μm; in (E), scale bars are 10 and 2 μm in the zooms; and in (H), scale bars are 10 and 5 μm in the zooms. See also Figure S7.