Like nanoscopic machines, protein assemblies carry out a multitude of cellular processes. Understanding their function and mechanics requires knowing their in situ structural organisation and dynamics, which are barely accessible to classical structural biology techniques (EM, NMR, crystallography). Super-resolution microscopy, and specifically single-molecule localisation microscopy (SMLM), is an ideal tool to study these assemblies in their natural cellular environment and to understand their modus operandi.
In our group, we push the limits of super-resolution microscopy by developing optical, biological, and computational methods.
To enable quantitative measurements, we have developed novel reference samples, taking advantage of the well-defined symmetry and stoichiometry of the nuclear pore complex (NPC). These standards allow quantification of the resolution of microscopes, labelling efficiencies, and the precise copy numbers of proteins in complexes.
To achieve the highest 3D resolution, we are developing new analysis tools and the new microscope technologies supercritical angle localisation microscopy and 4Pi-SMLM.
High-throughput super-resolution microscopy and comprehensive analysis software enable the acquisition of large datasets and their interpretation with powerful statistics.
The main biological question that drives technology development in my group is clathrin-mediated endocytosis. We achieved an initial breakthrough by applying our high-throughput super-resolution microscopy to determine the nanoscale distribution of 23 endocytosis proteins in over 100,000 yeast endocytic structures. As the super-resolution images contained timing markers, we could computationally reconstruct the dynamic molecular architecture of a forming endocytic vesicle from this massive dataset. This allowed us to discover how actin generates and transfers the force to pull in a membrane vesicle.
Our research vision is to develop the microscopy technologies that will allow us to visualise the structure and the dynamics of molecular machines in living cells on the nanoscale. This will add key technology to enable the emerging field of in situ structural biology, and, maybe most importantly, make the dimension of time accessible to structural analysis. By visualising the conformational and compositional changes macromolecular complexes undergo during their functional cycle, we will be able to see molecular machines in action and obtain unprecedented insights into the mechanisms of the core machinery of life.
To fulfil this vision, we will further develop our advanced microscopes for maximal 3D resolution and multicolour on native samples, and will develop computational tools to reconstruct dynamic protein assemblies from thousands of snapshot images. We will establish the novel MINFLUX super-resolution technology to directly image dynamic conformational changes of protein assemblies in living cells with nanometre spatial and millisecond temporal resolution. To highlight specific protein complexes in defined functional states inside electron densities, we will build seamless workflows for correlative super-resolution microscopy and electron tomography. We will drive the development of these technologies by investigating the dynamic structural organisation of the endocytic machinery in yeast and mammals.
We will continue to make these technologies available to our biological collaborators and as open source to the community to investigate structure–function relationships of other cellular machines.