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Here, we systematically compared the performance and cellular effects of fluorogenic jasplakinolide\u2010based probes, SiR\u2010actin and SiR\u2010XActin, with the genetically encoded Lifeact reporter in epithelial monolayers. Using an injury\u2010free wound healing assay combined with widefield images, particle image velocimetry, kymographs, and FUCCI\u2010based cell cycle tracking system, we assessed how probe choice, efflux inhibition, and genetic modification influence actin organization, migration, and proliferation. SiR\u2010XActin 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\u2010actin and Lifeact produced either probe\u2010 or cell line\u2010specific effects on migration persistence and wound closure, particularly under prolonged imaging. The use of verapamil, a commonly used phenylalkylamine\u2010derived calcium channel blocker, improved probe retention and migratory persistence without altering proliferation. In contrast, FUCCI\u2010expressing monolayers showed impaired motility independent of probe type, potentially reflecting cytoskeletal constraints associated with cell\u2010cycle reporters. Lifeact\u2010expressing cells exhibited an initial increase in migration rate followed by an incomplete wound closure, consistent with mild actin stabilization. Together, these findings identify SiR\u2010XActin as a minimally perturbative, high\u2010fidelity probe for monitoring actin filament dynamics during collective epithelial migration and highlight the importance of evaluating probe\u2013cell compatibility for live cytoskeletal studies.<\/jats:p>","DOI":"10.1002\/cm.70173","type":"journal-article","created":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T12:12:56Z","timestamp":1786450376000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Labeling Strategies for Monitoring Cytoskeleton Dynamics in Cultured Epithelial Cells"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5338-9812","authenticated-orcid":false,"given":"Victoria","family":"Levario\u2010Diaz","sequence":"first","affiliation":[{"name":"Chair of Cellular Biomechanics University of Bayreuth  Bayreuth Germany"},{"name":"Department of Cellular Biophysics Max Planck Institute for Medical Research  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lakshmi","family":"Nyapathi\u2010Gopinath","sequence":"additional","affiliation":[{"name":"Chair of Cellular Biomechanics University of Bayreuth  Bayreuth Germany"},{"name":"Department of Cellular Biophysics Max Planck Institute for Medical Research  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonah Luka","family":"Voigt","sequence":"additional","affiliation":[{"name":"Chair of Cellular Biomechanics University of Bayreuth  Bayreuth Germany"},{"name":"Department of Cellular Biophysics Max Planck Institute for Medical Research  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharina","family":"Weber","sequence":"additional","affiliation":[{"name":"Department of Cellular Biophysics Max Planck Institute for Medical Research  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marlene","family":"Maager","sequence":"additional","affiliation":[{"name":"Department of Cellular Biophysics Max Planck Institute for Medical Research  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6812-637X","authenticated-orcid":false,"given":"Veselin","family":"Nasufovic","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology Max Planck Institute for Medical Research  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elisabetta Ada","family":"Cavalcanti\u2010Adam","sequence":"additional","affiliation":[{"name":"Chair of Cellular Biomechanics University of Bayreuth  Bayreuth Germany"},{"name":"Department of Cellular Biophysics Max Planck Institute for Medical Research  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,11]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1146\/annurev.pharmtox.39.1.361","article-title":"Biochemical, Cellular, and Pharmacological Aspects of the Multidrug Transporter","volume":"39","author":"Ambudkar S. 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