Single-molecule localization based superresolution microscopy (SMLM) nowadays reaches a resolution sufficient to determine structures of protein assemblies in the cellular context. It is therefore a technique complementary to classical structural techniques such as x-ray crystallography or electron microscopy to investigate, how molecular machines are organized. In this talk, firstly I will introduce a new method for isotropic 3D SMLM and secondly I will report on our progress towards resolving a fundamental multi-protein machinery on the nanometer scale, namely the endocytotic machinery in S. cerevisiae.

Supercritical angle localization microscopy (SALM) is a 3D SMLM technique based on the principle of surface-generated fluorescence. This near-field fluorescence is strongly dependent on the distance of fluorophores from the coverslip and can therefore be used to estimate their axial positions. We established a robust and simple implementation of supercritical angle fluorescence detection for single-molecule localization microscopy, calibrated it using fluorescent bead samples, validated the method with DNA origami tetrahedra, and present proof-of-principle data on biological samples.

Endocytosis is a highly intricate cellular process, which involves the ordered recruitment of around 60 proteins. In yeast endocytosis, polymerizing actin provides the force to invaginate the membrane with a high efficiency and remarkable temporal and spatial regularity. We directly visualize the structural relation between polymerized actin and actin interacting proteins to ultimately understand how actin polymerization is regulated in situ. We discovered that several actin nucleation promoting factors and coat proteins show a striking ring-shaped organization even prior to actin polymerization. This lateral pre-patterning of the endocytic site provides an elegant explanation for an efficient force generation by the actin machinery and force transfer to the membrane.

By visualizing many protein pairs with dual-color superresolution microscopy, we are aiming to acquire a time-resolved localization map of the entire endocytic machinery, which would provide us with a comprehensive structural picture of endocytosis in yeast.


Seminar, June 14, 2017, 12:00. Seminar Room

Hosted by Prof. Stefan Wieser