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Coupling between clathrin-dependent endocytic budding and F-BAR-dependent tubulation in a cell-free system

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A Corrigendum to this article was published on 01 October 2010

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Abstract

Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped to elucidate the physical and molecular mechanisms involved in membrane transport1,2,3. To gain a better understanding of endocytosis, we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)4,5,6, revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked by GTPγS. Triggering fission by replacing GTPγS with GTP led not only to separation of clathrin-coated buds, but also to vesicle formation by fragmentation of the tubules. These results suggest a functional link between FBP17-dependent membrane tubulation and clathrin-dependent budding. They also show that clathrin spatially directs plasma membrane invaginations that lead to the generation of endocytic vesicles larger than those enclosed by the coat.

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Figure 1: Marked transformation of plasma membrane sheets induced by incubation with brain cytosol.
Figure 2: Localization and axial segregation of dynamin and FBP17 along the tubular invaginations.
Figure 3: Tubular invaginations require spatially coordinated action of clathrin, actin and FBP17.
Figure 4: Occurrence of membrane fission on replacement of GTPγS with GTP.
Figure 5: Schematic representation of the membrane invaginations described in this study and of their fission.

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  • 31 August 2010

    In the version of this letter initially published online, the author affiliations were incorrect. This error has been corrected in both the HTML and PDF versions of the letter.

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Acknowledgements

We thank Aurelien Roux and Yuxin Mao for advice and help; Lijun Liu, Frank Wilson and Christoph Rahner for critical technical assistance. This work was supported in part by the G. Harold and Leila Y. Mathers Charitable Foundation, the W.M. Keck Foundation and the National Institutes of Health grants (NS36251 and DK45735) to P.D.C., and National Institutes of Health grant (GM068518) to X.Z.; P.D.C. and X.Z. are Howard Hughes Medical Institute Investigators.

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M.W. and P.D.C. designed the experiments and wrote the manuscript; M.W. performed experiments. Experimental work was also contributed by B.H. (STORM), J.E.H. (electron microscopy), A.R. (electron microscopy) and M.G. (electron microscopy). B.H. and X.Z. also contributed to discussion and manuscript preparation.

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Correspondence to Pietro De Camilli.

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The authors declare no competing financial interests.

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Wu, M., Huang, B., Graham, M. et al. Coupling between clathrin-dependent endocytic budding and F-BAR-dependent tubulation in a cell-free system. Nat Cell Biol 12, 902–908 (2010). https://doi.org/10.1038/ncb2094

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