Journal: The Journal of Cell Biology

Article Title: Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells

doi: 10.1083/jcb.202210026

Figure Lengend Snippet: Net movement of dextran and EGF endosomes over time. (a) Airyscan confocal images of dextran-AF647 (magenta, left), Rab7-GFP (green, center), and their merge in HeLa cells fixed 2 min (top), 15 min (middle), and 30 min (bottom) after introduction of dextran to the cells. (b) Airyscan confocal images of EGF-AF647 (magenta, left), Rab7-GFP (green, center), and their merge in HeLa cells fixed 2 min (top), 15 min (middle), and 30 min (bottom) after introduction of EGF to the cells. (c) Airyscan confocal images of dextran-AF647 (magenta, left), LAMP1-GFP (green, center), and their merge in HeLa cells fixed 2 min (top), 15 min (middle), and 30 min (bottom) after introduction of dextran to the cells. (d) Airyscan confocal images of EGF-AF647 (magenta, left), LAMP1-GFP (green, center), and their merge in HeLa cells fixed 2 min (top), 15 min (middle), and 30 min (bottom) after introduction of EGF to the cells. (e) Quantification (mean ± SD) of the distance of dextran and EGF vesicles from the membrane in cells fixed 15 min after the introduction of AF647-labeled dextran or EGF. Each dot represents the mean distance of several endosomes from an individual cell ( n = 29 for dextran data, and n = 29 cells for EGF data, from n = 3 independent experiments). (f) Quantification (mean ± SD) of the distance of dextran and EGF vesicles from the membrane in cells fixed 30 min after the introduction of AF647-labeled dextran or EGF. Each dot represents the mean distance of several endosomes from an individual cell ( n = 26 cells for dextran data, and n = 37 cells for EGF data, from n = 3 independent experiments). (g) Plot of the number of Rab7 + dextran vesicles compared to the number of dextran vesicles (left), and plot of number of Rab7 + EGF vesicles compared with the number of EGF vesicles (right) at 2, 15, and 30 min after introduction of AF647-labeled dextran or EGF to HeLa cells. (h) Plot of the number of LAMP1 + dextran vesicles compared with the number of dextran vesicles (left), and plot of the number of LAMP1 + EGF vesicles compared with the number of EGF vesicles (right) at 2, 15, and 30 min after introduction of AF647-labeled dextran or EGF to HeLa cells. Scale bars in a–d: 10 µm; error bars in e–h represent SD.

Article Snippet: The following plasmids were used in this study: (i) mCherry-tubulin was a gift from Mariola Chacon, Technische Universität Dresden, Dresden, Germany; (ii) Gal4T-mCherry was a gift from Thomas Pucadyil, Indian Institute of Science Education and Research Pune, India; (iii) mCherry-DCTN1 was a gift from Kozo Tanaka, Tohoku University, Sendai, Japan; (iv) mCherry-Rab5 was a gift from Gia Voeltz (plasmid 49201; RRID:Addgene_49201; Addgene); (v) EGFP-Rab7A (in the pEGFP-C1 backbone) was a gift from Dr. Neftali Flores-Rodriguez (Sydney Microscopy & Microanalysis, University of Sydney, Sydney, Australia); (vi) LAMP1-mGFP was a gift from Esteban Dell’Angelica (plasmid 34831; RRID:Addgene_34831; Addgene); (vii) pEGFP-p150 was a gift from David Stephens (plasmid 36154; RRID:Addgene_36154; Addgene).

Techniques: Membrane, Labeling