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...

How do animal cells assemble into tissues and organs? A diverse array of tissue structures and shapes can be formed by organizing groups of cells into different polarized arrangements and by coordinating their polarity in space and time. Conserved design principles underlying this diversity are emerging from studies of model organisms and tissues. We discuss how conserved polarity complexes, signalling networks, transcription factors, membrane-trafficking pathways, mechanisms for forming lumens in tubes and other hollow structures, and transitions between different types of polarity, such as between epithelial and mesenchymal cells, are used in similar and iterative manners to build all tissues.The defining feature of metazoa is that their cells are organized into multicellular tissues and organs. Although almost every eukaryotic cell is spatially asymmetric or polarized, polarity must be coordinated in space and time for individual cells to form a tissue 1 . Cell polarity involves the asymmetric organization of most of the physical aspects of the cell, including the cell surface, intracellular organelles and the cytoskeleton 2,3 . Analysis of the polarization of unicellular eukaryotes, such as yeast, has yielded enormous insights into the mechanisms that underlie the polarity of individual cells 3 . Formation of a tissue, however, requires an ensemble cast; the emergent properties of the tissue result from the combined roles of the individual cells that are involved. Accordingly, several biological processes, including cell division, cell death, shape changes, cell migration and differentiation, must be coordinated with the polarity requirements of a tissue to form an organ 4 .Evolutionarily, epithelia are the most archetypal polarized tissues in metazoa, with ~60% of mammalian cell types being of epithelial or epithelial-derived origin 5 . Accordingly, the best studied polarized tissue is the simple epithelium of the mammalian intestine and kidney, the cells of which are columnar in shape (that is, they are taller than they are wide). The apical surfaces of these cells provide the luminal interface and are specialized to regulate the exchange of materials, such as nutrients from the intestine. The lateral surfaces of these cells contact adjacent cells and have specialized junctions and cell-cell adhesion structures 3,6 (FIG. 1a). The basal surfaces of these cells contact the underlying basement membrane, extracellular matrix (ECM) and, ultimately, underlying blood vessels. The basal and lateral surfaces are fairly similar in composition and organization and are often referred to together as the basolateral surface. The apical and basolateral surfaces, however, have very different NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript compositions. In vertebrates, tight junctions (TJs) are found at the apical-most portion of the lateral surfaces, where the TJs form barriers both between the apical and basolateral surfaces and between adjacent cells, limiting paracellular permeability...

Summary 1.There is continuing debate about the impact of agricultural practices on farmland wildlife. In particular, it has been postulated that a general decline in insect abundance linked with intensification of agriculture may have contributed to farmland bird decline. While some autecological studies have supported this hypothesis, larger-scale and longterm studies are needed. 2. Suction traps mounted on 12·2-m towers (Rothamsted-type) have been sampling aerial insects for nearly 40 years throughout the UK. Their catches are correlated over large spatial scales. We analysed insect catch data from a single suction trap run for 27 years in a rural location in Scotland, and showed that insect numbers have changed significantly over time, although non-linearly. The multivariate data set (numbers from the 12 common arthropod groups) was summarized using principal components analysis (PCA) to extract three components explaining 62% of the variation. 3. We also used PCA to describe agricultural change, using published agricultural data for eight measures of farming in Scotland. Arthropod abundance and principal component (PC) scores were significantly related to the agricultural PC scores as well to summary climatic measures. 4. Using Scottish data from the British Trust for Ornithology Common Birds Census, we extracted three PC to describe the time-dependent average densities of 15 common farmland birds in Scotland. Measures of bird density were significantly related to insect abundance and PC scores and, independently, to measures of agriculture and climate. 5. These data from a broad suite of species provide support for linked temporal change between farmland birds, invertebrate numbers and agricultural practice in Scotland. Although entirely correlative, the results are consistent with the view that agricultural change has influenced birds through changes in food quality or quantity. The work also shows how large-scale invertebrate sampling, in this case using suction traps, is useful for monitoring farmland biodiversity.

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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.

SUMMARY 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 down-regulate 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.

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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.