Extended Data Fig. 6: Molecular dissection and manipulation of AIS endocytic mechanisms.
From: Endocytosis in the axon initial segment maintains neuronal polarity
(a) GFP channel of the DMA-1 cell surface reporter in wild type and LRP protein mutant animals. (b) Quantification of GFP fluorescence from animals described in a. Wild type data is from Fig. 2k. (c) An SL2-GFP fusion at the C-terminus of the LRPL-1 endogenous locus showing LRPL-1 transcription in PVD. (d) Cell-specific expression of LRPL-1-GFP in PVD. Maximum Z projection of a 15 micron stack of the PVD membrane marker (left). Maximum Z projection of 0.65 micron stacks of the outlined regions on the left image (right). (e) Cell-specific LRPL-1-GFP fluorescence in PVD. (f) Endogenous DMA-1-RFP colocalizes with LRPL-1-GFP puncta in the AIS. (g) Cell-specific endogenous labelling of LRP-2-FLPon-GFP in PVD. Zoomed in region (indicated by the white box) is shown on the right. (h) Average LRP-2 fluorescence in neuronal subregions. (i) Model depicting two pathways for DMA-1 endocytosis. (j) Cell-specific endogenous LRP-2-FLPon-GFP localization in the AIS requires AP-2/APA-2 and DAB-1 function but not CAV-1. Average LRP-2-FLPon-GFP fluorescence in the (k) AIS and (l) cell body. (m) LRPL-1-GFP punctate localization in the AIS is dependent upon apa-2, dma-1, and lrp-2. Average LRPL-1-GFP fluorescence in the (n) AIS and the (o) cell body. (p) Cell-specific localization of wild type DMA-1-GFP or DMA-1 lacking its extracellular domain (DMA-1(ΔEC)) in the PVD neuron axon of wild type and lrp-2 loss of function mutant animals. (q) DMA-1-FLPon-GFP polarity index of animals described in p. (r) Cell-specific endogenous DMA-1-FLPon-GFP localization in the axon of dab-1 loss of function mutant animals. (s) DMA-1-FLPon-GFP polarity index of animals described in r. (t) Axonal regions of wild type and lrp-2 loss of function mutant animals cell-specifically expressing HPO-30-GFP. (u) HPO-30-GFP polarity index of animals described in t. (v) PVD axon labelled with a myristoylated GFP membrane marker in animals of the indicated genotype. (w) Axonal branches within the 50 microns distal to the AIS in animals described in v (n = 19 animals/genotype, except lrpl-1 lrp-2 for which n = 18 animals). (x) Chimera localization in induced human neurons fixed and stained at DIV14. (y) Chimera fluorescence in the specified neuronal region. (z) Chimera fluorescence in the axon of cells shown in x. Arrows designate the cropped region. Brightness was increased for better visibility, and all conditions were processed the same. Data are shown as mean ± s.e.m. N-values are indicated on bar graphs and represent the number of animals or cells. P values were calculated as follows: b, e, h, k, l, n, o, and q using a two-way ANOVA with Šidák multiple comparison test; s and u using an unpaired two-tailed t test; y using a two-way ANOVA with Tukey’s multiple comparison test. Scale bars, 20 µm (x), 10 µm (d left, g left), 5 µm (a, c, g right, p, r, t, v, z), 2 µm (j) 1 µm (d right, f, m).
