Visualizing the functional architecture of the endocytic machinery
Abstract
Clathrin-mediated endocytosis is an essential process that forms vesicles from the plasma membrane. Although most of the protein components of the endocytic protein machinery have been thoroughly characterized, their organization at the endocytic site is poorly understood. We developed a fluorescence microscopy method to track the average positions of yeast endocytic proteins in relation to each other with a time precision below 1 s and with a spatial precision of ~10 nm. With these data, integrated with shapes of endocytic membrane intermediates and with superresolution imaging, we could visualize the dynamic architecture of the endocytic machinery. We showed how different coat proteins are distributed within the coat structure and how the assembly dynamics of N-BAR proteins relate to membrane shape changes. Moreover, we found that the region of actin polymerization is located at the base of the endocytic invagination, with the growing ends of filaments pointing toward the plasma membrane.
Article and author information
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Reviewing Editor
- Suzanne R Pfeffer, Stanford University, United States
Version history
- Received: August 28, 2014
- Accepted: February 10, 2015
- Accepted Manuscript published: February 12, 2015 (version 1)
- Version of Record published: March 12, 2015 (version 2)
Copyright
© 2015, Picco et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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