High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein...

The self-assembly of protein complexes is at the core of many fundamental biological processes¹, ranging from the polymerization of cytoskeletal elements, such as microtubules², to viral capsid formation and organelle assembly³. To reach a comprehensive understanding of the underlying mechanisms of self-assembly, high spatial and temporal resolutions must be attained. This is complicated by the need to not interfere with the reaction during the measurement. As self-assemblies are often governed by weak interactions, they are especially difficult to monitor with high-speed atomic force microscopy (HS-AFM) due to the non-negligible tip-sample interaction forces involved in current methods. We have developed a HS-AFM technique, photothermal off-resonance tapping (PORT), which is gentle enough to monitor self-assembly reactions driven by weak interactions. We apply PORT to dissect the self-assembly reaction of SAS-6 proteins, which form a nine-fold radially symmetric ring-containing structure that seeds the formation of the centriole organelle. Our analysis reveals the kinetics of SAS-6 ring formation and demonstrates that distinct biogenesis routes can be followed to assemble a nine-fold symmetrical structure.