A molecular network for de novo generation of the apical surface and lumen

Abstract

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...

Tuba and N-WASP function cooperatively to position the central lumen during epithelial cyst morphogenesis

“…A major criticism of the Caco-2 system has been the use of cholera toxin to swell the lumen. 6 However, in our hands we observe swollen lumen without the addition of agents that may affect trafficking processes and, as a result, this system provides a reliable tool for dissecting protein localization and PAP placement.…”

“…Previous observations suggested that the PAP is laid down during the first cell division and is necessary to direct subsequent apical membrane targeting. 5,6 To test PAP localization we examined cysts at the twocell stage, the initial marking of the PAP being determined by aPKC immunofluorescence. aPKC showed diffuse cortical staining in isolated cells (not shown) but is observed to concentrate at juxtaposed membranes in two-cell control cysts ( Fig.…”

“…Incorrect placement of the PAP can have drastic results as cells are no longer able to coordinate apical membrane insertion in subsequent cell divisions, forming cell aggregates with multiple lumina. 5,6,10,25 Previously, the scaffolding protein Tuba has been shown to be required for lumenogenesis, being necessary for correct spindle orientation via Figure 4. a complex interrelationship exists between tuba and n-waSp at the pap and requires the polyproline region of n-waSp.…”

“…2 Subsequent cell divisions direct apical membrane and protein constituents to this point of initial apical membrane placement. 6 Coordinated luminal positioning enables the initial formation of a single hollow lumen that subsequently expands through polarized fluid secretion to separate apical membranes, such as occurs in the embryonic gastrointestinal tract, 7 or by apoptosis or autophagy of the central cells as is observed in mammary gland development. 8,9 Failure to establish initial luminal positioning causes defective lumenogenesis, often resulting in multiple, morphologically abnormal lumina.…”

“…8,9 Failure to establish initial luminal positioning causes defective lumenogenesis, often resulting in multiple, morphologically abnormal lumina. 5,6 Crucial to lumenal morphogenesis is then the mechanism(s) that mark the site where the PAP will form. Cdc42 signaling is increasingly implicated in this process, 2,10 with downstream consequences that include control of mitotic spindle orientation, 5 which itself influences PAP placement 5 and potentially regulation of cell-cell junctions.…”