Figure 5.
Dyn2 mutants defective in oligomerization or stimulated GTPase activity localizes to and stabilizes caveolae to the PM. (A) Representative image from TIRF movie of K44A Dyn2-GFP-Cav1-mCh cell. Red arrow highlights structure only positive for Cav1-mCh, yellow arrow highlights structure positive for both Cav1-mCh and K44A Dyn2-GFP, and green arrow highlights structures only positive for K44A Dyn2-GFP. Scale bar, 10 or 2 μm for magnification. (B) Quantification of percentage of caveolae positive for K44A Dyn2-GFP. Data from 10 cells are shown as scatter dot plot, mean ± SEM. (C) Caveolae track duration times and displacement lengths divided in pools of K44A Dyn2-GFP–negative (red) or –positive (orange). K44A Dyn2-GFP-Cav1-mCh cells were imaged on TIRF over 5 min. All Cav1-mCh spots were followed and scored whether they were positive or negative for Dyn2-GFP. Graph shows all Cav1-mCh tracks from three different cells. Analysis was performed using Imaris software, and data are presented as scatter dot plot, mean ± SEM. Significance was assessed using t test. (D) Representative image from TIRF movie of I684K Dyn2-GFP-Cav1-mCh cell. Red arrow highlights structure only positive for Cav1-mCh, yellow arrow highlights structure positive for both Cav1-mCh and I684K. Scale bar, 10 or 2 μm for magnification. (E) Quantification of Cav1-mCh track duration time in Dyn2-GFP-Cav1-mCh cells (Dyn2), K44A Dyn2-GFP-Cav1-mCh cells (K44A), and I684K Dyn2-GFP-Cav1-mCh cells (I684K) following 72 h depletion of endogenous Dyn2. Numbers were related to Dyn2-GFP-Cav1-mCh cells. Mean ± SEM from at least 19 cells per condition is shown. Significance was assessed using t test, ***P ≤ 0.001.

Dyn2 mutants defective in oligomerization or stimulated GTPase activity localizes to and stabilizes caveolae to the PM. (A) Representative image from TIRF movie of K44A Dyn2-GFP-Cav1-mCh cell. Red arrow highlights structure only positive for Cav1-mCh, yellow arrow highlights structure positive for both Cav1-mCh and K44A Dyn2-GFP, and green arrow highlights structures only positive for K44A Dyn2-GFP. Scale bar, 10 or 2 μm for magnification. (B) Quantification of percentage of caveolae positive for K44A Dyn2-GFP. Data from 10 cells are shown as scatter dot plot, mean ± SEM. (C) Caveolae track duration times and displacement lengths divided in pools of K44A Dyn2-GFP–negative (red) or –positive (orange). K44A Dyn2-GFP-Cav1-mCh cells were imaged on TIRF over 5 min. All Cav1-mCh spots were followed and scored whether they were positive or negative for Dyn2-GFP. Graph shows all Cav1-mCh tracks from three different cells. Analysis was performed using Imaris software, and data are presented as scatter dot plot, mean ± SEM. Significance was assessed using t test. (D) Representative image from TIRF movie of I684K Dyn2-GFP-Cav1-mCh cell. Red arrow highlights structure only positive for Cav1-mCh, yellow arrow highlights structure positive for both Cav1-mCh and I684K. Scale bar, 10 or 2 μm for magnification. (E) Quantification of Cav1-mCh track duration time in Dyn2-GFP-Cav1-mCh cells (Dyn2), K44A Dyn2-GFP-Cav1-mCh cells (K44A), and I684K Dyn2-GFP-Cav1-mCh cells (I684K) following 72 h depletion of endogenous Dyn2. Numbers were related to Dyn2-GFP-Cav1-mCh cells. Mean ± SEM from at least 19 cells per condition is shown. Significance was assessed using t test, ***P ≤ 0.001.

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