Movie 1, TIR-FM of a Cav1-Deficient Fibroblast Strongly Over-Expressing Cav1-GFP

Pelkmans & Zerial: Kinase-regulated quantal assemblies, kiss-and-run and recycling of caveolae

Supplementary movie legends

Movie 1, TIR-FM of a cav1-deficient fibroblast strongly over-expressing Cav1-GFP. Note the smaller subunits with high lateral mobility.

Movie 2, TIR-FM of a HeLa cell stably expressing Cav1-GFP. Frame intervals are 500 ms. 2a, Detailed recording of another HeLa cell expressing Cav1-GFP.

Movie 3, 3a, 3b, TIRF imaging of a 25x25 pixel region of interest of the plasma membrane in which docking of caveolar vesicles occurs. Time between images is 17ms.

Movie 4, Simultaneous dual colour TIR-FM of HeLa cells stably expressing Cav1-mRFP and having bound FITC-ChTxB (methods) at neutral pH. On left half of the images the FITC signal is projected and on the right half the mRFP signal. Note that due to high laser power to detect sufficient FITC signal, the extent of bleaching of structures permanently fixed to the plasma membrane is quite large. Therefore, docking structures appear brighter then fixed structures at later time-points in the sequence. 4a, zoomed in on a docking caveolar vesicle directly after changing the extra-cellular pH to 4.0 (methods). Note that the FITC signal on the right is rapidly quenched after docking of the caveolar vesicle (seen on the right) and before the caveolar vesicle detaches from the plasma membrane, indicating the temporary fusion of the caveolar vesicle with the plasma membrane, leading to exposure of the lumen of the caveolar vesicle to extra-cellular protons. 4b,c and d: similar to 4a, except that for b and c the FITC signal is on the right and the Cav1-mRFP signal on the left.

Movie 5, Combined TIR-FM (green) and Epi-FM (red) sequence of a HeLa cell stably expressing Cav1-GFP. Note that the signal in Epi-FM does not represent the total cellular pool of Cav1-GFP, due to the high NA of our objective. Some vesicles undergo directional motion in Epi-FM, but most vesicles repeatedly appear in TIR-FM and do not travel far below the cell surface in Epi-FM. a, Zoomed in on a few caveolar vesicles displaying kiss-and-run interactions. Note the repeated docking within a small area. b, Note the caveolar vesicle located in the right bottom. In the movie, it will detach and rapidly move in a directional manner diagonally to the upper right corner where it transiently docks on the plasma membrane, before detaching again and interacting with another caveolar vesicle close-by, underneath the plasma membrane.

Movie 6, TIR-FM sequence of a Cav1-GFP expressing HeLa cell silenced for ARAF1. Note the extensive, homogeneous staining of the plasma membrane, which appears to consist of rapidly diffusing smaller Cav1-GFP-positive entities, and a reduced number of discrete spots.

Movie 7, TIR-FM sequence of a Cav1-GFP expressing HeLa cell silenced for SRC. Note the appearance of large Cav1-GFP-positive aggregates and the strongly reduced kiss-and-run dynamics.

Movie 8, TIR-FM sequence of a Cav1-GFP expressing HeLa cell silenced for KIAA0999. While the overall appearance of caveolar structures seems not to deviate from the normal pattern (compare to Fig. 1), the number of kiss-and-run events is strongly reduced.

Movie 9, TIR-FM sequence of a Cav1-GFP expressing HeLa cell silenced for DYRK3. A normal number of caveolar vesicles is visible, in addition to some diffuse staining, similar to ARAF1-silenced cells. The dynamics of the caveolar vesicles is strongly increased.

Movie 10, TIR-FM sequence of a Cav1-GFP expressing HeLa cell treated with 1 µM Okadaic Acid. Note the extensive kiss-and-run dynamics of caveolar structures.

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