Journal: The Journal of Cell Biology

Article Title: Regulation of exosome secretion by Rab35 and its GTPase-activating proteins TBC1D10A–C

doi: 10.1083/jcb.200911018

Figure Lengend Snippet: Rab35 functions in recruitment of the endosomal vesicle to the plasma membrane. (A) Control (Ctl) or siRNA against Rab35 was delivered into cells by nucleofection, and the cells were imaged by immunofluorescence microscopy to detect PLP-myc. (B and C) Vesicle number and size are displayed in a histogram. Note that vesicle size was unaffected, whereas the number of vesicles increased after Rab35 knockdown ( n = ∼80 cells from three independent experiments). (D) To analyze vesicular movement, PLP-myc was cotransfected with wild-type or mutant EGFP-tagged Rab35, cells were stained with LysoTracker red DND-99, and time-lapse images were acquired at 1 frame every 2 s at 37°C. The motility of LysoTracker-labeled vesicles was slightly reduced when active GTP-locked Rab35 Q67A was expressed ( n = ∼1,800 vesicles from three independent experiments). (E and F) The mean number of LysoTracker green DND-26–labeled vesicles in a field of 12.7 µm × 12.7 µm (unit area) was determined in the evanescent excitation field. Expression of the GTP-locked Rab35 Q67A increased the number of vesicles in the TIRF evanescence field as compared with the GDP-locked Rab35 S22N ( n = 70 cells from three independent experiments; mean ± SD; ***, P < 0.001; Welch’s two-sample t test). (G) The mobility of LysoTracker green DND-26–labeled vesicles was determined by time-lapse TIRF microscopy and analyzed by calculating the temporal correlation coefficient between pairs of images separated by time. The degree of temporal colocalization is inversely related to vesicle motility ( n = ∼35 videos from three independent experiments; mean ± SEM). (H) Representative traces of intracellular Ca 2+ concentration (top) and membrane capacitance (bottom) in response to flash photolysis of caged Ca 2+ (indicated by arrows) in cells expressing wild-type (black trace) and GDP-locked Rab35 S22N (gray trace). Error bars represent SEM. (I) A decrease in capacitance during the exocytotic burst (0–1 s) and an increase in time constant of the slow compartment were observed in cells expressing Rab35 S22N (*, P < 0.05; ***, P < 0.001; Mann-Whitney test; mean ± SEM). Bars: (A) 10 µm; (E) 5 µm.

Article Snippet: Cells were bathed in extracellular solution (140 mM NaCl, 2.8 mM KCl, 4 mM CaCl 2 , 41 mM MgCl 2 , 10 mM Na-Hepes, and 2 mg/ml glucose, pH 7.2; osmolarity ∼310 mosM) and patched using a pipette solution containing the photolysable Ca 2+ chelator NP-EGTA (110 mM Cs-glutamate, 20 mM Cs-Hepes, 2 mM Mg-ATP, 8 mM NaCl, 0.3 mM Na-GTP, 10 mM NP-EGTA, 9 mM CaCl 2 , 400 μM Fura-4F [Invitrogen], 400 μM Furaptra [Invitrogen], pH 7.2; osmolarity, ∼300 mosM).

Techniques: Clinical Proteomics, Membrane, Control, Immunofluorescence, Microscopy, Knockdown, Mutagenesis, Staining, Labeling, Expressing, Concentration Assay, MANN-WHITNEY