Extended Data Fig. 3: Endocytosis of dendritic receptors in the AIS of cultured mouse and rat neurons.
From: Endocytosis in the axon initial segment maintains neuronal polarity
(a, b) Confocal images of mouse neurons fixed and stained at DIV14 for the indicated endogenous proteins, including MAP2 (dendrites) and Tuj1 (all neurites). AIS zoom is shown on the bottom, and brightness is increased for better visibility. (c) AP-2 labelled structure containing TfR from the AIS of the image shown in b (white arrow). Brightness is increased for better visibility. (d) Confocal images of mouse neurons fixed and stained at DIV14 for the indicated endogenous proteins. AIS zoom is shown on the bottom, and brightness is increased for better visibility. (e) Clathrin-coated structure containing TfR in the AIS of the image shown in d (white arrow). Brightness is increased for better visibility. (f) Neurons transfected with TfR-SEP and mScarlet-NavII-III, fixed at DIV10, and stained with anti-neurofascin (NF) antibody (clone A12/18) and anti-mScarlet antibody. Intense staining of both mScarlet-NavII-III and NF defines the AIS. 18/20 transfected neurons (from 3 cultures) showed colocalization with endogenous NF. (g) Scission/endocytic example events from Fig. 2m, as defined as the appearance of a TfR-SEP cluster (i.e. an endocytic vesicle) at pH 5.5 (time 0) and corresponding to a preexisting cluster at pH 7.4 (time −2). Note that these vesicles remain visible at pH 5.5 for 3 frames or more. Contrast is increased for pH 5.5 frames for better visibility. (h) Same analysis as Fig. 2m for neurons transfected with TfR-SEP and incubated with anti-NF antibody (clone A12/18). (i) Frequencies of endocytic events in the AIS (20 recordings from 4 different cultures) and selected dendrites (12 recordings). Event frequency is similar between both AIS visualization methods: for neurons expressing mScarlet-NavII-III, the frequency is 0.080 ± 0.015 events/μm2/min (n = 21) and for neurons stained with anti-NF antibody, the frequency is 0.073 ± 0.012 events/μm2/min (n = 20); P = 0.72 using an unpaired t test. (j) Hippocampal neurons (untransfected and TfR-SEP transfected) with surface bound or internalized transferrin ligand labelled with Alexa647, fixed and labelled with anti-transferrin receptor antibody (anti-TfR, clone H68.4). (k) Fluorescence intensity of surface transferrin ligand labeled with Alexa647 bound at 4 °C (surface) is 2.19 fold higher in TfR-SEP transfected neurons: 1266 ± 12 arbitrary units (a.u). in control (n = 32) versus 2773 ± 341 a.u. in TfR-SEP transfected neurons (n = 30) across 3 cultures. (l) Fluorescence intensity of internalized transferrin ligand labelled with Alexa647 is 2.64 fold higher in TfR-SEP transfected neurons: Control (2238 ± 174 a.u., n = 33) versus TfR-SEP (5911 ± 568 a.u., n = 34) across 3 cultures. P values in i, k, and l were calculated with a two-tailed unpaired t test. All data are shown as mean ± s.e.m. N-values are indicated on bar graphs and represent the number of neuronal regions scored for each condition. Scale bars, 20 µm (a, b top, d top, j), 10 µm (f, h), 5 µm (b bottom, d bottom) and 1 µm (c, e).
