To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is masterminded by polarity complexes, which are thought to direct downstream events such as polarized membrane traffic, though this interconnection is not well understood. We report that Rab11a regulates apical traffic and lumen formation via the Rab GEF Rabin8, and its target Rab8a. Rab8a/11a act via the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation via the Cdc42 GEF, Tuba. These components assemble at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical exocytosis during lumen formation, revealing a novel interplay of the machineries of vesicular transport and polarization.Most internal epithelial organs consist of a monolayer of polarized epithelial cells surrounding a central lumen. Polarization requires the interaction of the signaling complexes and scaffolds that define cortical domains with the polarized membrane sorting machinery 1 . In yeast, traffic from the trans-Golgi network to the surface is regulated by Ypt32p and Sec4p 2 , homologs of mammalian Rab11 and Rab8, respectively. These are linked by Sec2p (homolog of mammalian Rabin8), a guanine nucleotide exchange factor (GEF) for Sec4p, which is recruited by Ypt32p. Sec2p and Sec4p in turn interact with the exocyst, which docks vesicles to the surface 3 .Definition of cortical domains in metazoa involves a complex of Par3, Par6, atypical PKC (aPKC), and the GTPase Cdc42 4 . This complex is a master regulator of polarity, conventionally depicted upstream of membrane trafficking machinery. How this complex interfaces with membrane transport is poorly understood.Here we show a molecular mechanism for lumen and apical surface formation, linking Rab8a/ 11a, exocyst, annexin2, Cdc42 and its GEF Tuba, and the Par3/aPKC complex. This novel NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 November 8. Published in final edited form as:Nat Cell Biol. 2010 November ; 12(11): 1035-1045. doi:10.1038/ncb2106. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript pathway shows how the membrane traffic and cortical polarity machineries cooperate to generate the apical surface and lumen. RESULTS Apical polarization during lumen formationUpon plating into 3D culture, individual MDCK cells proliferate and assemble into cyst structures -a polarized spherical monolayer surrounding a central lumen. Lumenogenesis requires the apical membrane determinant gp135/podocalyxin 5 (PCX in figures). Initially, MDCK aggregates have podocalyxin at the ECM-contacting surface (Fig. 1a, 12 h; Fig. S1a), before polarity inversion occurs, with β-catenin and Na/K-ATPase at cell-cell junctions and podocalyxin now at the lumen (Fig. 1a, 24-48 h, arrows; Fig. S1d) 6,7 . Early lumens occur at a site previously termed the "Pre-Apical Patch" (PAP), where opposing plasma membranes are separated, but the po...

PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signaling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein b-Arrestin1. Because b-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42-dependent morphogenic processes through a b-Arrestin1-ARHGAP21 complex. Here, we show that PTEN knockdown (KD) impairs b-Arrestin1 membrane localization, b-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN deficiency were phenocopied by b-Arrestin1 KD or inhibition of b-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of b-Arrestin1, ARHGAP21 and Cdc42.

After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related to advanced stages of tumour progression and invasiveness. But the key roles of these proteins in crosstalk with the Hippo and liver kinase B1 (LKB1)-AMPK pathways and in epithelial function and proliferation indicate that they may also be associated with the early stages of tumorigenesis. For example, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.