Journal: Molecular Biology of the Cell
Article Title: Rab11 regulates autophagy at dendritic spines in an mTOR- and NMDA-dependent manner
doi: 10.1091/mbc.e23-02-0060
Figure Lengend Snippet: FIGURE 2: mTOR inhibition targets Rab11 to Atg9A-positive reservoirs. (A) Representative Airyscan images of dendrites of neurons that were transfected with a plasmid encoding EGFP (blue) on DIV22 to allow accurate dendritic spine tracing and immunostained the next day for native Rab11 (magenta) and Atg9A, Hook3, or syntaxin 12 (stx12; green). The overlay channel shows Rab11 (magenta), Atg9A, Hook3, or stx12 (green), and the EGFP channel as an outline. The plasmid was expressed overnight. The neurons were incubated with INK128 (300 nM) for 20 min, fixed, and immunostained. Scale bar = 2.5 µm. The rightmost photomicrographs represent individual dendritic spines indicated in
Article Snippet: For Western blot (WB), immunofluorescence (IF), and immunoprecipitation (IP), the following primary antibodies were used: rabbit antiphospho-Akt (Ser473; Cell Signaling Technology, catalogue no. 4060, 1:1000 for WB), mouse anti-Akt (Cell Signaling, catalogue no. 2920, 1:1000 for WB), rabbit anti-Atg9A antibody (Thermo Fisher Scientific, catalogue no. PA5-21043, 1:200 for IF, 1:100 for WB), rabbit anti-caspase 3 antibody (Cell Signaling, catalogue no. 9662; 1:1000 for WB), mouse monoclonal anti-FLAG(R) M2 antibody (Sigma, F1804, 1:200 for IF) rabbit anti-phospho-GluA1 (Ser845; Millipore, catalogue no. 04-1073, 1:500 for WB), mouse anti-GluA1 (Santa Cruz Biotechnology, catalogue no. sc-55509, 1:100 for WB), rabbit anti-Hook3 antibody (Proteintech, catalogue no. 15457-1-AP, 1:100 for IF), rabbit anti-LC3B antibody (Thermo Fisher Scientific, catalogue no. PA1-46286, 1:200 for WB), rabbit anti-LC3B antibody (Sigma Aldrich, catalogue no. L7543, 1:2000 for WB), rabbit antiSQSTM1/p62 polyclonal antibody (Cell Signaling, catalogue no. 9662; 1:1000 for WB), rabbit anti-syntaxin 12 antibody (Proteintech, catalogue no. 14259-1-AP, 1:100 for IF), mouse anti-Rab11 antibody (BD Biosciences, catalogue no. 610656, Clone 47/Rab11, 1:50 for IF), and rabbit anti-Rab11a antibody (Thermo Fisher Scientific, catalogue no. 71-5300, 5 μg for IP), rabbit anti-phospho ribosomal protein S6 (Ser235/236; Cell Signaling Technology, catalogue no. 4858, 1:1000 for WB), mouse antiribosomal protein S6 (Cell Signaling Technology, catalogue no. 2317, 1:1000 for WB), mouse anti-αtubulin (Sigma-Aldrich, catalogue no. T5168, 1:5000 for WB).
Techniques: Inhibition, Transfection, Plasmid Preparation, Incubation
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![Dextran as endosomal cargo marker and EGFR’s interaction with Hook proteins. (a) Spinning disk microscopy images of mCherry-Rab5 (left) and dextran-A647 (right). The signal-to-noise ratio was higher in cells with endocytosed dextran, allowing us to track dextran vesicles with high spatio-temporal resolution and perform dual channel imaging along with single molecules of dynein. (b) Spinning disk microscopy image from a 60-s-long time-lapse video of mCherry-Rab5 (green) and dextran-A647 (magenta) in cells (left) and the corresponding kymograph (right). Yellow arrowheads point to colocalized Rab5 and dextran, indicating dextran vesicles were a proxy for early endosomal compartments. In images acquired within 60 min after a 10-min pulse of dextran, 63 ± 14% of the dextran vesicles were associated with a Rab5 punctae ( n = 1 25 dextran vesicles from n = 1 independent experiment with 17 cells). (c) Immunofluorescence images of EGFR (left, green), Hook1 (middle, magenta), and their merge (right) obtained using Airyscan confocal microscopy. The inset (marked with a white box) is depicted at the bottom of the images. EGFR and <t>Hook3</t> channel insets are depicted as intensity maps and the white arrowheads point to EGFR punctae, some of which colocalize with Hook1. (d) Immunofluorescence images of EGFR (left, green), Hook3 (middle, magenta), and their merge (right) obtained using Airyscan confocal microscopy. The inset (marked with a white box) is depicted at the bottom of the images. EGFR and Hook3 channel insets are depicted as intensity maps and the white arrowheads point to EGFR punctae, some of which colocalize with Hook3. (e) Plot of the probability of co-occurrence of EGFR with Hook1 and Hook3, showing a slightly higher probability of Hook1 being found on EGFR vesicles compared with Hook3. We confirmed that the colocalization of EGFR with Hook1 and Hook3 was not coincidental by calculating the probability of co-occurrence after flipping the EGFR channel horizontally and proceeding with our analysis. For both Hook1 and Hook3, the colocalization probability with EGFR reduced significantly with the flipped image (flipped EGFR with Hook1: 0.3 ± 0.2 [mean ± SD]; with Hook3: 0.2 ± 0.1 [mean ± SD]; both P < 10 −4 two-sample Kolmogorov–Smirnov test), indicating that the colocalization probability calculated from the original image is a true representation. n = ∼25 cells across three independent experiments. (f) Spinning disk microscopy image from a 10-s-long time-lapse video of dextran-A647 in cells treated with 10 µM nocodazole (left) and the corresponding kymograph (right). The kymograph shows abrogation of directed transport, as expected, upon MT depolymerization. (g) Mean squared displacement (MSD) analysis of dextran vesicles tracked in cells treated with 10 µM nocodazole for >30 min. The MSD data of dextran vesicles was fit to <x2> = 4Dt + c , and the intercept c was estimated to 0.0008. The diffusion coefficient D was 0.003 µm 2 /s, indicating that even in the absence of MTs, intracellular crowding likely prevented the dextran vesicles from diffusing away. ( n = 804 dextran vesicles from n = 1 independent experiment with 24 cells.) Error bars represent SEM. (h) Histogram of net movement of dextran (gray) and EGF (brown) endosomes, indicating that EGF-containing endosomes undertook more net minus end–directed movements in these 3-min time-lapse videos. (i) Probability distribution ( Ρ + (τ) ) of the plus end–directed runs for dextran (gray) and EGF (brown) vesicles. The plus end run time for both dextran and EGF were calculated to be 0.6 ± 0.2 s. (j) Quantification of the mean pixel intensity of Hook1 (light gray) and Hook3 (dark gray) in NC and Hook1/Hook3 siRNA cells. siRNA of Hook1 resulted in a reduction of Hook1 by 33.9% and reduction of Hook3 by 37.9% ( n > 100 cells across n = 2 independent experiments). (k) Representative images of EGF in control cells (left, “NC”), and cells with Hook1 siRNA (middle) and Hook3 siRNA (right), fixed 20 min after the addition of fluorescent EGF. Dashed lines indicate cell boundaries. (l) Plots of mean dispersion of EGF vesicles (left) and displacement between the center of mass of EGF vesicles and the cell centroid (right) in NC, Hook1 siRNA, and Hook3 siRNA cells. “n.s.” represents no significant difference and ** represents P < 0.01 ( n > 45 cells across n = 3 independent experiments, Kruskal–Wallis test). In a, b, f, and k, “N” marks the location/direction of the nucleus. Error bars in e, j, and l represent SD.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8859/pmc10798859/pmc10798859__JCB_202210026_FigS4.jpg)
