Abstract
A functional genomics approach has revealed that caveolae/raft-mediated endocytosis is subject to regulation by a large number of kinases1. Here we explore the role of some of these kinases in caveolae dynamics. We discover that caveolae operate using principles different from classical membrane trafficking. First, each caveolar coat contains a set number (one ‘quantum’) of caveolin-1 molecules. Second, caveolae are either stored as in stationary multi-caveolar structures at the plasma membrane, or undergo continuous cycles of fission and fusion with the plasma membrane in a small volume beneath the surface, without disassembling the caveolar coat. Third, a switch mechanism shifts caveolae from this localized cycle to long-range cytoplasmic transport. We have identified six kinases that regulate different steps of the caveolar cycle. Our observations reveal new principles in caveolae trafficking and suggest that the dynamic properties of caveolae and their transport competence are regulated by different kinases operating at several levels.
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Acknowledgements
We thank K. Anderson and S. Diez for help with TIR-FM, and M. Drab for Cav1-/- fibroblasts. D. Dorris, R. Günther, I. Baines and the Max Planck Institute for Molecular Cell Biology and Genetics are acknowledged for having made possible the kinome screen of endocytosis. We thank D. Meder, K. Simons, A. Helenius and Y. Kalaidzidis for discussions and critical reading of the manuscript. L.P. would like to thank A. Helenius for support. This work was supported by grants from The Max Planck Society ‘RNAi interference’ initiative and the Bunderministerium für Bildung und Forschung. L.P. is a Marie Curie Fellow.
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Supplementary information
Supplementary Figure Legends
Legends to accompany Supplementary Figures S1-S4 (DOC 38 kb)
Supplementary Figure S1
Determination of caveolar structure size and number of Cav1-GFP molecules. (JPG 485 kb)
Supplementary Figure S2
TIR-FM image of a HeLa cell stably expressing Cav1-mRFP and intensity-time plots of TIR-FM time-lapse sequences (200ms intervals) of caveolar vesicles. (JPG 596 kb)
Supplementary Figure S3
An experiment in which dual colour Epi-FM and TIR-FM is combined with pH quenching (recorded with 300ms intervals). (JPG 269 kb)
Supplementary Figure S4
Imaging of Cav1-GFP in cells. (JPG 249 kb)
Supplementary Methods
Additional methods section of the manuscript. (DOC 53 kb)
Supplementary Video S1
TIR-FM of a cav1-deficient fibroblast strongly over-expressing Cav1-GFP. (AVI 11729 kb)
Supplementary Video S2
TIR-FM of a HeLa cell stably expressing Cav1-GFP. Frame intervals are 500 ms. (MOV 7815 kb)
Supplementary Video S2a
Detailed recording of another HeLa cell expressing Cav1-GFP. (MOV 10681 kb)
Supplementary Video S3
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. (MOV 1370 kb)
Supplementary Video S3a
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. (MOV 1370 kb)
Supplementary Video S3b
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. (MOV 1370 kb)
Supplementary Video S4
Simultaneous dual colour TIR-FM of HeLa cells stably expressing Cav1-mRFP and having bound FITC-ChTxB (methods) at neutral pH. (MOV 20652 kb)
Supplementary Video S4a
Video zoomed in on a docking caveolar vesicle directly after changing the extra-cellular pH to 4.0 (methods). (AVI 11726 kb)
Supplementary Video S4b
Same circumstances as 4a, except that the FITC signal is on the right and the Cav1-mRFP signal on the left. (AVI 15633 kb)
Supplementary Video S4c
Same circumstances as 4a, except that the FITC signal is on the right and the Cav1-mRFP signal on the left. (AVI 11491 kb)
Supplementary Video S4d
Video zoomed in on a docking caveolar vesicle directly after changing the extra-cellular pH to 4.0 (methods). (MOV 5061 kb)
Supplementary Video S5
Combined TIR-FM (green) and Epi-FM (red) sequence of a HeLa cell stably expressing Cav1-GFP. (MOV 6438 kb)
Supplementary Video S5a
Zoomed in on a few caveolar vesicles displaying kiss-and-run interactions. (MOV 4577 kb)
Supplementary Video S5b
The caveolar vesicle located in the right bottom 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. (MOV 2639 kb)
Supplementary Video S6
TIR-FM sequence of a Cav1-GFP expressing HeLa cell silenced for ARAF1. (AVI 3912 kb)
Supplementary Video S7
TIR-FM sequence of a Cav1-GFP expressing HeLa cell silenced for SRC. (AVI 11729 kb)
Supplementary Video S8
TIR-FM sequence of a Cav1-GFP expressing HeLa cell silenced for KIAA0999. (AVI 11729 kb)
Supplementary Video S9
TIR-FM sequence of a Cav1-GFP expressing HeLa cell silenced for DYRK3. (AVI 11729 kb)
Supplementary Video S10
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. (MOV 10429 kb)
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Pelkmans, L., Zerial, M. Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae. Nature 436, 128–133 (2005). https://doi.org/10.1038/nature03866
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DOI: https://doi.org/10.1038/nature03866
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