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bioRxiv · 10.64898/2026.11.05.764262

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

Nobles, A. D.·Rutkowski, D. M.·Belden, C. R.·Haney, L. M.·Davenport, A. E.·Vavylonis, D.·Henty-Ridilla, J. L.
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Abstract

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.

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Nobles, A. D., Rutkowski, D. M., Belden, C. R., Haney, L. M., Davenport, A. E., Vavylonis, D., Henty-Ridilla, J. L.. 2026-11-06. Breaking the bottleneck: semi-automated quantification of actin filament dynamics. https://doi.org/10.64898/2026.11.05.764262

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