Bulk exocytosis of large intracellular apical precursor organelles establishes apical domain identity during de novo lumen formation

The creation of a microvilli-rich apical luminal domain is a key event in the development of epithelial tissues. De novo lumenogenesis, in which epithelial cells establish apical identity by directing apical cargo to an apical membrane initiation site (AMIS), has been studied extensively. However, the mechanisms that govern the formation of the AMIS and its progression into a luminal precursor remain poorly understood. In this study, we employed super-resolution imaging, light and electron microscopy, and time-resolved proximity proteomics to explore the spatial, temporal, and molecular processes involved in apical lumen initiation in MDCK-II cells. Interestingly, we discovered that, in both 2D and 3D cultures, the formation of the apical cell cortex begins with the fusion of large intracellular apical precursor organelles called vacuolar apical compartments (VACs) at developing cell junctions. Time-resolved proteomics and high-resolution imaging indicate that the exocytosis of VACs and lumen initiation is temporally coordinated with the formation and maturation of the apical junctional complex. Furthermore, we report that loss of the Crumbs complex proteins Pals1 or PatJ perturbs the organisation of the apical cell cortex and impedes VAC exocytosis at the AMIS, resulting in severe defects in lumen morphogenesis. Overall, our findings define a temporally resolved protein network for de novo cell polarization and highlight Pals1/PatJ as key regulators of apical domain initiation. We further propose that VACs represent a previously unrecognized apical transport carrier that plays a crucial role in the rapid establishment of apical domain identity.