Labeling Strategies for Monitoring Cytoskeleton Dynamics in Cultured Epithelial Cells

ABSTRACT

Collective cell migration relies on coordinated cytoskeletal remodeling, yet the impact of live‐cell actin filament probes on these dynamics remains poorly characterized. Here, we systematically compared the performance and cellular effects of fluorogenic jasplakinolide‐based probes, SiR‐actin and SiR‐XActin, with the genetically encoded Lifeact reporter in epithelial monolayers. Using an injury‐free wound healing assay combined with widefield images, particle image velocimetry, kymographs, and FUCCI‐based cell cycle tracking system, we assessed how probe choice, efflux inhibition, and genetic modification influence actin organization, migration, and proliferation. SiR‐XActin provided robust labeling of actin filaments with minimal perturbation in migration rate, directionality, or cell cycle progression, enabling stable visualization of cytoskeletal dynamics in monolayers. In contrast, SiR‐actin and Lifeact produced either probe‐ or cell line‐specific effects on migration persistence and wound closure, particularly under prolonged imaging. The use of verapamil, a commonly used phenylalkylamine‐derived calcium channel blocker, improved probe retention and migratory persistence without altering proliferation. In contrast, FUCCI‐expressing monolayers showed impaired motility independent of probe type, potentially reflecting cytoskeletal constraints associated with cell‐cycle reporters. Lifeact‐expressing cells exhibited an initial increase in migration rate followed by an incomplete wound closure, consistent with mild actin stabilization. Together, these findings identify SiR‐XActin as a minimally perturbative, high‐fidelity probe for monitoring actin filament dynamics during collective epithelial migration and highlight the importance of evaluating probe–cell compatibility for live cytoskeletal studies.