Probing self-assembly dynamics by high speed-atomic force microscopy

Cellular life harbours a fascinating variety of complex processes and we are still at the beginning of our understanding of these processes. Atomic-scale reconstructions using crystallography or electron microscopy approaches have unveiled great views on cellular components such as proteins and higher-order proteinaceous assemblies. However, these static techniques do not reveal the dynamics of the studied constructs. Using High Speed-Atomic Force Microscopy (HS-AFM) we are now able to scrutinize the dynamics of molecular processes at the nanometre scale, in real time, in liquid. I will start off with discussing the principles and background of HS-AFM and discuss practicalities such as surface treatment and experimental approach. Next I will dive into the applications. Thereby, I will show how we are using the HS-AFM technique to study the fascinating physics of sub-cellular dynamics and biomimetic assembly processes. This will be illustrated by discussing assembly (and disassembly) of ESCRT-III protein complexes and HS-AFM visualization of the dynamics of supramolecular polymerization of synthetic self-replicators. Furthermore, the formation dynamics of 2D capsid protein lattices of human immunodeficiency virus (HIV) will be discussed, particularly revealing how complex the kinetics of viral self-assembly can be, with multiple assembly pathways and continuously occurring assembly and disassembly events. Finally, studies of nucleus formation and growth of hepatitis B virus (HBV) capsid protein complexes are shown revealing real time binding of capsid proteins and the dynamics of assembly initiation of HBV. Combining the insights from the static atomic-scale reconstructions with the dynamic molecular-scale HS-AFM experiments we are now finally able to provide a comprehensive view