Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy

Abstract

Actin filaments are a major component of the cytoskeleton involved in many basic processes such as cell migration, cell adhesion, cell division and muscle contraction. The most important property of actin is that it forms filaments (F-actin) from globular subunits (G-actin). In the 1960s, Oosawa and co-workers developed a model for the G- to F-actin transition where the rate-limiting step is the formation of a stable actin nucleus, later determined to consist of 2-4 actin monomers. Nuclei are elongated by the addition of monomers to the filament ends. This model is widely accepted, although the molecular details could never be visualised experimentally. Here, we used interferometric scattering microscopy (iSCAT) to monitor nucleation and elongation of actin filaments at the single-molecule level. Building on recent improvements in label-free single molecule sensitivity and mass accuracy, we could reveal the polydispersity of G-actin at concentrations relevant to nucleation and filament growth, showing signatures of small protein oligomers in line with the nuclei sizes discussed above. Using the same approach, we could monitor the nanoscopic arrival of individual subunits dynamically attaching to and detaching from the filament tip during polymerisation. Contrary to the standard model, where actin grows exclusively from monomers, we also found signatures of larger oligomers being added to the filament. Together with our polydispersity measurements, these results point towards a nucleation and growth mechanism for actin filaments based on monomers and small oligomers, rather than exclusively monomers. These results demonstrate the potential of iSCAT for label-free single-molecule imaging and for investigating the mechanisms of mesoscopic dynamics in solution.