TY - JOUR TI - Visualizing the functional architecture of the endocytic machinery AU - Picco, Andrea AU - Mund, Markus AU - Ries, Jonas AU - Nédélec, François AU - Kaksonen, Marko A2 - Pfeffer, Suzanne R VL - 4 PY - 2015 DA - 2015/02/12 SP - e04535 C1 - eLife 2015;4:e04535 DO - 10.7554/eLife.04535 UR - https://doi.org/10.7554/eLife.04535 AB - 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. KW - endocytosis KW - actin KW - live-cell imaging JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -