Breaking the bottleneck: semi-automated quantification of actin filament dynamics
- 1SUNY Upstate Medical University
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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BibTeX
@misc{nobles2026breaking,
title = {{Breaking the bottleneck: semi-automated quantification of actin filament dynamics}},
author = {A. D. Nobles and D. M. Rutkowski and C. R. Belden and L. M. Haney and A. E. Davenport and D. Vavylonis and J. L. Henty-Ridilla},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.05.764262},
url = {https://www.biorxiv.org/content/10.64898/2026.11.05.764262v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Breaking the bottleneck: semi-automated quantification of actin filament dynamics
AU - A. D. Nobles
AU - D. M. Rutkowski
AU - C. R. Belden
AU - L. M. Haney
AU - A. E. Davenport
AU - D. Vavylonis
AU - J. L. Henty-Ridilla
PY - 2026
DA - 2026/11/05
PB - bioRxiv
DO - 10.64898/2026.11.05.764262
UR - https://www.biorxiv.org/content/10.64898/2026.11.05.764262v1
AB - 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.
ER -