Figure 1: Microtubules imaged with highest 3D resolution reveal the hollow volume within the antibody labelled tubule. Scale bars: 1 µm and 100 nm (inset).

Figure 1: Microtubules imaged with highest 3D resolution reveal the hollow volume within the antibody labelled tubule. Scale bars: 1 µm and 100 nm (inset).

The Ries group studies nanoscale multi-protein machineries in their functional cellular context, and elucidates their dynamic structural organisation using tailor-made superresolution microscopy technologies.

Previous and current research

Like nanoscopic machines, protein complexes and assemblies carry out a variety of essential cellular processes. Understanding their function and mechanics requires knowing their in situ structural organisation, which is barely accessible to classical structural biology techniques (EM, NMR, crystallography). Recently developed superresolution microscopy techniques however are ideal tools that allow us to study these assemblies in their natural cellular environment and to understand their modus operandi.

In our group, we push the limits of the technology by developing optical, biological and computational methods for superresolution microscopy:

We are developing novel techniques to achieve highest 3D resolution of single fluorophores using Supercritical Angle Localization Microscopy and correlative superresolution and electron microscopy, and are implementing quantitative superresolution imaging based on counting reference standards. Lattice-light sheet microscopy allows us to dynamically visualise structures in thick samples and organisms.

High-content superresolution microscopy and a computational analysis framework enables the acquisition of large datasets of structures with powerful statistics.

In the past, we introduced nanobodies as versatile superresolution labels and pioneered superresolution microscopy in yeast, where strain libraries with tags and mutations allow system-wide superresolution studies.

We then apply our newly developed technologies to exciting cell biological systems:

In one project, we study the complex and dynamic protein machinery that performs clathrin-mediated endocytosis in yeast. Using automated high-content superresolution imaging and quantitative data analysis, we determined how more than a dozen endocytic proteins are organised at the nanoscale. The architectural principles we discovered allowed us to understand how the endocytic machinery achieves remarkably high regularity and efficiency. Our vision is to reconstruct the time-resolved distributions of all endocytic proteins and integrate this data with mathematical modelling, to understand key aspects of the endocytic mechanism, including how the machinery assembles, how membrane curvature is induced and how vesicle scission is mediated.

In other projects, we are studying the structure of the kinetochore, we are investigating intracellular aggregation of Parkinsons’ alpha-synuclein and we aim at reconstructing the 3D organisation of DNA in a cell nucleus.

Future projects and goals

Our vision is to further extend superresolution microscopy as a tool for structural cell biology in situ. We aim to push its limits on all fronts to establish a technique which combines nanometre 3D resolution with maximum labelling efficiencies, absolute measurements of protein copy numbers, precise multi-colour measurements, high-throughput for large scale statistics and novel data analysis approaches, to address the vast array of exciting biological questions at the nanoscale, which are becoming accessible only now.

Figure 2: Left: dual-colour diffraction limited image of endocytic proteins in yeast. Right: Dynamic 3-color reconstruction of the endocytic machinery showing the membrane (green), actin (red) and an actin nucleation promoting factor (blue).

Figure 2: Left: dual-colour diffraction limited image of endocytic proteins in yeast. Right: Dynamic 3-color reconstruction of the endocytic machinery showing the membrane (green), actin (red) and an actin nucleation promoting factor (blue). Scale bar 100 nm.