Figure 2.
Dyn2-GFP increases PM duration of caveolae but does not influence their mobility. (A) Immunoblots of Dyn2-GFP-Cav1-mCh cells treated with concentrations of doxycycline as indicated. Doxycycline concentration used for further experiments was 1.0 ng/ml. (B) Representative image from TIRF movie of Dyn2-GFP-Cav1-mCh cell. Red arrow highlights structure only positive for Cav1-mCh, yellow arrow highlights structure positive for both Cav1-mCh and Dyn2-GFP, and green arrow highlights structures only positive for Dyn2-GFP. Scale bar, 10 and 2 μm for magnification. (C) Quantification of percentage of caveolae positive for Dyn2-GFP. Data from 10 cells are shown as scatter dot plot, mean ± SEM. (D) Top, TIRF image series of stable caveolae positive for both Cav1-mCh and Dyn2-GFP. Time in seconds as indicated. Scale bar, 2 μm. Bottom, kymograph showing stable association of Cav1-mCh and Dyn2-GFP to the plasma membrane during 300 s in TIRF movie. (E and F) Quantification of the fluorescence intensity in arbitrary units (a.u) of mCh and GFP of caveolae positive for both Cav1-mCh and Dyn2-GFP over time. (E) Fluorescence intensity from 10 stable caveolae over 300 s, mean ± SEM (shaded area). (F) Fluorescence intensity of fissioned caveolae, time point 0 s represent the last frame of caveolae before fission. Mean ± SEM (shaded area) from nine fissioned caveolae). (G) Quantification of Cav1-mCh track duration time, displacement length and mean speed in Cav1-mCh cells (gray) and Dyn2-GFP-Cav1-mCh cells (black). Analysis was performed using Imaris software, and track mean from at least 10 cells per condition is shown ± SEM. (H) Caveolae track duration times and displacement lengths divided in pools of Dyn2-GFP negative (red) or positive (orange). Dyn2-GFP-Cav1-mCh cells were imaged on TIRF over 5 min. Cav1-mCh spots were followed throughout the time series and scored whether they were positive or negative for Dyn2-GFP. Scatter dot plots show all Cav1-mCh tracks from three different cells. Analysis was performed using Imaris software, and data are shown as mean ± SEM. Significance was assessed using t test, *P ≤ 0.05, **P ≤ 0.01. Source data are available for this figure: SourceData F2.

Dyn2-GFP increases PM duration of caveolae but does not influence their mobility. (A) Immunoblots of Dyn2-GFP-Cav1-mCh cells treated with concentrations of doxycycline as indicated. Doxycycline concentration used for further experiments was 1.0 ng/ml. (B) Representative image from TIRF movie of Dyn2-GFP-Cav1-mCh cell. Red arrow highlights structure only positive for Cav1-mCh, yellow arrow highlights structure positive for both Cav1-mCh and Dyn2-GFP, and green arrow highlights structures only positive for Dyn2-GFP. Scale bar, 10 and 2 μm for magnification. (C) Quantification of percentage of caveolae positive for Dyn2-GFP. Data from 10 cells are shown as scatter dot plot, mean ± SEM. (D) Top, TIRF image series of stable caveolae positive for both Cav1-mCh and Dyn2-GFP. Time in seconds as indicated. Scale bar, 2 μm. Bottom, kymograph showing stable association of Cav1-mCh and Dyn2-GFP to the plasma membrane during 300 s in TIRF movie. (E and F) Quantification of the fluorescence intensity in arbitrary units (a.u) of mCh and GFP of caveolae positive for both Cav1-mCh and Dyn2-GFP over time. (E) Fluorescence intensity from 10 stable caveolae over 300 s, mean ± SEM (shaded area). (F) Fluorescence intensity of fissioned caveolae, time point 0 s represent the last frame of caveolae before fission. Mean ± SEM (shaded area) from nine fissioned caveolae). (G) Quantification of Cav1-mCh track duration time, displacement length and mean speed in Cav1-mCh cells (gray) and Dyn2-GFP-Cav1-mCh cells (black). Analysis was performed using Imaris software, and track mean from at least 10 cells per condition is shown ± SEM. (H) Caveolae track duration times and displacement lengths divided in pools of Dyn2-GFP negative (red) or positive (orange). Dyn2-GFP-Cav1-mCh cells were imaged on TIRF over 5 min. Cav1-mCh spots were followed throughout the time series and scored whether they were positive or negative for Dyn2-GFP. Scatter dot plots show all Cav1-mCh tracks from three different cells. Analysis was performed using Imaris software, and data are shown as mean ± SEM. Significance was assessed using t test, *P ≤ 0.05, **P ≤ 0.01. Source data are available for this figure: SourceData F2.

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