Anirban Datta

PTEN-Mediated Apical Segregation of Phosphoinositides Controls Epithelial Morphogenesis through Cdc42

Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. We show that PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. We conclude that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation.

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

To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is regulated by polarity complexes that are hypothesized to direct downstream events, such as polarized membrane traffic, although this interconnection is not well understood. We have found that Rab11a regulates apical traffic and lumen formation through the Rab guanine nucleotide exchange factor (GEF), Rabin8, and its target, Rab8a. Rab8a and Rab11a function through the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation through 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 an interaction between the machineries of vesicular transport and polarization.

Epithelial polarity and tubulogenesis in vitro

The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.

Molecular Regulation of Lumen Morphogenesis

The asymmetric polarization of cells allows specialized functions to be performed at discrete subcellular locales. Spatiotemporal coordination of polarization between groups of cells allowed the evolution of metazoa. For instance, coordinated apical-basal polarization of epithelial and endothelial cells allows transport of nutrients and metabolites across cell barriers and tissue microenvironments. The defining feature of such tissues is the presence of a central, interconnected luminal network. Although tubular networks are present in seemingly different organ systems, such as the kidney, lung, and blood vessels, common underlying principles govern their formation. Recent studies using in vivo and in vitro models of lumen formation have shed new light on the molecular networks regulating this fundamental process. We here discuss progress in understanding common design principles underpinning de novo lumen formation and expansion.

Rab GTPase-Myo5B complexes control membrane recycling and epithelial polarization.

The Rab GTPases are the largest family of proteins regulating membrane traffic. Rab proteins form a nidus for the assembly of multiprotein complexes on distinct vesicle membranes to regulate particular membrane trafficking pathways. Recent investigations have demonstrated that Myosin Vb (Myo5B) is an effector for Rab8a, Rab10, and Rab11a, all of which are implicated in regulating different pathways for recycling of proteins to the plasma membrane. It remains unclear how specific interactions of Myo5B with individual Rab proteins can lead to specificity in the regulation of alternate trafficking pathways. We examined the relative contributions of Rab/Myo5B interactions with specific pathways using Myo5B mutants lacking binding to either Rab11a or Rab8a. Myo5B Q1300L and Y1307C mutations abolished Rab8a association, whereas Myo5B Y1714E and Q1748R mutations uncoupled association with Rab11a. Expression of Myo5B tails containing these mutants demonstrated that Rab11a, but not Rab8a, was required for recycling of transferrin in nonpolarized cells. In contrast, in polarized epithelial cyst cultures, Myo5B was required for apical membrane trafficking and de novo lumen formation, dependent on association with both Rab8a and Rab11a. These data demonstrate that different combinations of Rab GTPase association with Myo5B control distinct membrane trafficking pathways.

The Cdc42 GEF Intersectin 2 controls mitotic spindle orientation to form the lumen during epithelial morphogenesis

Intersectin 2 localizes to centrosomes, where it regulates Cdc42 and helps to orient the apical surface correctly during cyst formation. (See also companion paper from Qin et al., in this issue.)

Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin–Darby canine kidney cells in three‐dimensional collagen gel culture, blockade of β1‐integrin by the AIIB2 antibody or expression of dominant‐negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA–ROCK I–myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells

The formation of epithelial tissues requires both the generation of apical–basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.

A Molecular Switch for the Orientation of Epithelial Cell Polarization

The formation of epithelial tissues containing lumens requires not only the apical-basolateral polarization of cells, but also the coordinated orientation of this polarity such that the apical surfaces of neighboring cells all point toward the central lumen. Defects in extracellular matrix (ECM) signaling lead to inverted polarity so that the apical surfaces face the surrounding ECM. We report a molecular switch mechanism controlling polarity orientation. ECM signals through a β1-integrin/FAK/p190RhoGAP complex to downregulate a RhoA/ROCK/Ezrin pathway at the ECM interface. PKCβII phosphorylates the apical identity-promoting Podocalyxin/NHERF1/Ezrin complex, removing Podocalyxin from the ECM-abutting cell surface and initiating its transcytosis to an apical membrane initiation site for lumen formation. Inhibition of this switch mechanism results in the retention of Podocalyxin at the ECM interface and the development instead of collective front-rear polarization and motility. Thus, ECM-derived signals control the morphogenesis of epithelial tissues by controlling the collective orientation of epithelial polarization.

Polarity proteins PAR6 and aPKC regulate cell death through GSK-3β in 3D epithelial morphogenesis

Epithelial cells are polarized, with an apical surface facing a lumen or outer surface and a basolateral surface facing other cells and extracellular matrix (ECM). Hallmarks of epithelial carcinogenesis include loss of polarity, as well as uncontrolled proliferation and resistance to apoptosis. Are these features controlled by a common molecular mechanism? The partitioning-defective 3 (PAR3)-PAR6-atypical PKC (aPKC) complex is a master regulator that controls polarization in many animal cells. Here we show that PAR6 is involved in apoptosis by regulating aPKC and glycogen synthase kinase 3β (GSK-3β) activity. During epithelial morphogenesis in 3D culture of Madin-Darby canine kidney (MDCK) cells, expression of an N-terminally deleted PAR6 (PAR6ΔN) leads to a significant increase in caspase-dependent cell death by downregulating aPKC activity. Accordingly, inhibition of aPKC in wild-type (WT) MDCK cells with either a cell-permeable PKCζ pseudosubstrate or RNAi promotes apoptosis, which suggests that PAR6 regulates apoptosis via an aPKC-mediated pathway. GSK-3β, a substrate of aPKC, is hyper-activated by expressing PAR6ΔN. GSK-3β inhibitors block PAR6ΔN-induced apoptosis while expression of constitutively active GSK-3β (S9A) promotes apoptosis, which is rescued by ectopic expression of aPKC. We conclude that a PAR6-aPKC-GSK-3β mechanism links cell polarity and apoptosis.