Fig. 4: Antagonizing DMA-1 AIS endocytosis and identifying dendritic receptor post-endocytic targeting to RAB-7 positive late endosomes.
From: Endocytosis in the axon initial segment maintains neuronal polarity
a, Localization of endogenous DMA-1–FLPon–GFP and DMA-1–NF-186–FLPon–GFP chimeras in the C. elegans PVD neuron. Scale bar, 10 µm. b,c, AIS fluorescence (b) and polarity index (c) of chimaeras from C. elegans animals described in a. d, Axonal region of C. elegans animals expressing DMA-1–NF-186 chimaeras. Scale bar, 5 µm. e, Axonal branches in the 50 µm distal to the AIS in C. elegans animals described in d. f, Escape behaviour in C. elegans animals expressing DMA-1-NF-186 chimaeras in response to a harsh touch stimulus. g, Endogenous DMA-1–RFP is concentrated into GFP–FLPon–RAB-7 positive puncta in the AIS of the C. elegans PVD neuron. Scale bar, 1 µm. h, Manders’ overlap coefficient analysis for endogenous DMA-1 and endogenous Rab proteins in the PVD neuron. i, SER-1–GFP localization in the PVD neuron. Scale bar, 10 µm. j,k, SER-1–GFP is concentrated into AP-2-labelled clathrin-coated structures (j) and RAB-7-labelled late endosomes in the AIS (k) of the PVD neuron. Scale bars, 1 µm. l, SER-1–GFP localization in the dendrite of wild-type and apa-2 mutant animals. Scale bar, 2 µm. m, SER-1–GFP polarity index for C. elegans animals described in l. n, A model depicting the steps of polarized receptor endocytic clearance from the AIS. Axonal (red) and dendritic (blue) transmembrane proteins: (1) diffuse laterally into the AIS; (2) are trapped in the AIS by a diffusion barrier; (3) are captured for endocytosis by binding the clathrin-mediated endocytic machinery; (4) are targeted to RAB-7-positive late endosomes; and (5) are degraded through lysosomal pathways. Model created with BioRender.com. Data are mean ±s.e.m. n represents the number of individual C. elegans animals for each condition. b,c,f,h, One-way ANOVA with Dunnett’s test. m, Two-tailed unpaired t-test with Welch’s correction.
