Abstract
It is unclear whether the establishment of apical–basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.
Highlights
It is unclear whether the establishment of apical–basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface
To study the physiological role of IRSp53 in epithelial morphogenesis, we examined its expression (Supplementary Fig. 1A) and localization in several epithelial tissues characterized by glandular or tubular structures (Fig. 1a–d)
To determine whether IRSp53 is involved in the establishment of apical–basal polarity and lumen formation, IRSp53 was depleted in Madin Darby canine kidney (MDCK) cells using CRISPR/Cas[9], shRNA24, and Short interfering RNA (siRNA), and in Caco-2 cells using CRISPR/Cas[9]
Summary
It is unclear whether the establishment of apical–basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Polarization requires interactions between the signaling complexes and scaffolds that define cortical domains with membrane-sorting machinery[1] This architectural organization and the morphogenesis processes that underlie it can be reproduced by plating cells on pliable matrigel in a matrigel-containing medium that provides the mechanochemical cues for formation of a polarized hollow sphere (cyst). Of the AMIS is mediated by both microtubules and branched actin filaments, which promote recruitment and anchoring of vesicles[5] Transmembrane proteins, such as podocalyxin (PODXL; classical apical marker, known as GP135). Crumbs[3], are transcytosed from the plasma membrane facing the extracellular matrix (ECM) toward the first cell–cell contact site[6] This occurs via RAB11-RAB8 endo/exosomes trafficking and through a direct anchoring with RAB35 at the AMIS6,7. Coordination between actin cytoskeletal dynamics and membrane trafficking is essential for the initiation of a polarized central lumen
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