Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

Abstract

Although lumen generation has been extensively studied through so-called cyst-formation assays in Madin-Darby canine kidney (MDCK) cells, an underlying mechanism that leads to the initial appearance of a solitary lumen remains elusive. Lumen formation is thought to take place at early stages in aggregates containing only a few cells. Evolutionarily conserved polarity protein complexes, namely the Crumbs, Par, and Scribble complexes, establish apicobasal polarity in epithelial cells, and interference with their function impairs the regulated formation of solitary epithelial lumina. Here, we demonstrate that MDCK cells form solitary lumina during their first cell division. Before mitosis, Crumbs3a becomes internalized and concentrated in Rab11-positive recycling endosomes. These compartments become partitioned in both daughter cells and are delivered to the site of cytokinesis, thus forming the first apical membrane, which will eventually form a lumen. Endosome trafficking in this context appears to depend on the mitotic spindle apparatus and midzone microtubules. Furthermore, we show that this early lumen formation is regulated by the apical polarity complexes because Crumbs3 assists in the recruitment of aPKC to the forming apical membrane and interference with their function can lead to the formation of a no-lumen or multiple-lumen phenotype at the two-cell stage.

ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture

“…This is consistent with the placement of tight junctions at apicobasal interfaces in mature epithelial monolayers (Bryant et al, 2010) as well as with the association of many polarity proteins with the apical junctional complex and barrier development (Schluter et al, 2009;Shin et al, 2005;Straight et al, 2004). Tight junction proteins might, therefore, contribute to lumen formation.…”

P114RhoGEF governs cell motility and lumen formation during tubulogenesis via ROCK-myosin II pathway

“…Interference at any step resulted in a striking phenotype of multiple lumens, which are discontinuous and scattered throughout the length of the tubule, rather than a single lumen that is continuous with the central lumen of the cyst. The mechanism of lumen formation in 3D MDCK cysts has been extensively studied (Bañón-Rodríguez et al, 2014;Bryant et al, 2010;Datta et al, 2011;Gálvez-Santisteban et al, 2012;Jaffe et al, 2008;Rodriguez-Fraticelli and Martin-Belmonte, 2014;Roland et al, 2011;Schluter et al, 2009;Yu et al, 2008). There are similarities, at least superficially, between lumen formation in cysts and tubules.…”

“…Correctly oriented cell division is required for formation of a single lumen during cystogenesis of several epithelial cell lines (Jaffe et al, 2008;Schluter et al, 2009). To test whether ROCK affects cell orientation, we measured the angle of cell division relative to the long axis of the tube during tubulogenesis.…”

Phosphoinositides in Cell Architecture

“…Last, a role for Cdc42 activity in mitotic spindle positioning and lumen formation has recently been described (Jaffe et al 2008;Schluter et al 2009;Rodriguez-Fraticelli et al 2010). Intersectin 2, a Cdc42-specific GEF, was proposed to regulate Cdc42-dependent spindle positioning and lumen formation; however, there are probably other regulators of Cdc42, because RNAi of Intersectin 2 does not recapitulate the strong apical membrane biogenesis defects and accumulation of vacuoles seen when total Cdc42 levels are reduced.…”