Labeling Strategies for Monitoring Cytoskeleton Dynamics in Cultured Epithelial Cells

Area of Science:

• Cell Biology
• Biophysics

Background:

• Collective cell migration is crucial for development and disease.
• Understanding cytoskeletal dynamics, particularly actin filaments, is key.
• The impact of live-cell actin probes on these processes is not fully understood.

Purpose of the Study:

• To systematically compare fluorogenic jasplakinolide-based probes (SiR-actin, SiR-XActin) and the Lifeact reporter.
• To evaluate probe performance and cellular effects in epithelial monolayers during migration.
• To assess the influence of probe choice, efflux inhibition, and genetic modification on actin dynamics, migration, and proliferation.

Main Methods:

• Injury-free wound healing assay.
• Widefield imaging, particle image velocimetry, and kymography.
• FUCCI-based cell cycle tracking and verapamil treatment.

Main Results:

• SiR-XActin demonstrated robust actin labeling with minimal perturbation to migration rate, directionality, and cell cycle.
• SiR-actin and Lifeact showed probe- or cell line-specific effects on migration persistence and wound closure.
• Verapamil enhanced probe retention and migratory persistence without affecting proliferation.
• FUCCI expression impaired cell motility, and Lifeact induced mild actin stabilization.

Conclusions:

• SiR-XActin is a high-fidelity, minimally perturbative probe for live imaging of actin dynamics in collective epithelial migration.
• Probe-cell compatibility is critical for accurate live cytoskeletal studies.
• Careful selection of actin probes is necessary to avoid artifacts in migration and proliferation studies.