Abstract
Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit–Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.
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Acknowledgements
We thank S. Mayor and M. Rao for helpful discussions and sharing unpublished data, M. McNiven for advice, and J.-B. Sibarita and B. Stechmann for assistance with experiments. The following colleagues are acknowledged for providing materials: T. Kirchhausen, M. Bornens, M. McNiven and A. Smith. Our laboratories were supported by: Ligue Nationale contre le Cancer, Association de Recherche Contre le Cancer, Curie Institute (PIC Vectorisation), European Commission (SoftComp), CNRS (ACI Dynamique et réactivité des assemblages biologiques) and the Human Frontier Science Program Organization. W.R. holds a postdoctoral fellowship from the CNRS, and L.B. is supported by a grant from the Direction Générale pour l'Armement (DGA).
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Supplementary Information
The file contains Supplementary Figures 1-14 with Legends, Legends for Supplementary Movies 1-11 and additional references. (PDF 8199 kb)
Supplementary Movie 1
The file contains Supplementary Movie 1 which shows confocal live cell imaging of tubule growth after injection of 20 nM Alexa488-labeled STxB onto energy-depleted HeLa cells at 37°C. (MOV 1382 kb)
Supplementary Movie 2
The file contains Supplementary Movie 2 which shows confocal live cell imaging of tubule growth at 37°C following binding of 20 nM Alexa488-labeled STxB onto energy-depleted HeLa cells at 4°C. (MOV 1702 kb)
Supplementary Movie 3
The file contains Supplementary Movie 3 which shows confocal live cell imaging of tubule growth at 37°C following binding of 20 nM Alexa488-labeled STxB onto energy-depleted HeLa cells at 4°C. (MOV 3982 kb)
Supplementary Movie 4
The file contains Supplementary Movie 4 which shows evanescent field live cell imaging of tubule scission at 37°C after wash out of energy poisons. (MOV 2105 kb)
Supplementary Movie 5
The file contains Supplementary Movie 5 which shows confocal real time imaging experiment showing tubule formation on a GUV composed of DOPC/cholesterol/Gb3 after injection of Cy3-labeled STxB. (MOV 6177 kb)
Supplementary Movie 6
The file contains Supplementary Movie 6 which shows confocal real time imaging showing tubule dynamics at the equatorial plane of a GUV composed of DOPC/cholesterol/Gb3 at steady state. (MOV 668 kb)
Supplementary Movie 7
The file contains Supplementary Movie 7 which shows spinning-disk confocal real time imaging experiment showing tubule dynamics at steady state in 3D-projection at the equatorial plane of a GUV composed of DOPC/cholesterol/Gb3 during incubation with Cy3-labeled STxB. (MOV 1600 kb)
Supplementary Movie 8
The file contains Supplementary Movie 8 which shows confocal real time imaging experiment at the equatorial plane of a GUV composed of DOPC/cholesterol/Gb3 in the absence of STxB. (MOV 2159 kb)
Supplementary Movie 9
The file contains Supplementary Movie 9 which shows confocal real time imaging experiment on a GUV composed of DOPC/cholesterol/Gb3 onto which the Cy3-labeled W34A mutant was injected. (MOV 3433 kb)
Supplementary Movie 10
The file contains Supplementary Movie 10 which shows confocal real time imaging experiment on a GUV composed of DOPC/cholesterol and Gb3 with a single unsaturated C22:1 acyl chain. (MOV 2371 kb)
Supplementary Movie 11
The file contains Supplementary Movie 11 which shows confocal real time imaging experiment on a GUV composed of DOPC/cholesterol and Gb3 with a saturated C22:0 acyl chain. (MOV 1656 kb)
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Römer, W., Berland, L., Chambon, V. et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450, 670–675 (2007). https://doi.org/10.1038/nature05996
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DOI: https://doi.org/10.1038/nature05996
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