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Early hallmarks include the trafficking and enrichment of polarity molecules to form the apical membrane (AM) or cell\u2013cell junctions, and the apical positioning of the centrosome. However, the dependencies among polarity molecules, AM formation, and centrosome positioning remain poorly understood. When cultured in Matrigel, de novo polarization of a single epithelial cell is often coupled with cytokinesis. During mitotic exit, centrosomes move to the future AM site, raising questions about their role in polarization. We perturbed centrosomes and polarity regulators in Matrigel-cultured cells and manipulated polarity direction using suspension culture to examine the relationships among polarization features. Surprisingly, centrosomal microtubules may not be necessary for centrosome positioning or AM formation, but their absence reduces the efficiency of AM formation. The polarity regulator Par3, rather than AM component trafficking, influences centrosome positioning. In suspension culture, centrosomes migrate in the direction opposite to AM formation. Taken together, our findings define the hierarchical relationships among several polarization features and show that centrosome-based polarity is not universal in epithelial cells, providing new insights into the mechanisms of epithelial polarization.<\/jats:p>","DOI":"10.7554\/elife.101088","type":"journal-article","created":{"date-parts":[[2024,10,10]],"date-time":"2024-10-10T14:25:17Z","timestamp":1728570317000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.101088","source":"Crossref","is-referenced-by-count":0,"title":["Centrosome migration and apical membrane formation during epithelial polarization in MDCK cysts"],"prefix":"10.7554","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-2455-3880","authenticated-orcid":true,"given":"Po-Kai","family":"Wang","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05bxb3784","id-type":"ROR","asserted-by":"publisher"}],"name":"Program in Molecular Medicine, National Yang Ming Chiao Tung University and 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