Breaking the bottleneck: semi-automated quantification of actin filament dynamics

Actin reconstitution assays visualized by total internal reflection fluorescence (TIRF) microscopy have been crucial for defining mechanisms of filament assembly, disassembly, and higher-order organization. Yet quantitative analysis remains slow, labor-intensive, and subject to investigator bias. We developed SMAUG (Single filament Measurement and Analysis Using a GUI), a semi-automated image analysis interface that extends TSOAX filament segmentation and tracking with user-verified trace analysis of time-lapse TIRF images. SMAUG generates kymographs and quantifies filament nucleation, elongation, pausing, junctions, and filament-associated fluorescence while retaining measurements at the individual filament level. We tested the approach across actin reconstitution assays producing concentration-dependent polymerization, formin-stimulated elongation, capping-induced arrest, Arp2/3 branching, and fascin-mediated bundling. SMAUG reproduced manual measurement while reducing analysis time and revealed where dense networks and bundled filaments require user validation. Together, TSOAX and SMAUG provide a semi-automated approach for quantitative analysis of actin filament dynamics.