Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B-dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity.     

Discs Lost, a novel multi-PDZ domain protein, establishes and maintains epithelial polarity

Polarization of epithelial cells depends on a hierarchical process whereby specific membrane-associated proteins become targeted to specialized membrane domains. Here, we describe a novel Drosophila protein, Discs Lost (DLT), that plays a crucial role in the polarization of embryonic epithelia during cellular blastoderm formation. At subsequent stages of development, DLT interacts with the apical determinant Crumbs (CRB) and the laterally localized protein Neurexin IV (NRX IV). Mutations in dlt or double-stranded RNA interference lead to aberrant localization of CRB and NRX IV and cause a concomitant loss of epithelial cell polarity. Hence, DLT is required to establish and maintain cell polarity and participates in different molecular complexes that define apical and lateral membrane domains.     

Polarized sorting and trafficking in epithelial cells

The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.     

Apicobasal polarity in the kidney

The apicobasal polarization of epithelia is critical for many aspects of kidney function. Over the last decade there have been major advances in our understanding of the mechanisms that underlie this polarity. Critical to this understanding has been the identification of protein complexes on the apical and basolateral sides of epithelial cells that act in a mutually antagonistic manner to define these domains. Concomitant with the creation of apical and basolateral domains is the formation of highly specialized cell-cell junctions including adherens junctions and tight junctions. Recent research points to variability in the polarity and junctional complexes amongst different species and between different cell types of the kidney. Defects in apicobasal polarity are prominent in several disorders including acute renal failure and polycystic kidney disease.     

Apical targeting in polarized epithelial cells: There's more afloat than rafts

Most metazoan cells are 'polarized'. A crucial aspect of this polarization is that the plasma membrane is divided into two or more domains with different protein and lipid compositions or example, the apical and basolateral domains of epithelial cells or the axonal and somatodendritic domains of neurons. This polarity is established and maintained by highly specific vesicular membrane transport in the biosynthetic, endocytic and transcytotic pathways. Two important concepts, the 'SNARE' and the 'raft' hypotheses, have been developed that together promise at least a partial understanding of the underlying general mechanisms that ensure the necessary specificity of these pathways.     

Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly

Cellular polarization involves the generation of asymmetry along an intracellular axis. In a multicellular tissue, the asymmetry of individual cells must conform to the overlying architecture of the tissue. However, the mechanisms that couple cellular polarization to tissue morphogenesis are poorly understood. Here, we report that orientation of apical polarity in developing Madin-Darby canine kidney (MDCK) epithelial cysts requires the small GTPase Rac1 and the basement membrane component laminin. Dominant-negative Rac1 alters the supramolecular assembly of endogenous MDCK laminin and causes a striking inversion of apical polarity. Exogenous laminin is recruited to the surface of these cysts and rescues apical polarity. These findings implicate Rac1-mediated laminin assembly in apical pole orientation. By linking apical orientation to generation of the basement membrane, epithelial cells ensure the coordination of polarity with tissue architecture.     

Integrins contribute to the establishment and maintenance of cell polarity in the follicular epithelium of the Drosophila ovary

The generation of epithelial cell polarity is a key process during development. Although the induction and orientation of cell polarity by cell-cell and cell-extracellular matrix (ECM) interactions is well established, the molecular mechanisms by which signals from the ECM control cell polarity in developing epithelial tissues remain poorly understood. Here, we have used the follicular epithelium of the Drosophila ovary to investigate the role that integrins, the main cell-ECM receptors, play in the establishment of apicobasal polarity. Mature follicle cells have an apical side facing the germ line and a basal side in contact with a basement membrane. Our results show that integrins - presumably via interactions with the basement membrane - play a reinforcing role in follicle cell polarization, as they are required to establish and/or maintain follicle cell membrane asymmetry only when contact with the germ line is prevented. We suggest that the primary cue for polarization of the follicular epithelium is contact with the germline cells. In addition, while interfering with apical and lateral polarization cues leads to apoptosis, we show here that inhibition of contact with the basement membrane mediated by integrins does not affect cell survival. Finally, we provide evidence to suggest that integrins are required to orientate epithelial polarity in vivo.     

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.     

The dual role of annexin II in targeting of brush border proteins and in intestinal cell polarity

Functional intestinal epithelium relies on complete polarization of enterocytes marked by the formation of microvilli and the accurate trafficking of glycoproteins to relevant membrane domains. Numerous transport pathways warrant the unique structural identity and protein/lipid composition of the brush border membrane. Annexin II (Ca(2+)-dependent lipid-binding protein) is an important component of one of the apical protein transport machineries, which involves detergent-resistant membranes and the actin cytoskeleton. Here, we investigate in intestinal Caco-2 cells the contribution of annexin II to the sorting and transport of brush border hydrolases and role in intestinal cell polarity. Downregulation of annexin II in Caco-2-A4 cell line results in a severe reduction of the levels of the brush border membrane resident enzyme sucrase isomaltase (SI) as well as structural components such as ezrin. This reduction is accompanied by a redistribution of these proteins to intracellular compartments and a striking morphological transition of Caco-2 cells to rudimentary epithelial cells that are characterized by an almost flat apical membrane with sparse and short microvilli. Concomitant with this alteration is the redistribution of the intermediate filament protein keratin 19 to the intracellular membranes in Caco-2-A4 cells. Interestingly, keratin 19 interacts with annexin II in wild type Caco-2 cells and this interaction occurs exclusively in lipid rafts. Our findings suggest a role for annexin II and K19 in differentiation and polarization of intestinal cells.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

Microtubule organization and function in epithelial cells

Microtubules are essential for many aspects of polarity in multicellular organisms, ranging from the asymmetric distribution of cell-fate determinants in the one-cell embryo to the transient polarity generated in migrating fibroblasts. Epithelial cells exhibit permanent cell polarity characterized by apical and basolateral surface domains of distinct protein and lipid composition that are segregated by tight junctions. They are also endowed with a microtubule network that reflects the asymmetry of their cell surface: microtubule minus-ends face the apical- and microtubule plus-ends the basal domain. Strikingly, the formation of distinct surface domains during epithelial differentiation is accompanied by the re-organization of microtubules from a uniform array focused at the centrosome to the noncentrosomal network that aligns along the apico-basolateral polarity axis. The significance of this coincidence for epithelial morphogenesis and the signaling mechanisms that drive microtubule repolymerization in developing epithelia remain major unresolved questions that we are only beginning to address. Studies in cultured polarized epithelial cells have established that microtubules serve as tracks that facilitate targeted vesicular transport. Novel findings suggest, moreover, that microtubule-based transport promotes protein sorting, and even the generation of transport carriers in the endo- and exocytic pathways.     

Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

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.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

Integrated activity of PDZ protein complexes regulates epithelial polarity

Polarized cells contain numerous membrane domains, but it is unclear how the formation of these domains is coordinated to create a single integrated cell architecture. Genetic screens of Drosophila melanogaster embryos have identified three complexes, each containing one of the PDZ domain proteins--Stardust (Sdt), Bazooka (Baz) and Scribble (Scrib)--that control epithelial polarity and formation of zonula adherens. We find that these complexes can be ordered into a single regulatory hierarchy that is initiated by cell adhesion-dependent recruitment of the Baz complex to the zonula adherens. The Scrib complex represses apical identity along basolateral surfaces by antagonizing Baz-initiated apical polarity. The Sdt-containing Crb complex is recruited apically by the Baz complex to counter antagonistic Scrib activity. Thus, a finely tuned balance between Scrib and Crb complex activity sets the limits of the apical and basolateral membrane domains and positions cell junctions. Our data suggest a model in which the maturation of epithelial cell polarity is driven by integration of the sequential activities of PDZ-based protein complexes.     

A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity

During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.     

Interactions between the crumbs,lethal giant larvae and bazooka pathways in epithelial polarization

Several protein complexes that are involved in epithelial apicobasal polarity have been identified. However, the mechanism by which these complexes interact to form an integrated polarized cell morphology remains unclear. Crumbs (Crb) and Lethal giant larvae (Lgl) are components of distinct complexes that regulate epithelial polarization in Drosophila melanogaster, but may not interact directly as they localize to the apical and basolateral membrane, respectively. Nevertheless, a genetic screen identifies marked functional interactions between crb and lgl. These interactions extend to other genes within the crb (stardust, sdt) and lgl (discs large, dlg; scribble, scrib) pathways. Our findings suggest that the crb and lgl pathways function competitively to define apical and basolateral surfaces. They also suggest that in the absence of lgl pathway activity, the crb pathway is not required to maintain epithelial polarity. Moreover, we show that crb and lgl cooperate in zonula adherens formation early in development. At later stages, epithelial cells in these mutants acquire normal polarity, indicating the presence of compensatory mechanisms. We find that bazooka (baz) functions redundantly with crb/sdt to support apical polarity at mid- to late-embryogenesis. Despite regaining cell polarity, however, epithelial cells in crb and lgl pathway mutants fail to re-establish normal overall tissue architecture, indicating that the timely acquisition of polarized cell structure is essential for normal tissue organization.     

Insane in the apical membrane: Trafficking events mediating apicobasal epithelial polarity during tube morphogenesis

The creation of cellular tubes is one of the most vital developmental processes, resulting in the formation of most organ types. Cells have co‐opted a number of different mechanisms for tube morphogenesis that vary among tissues and organisms; however, generation and maintenance of cell polarity is fundamental for successful lumenogenesis. Polarized membrane transport has emerged as a key driver not only for establishing individual epithelial cell polarity, but also for coordination of epithelial polarization during apical lumen formation and tissue morphogenesis. In recent years, much work has been dedicated to identifying membrane trafficking regulators required for lumenogenesis. In this review we will summarize the findings from the past couple of decades in defining the molecular machinery governing lumenogenesis both in 3D tissue culture models and during organ development in vivo.      

Lipid polarity is maintained in absence of tight junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.     

Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells

Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.     

Junctions as Organizing Centers in Epithelial Cells? A Fly Perspective

Junctions isolate the apical from the basal–lateral surfaces and are required for the polarization of epithelial cells. Genetics of junction formation in Drosophila has identified numerous transmembrane and scaffolding proteins that assemble in membrane-associated complexes. This review focuses on recent findings suggesting that junctions are autonomous organizing centers for the establishment and maintenance of apical-basal polarity in epithelial cells as well as other polarized processes such as the orientation of the division apparatus.