Labeling Strategies for Monitoring Cytoskeleton Dynamics in Cultured Epithelial Cells.

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.