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

Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1-occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation.

FIP5 phosphorylation during mitosis regulates apical trafficking and lumenogenesis.

Apical lumen formation is a key step during epithelial morphogenesis. The establishment of the apical lumen is a complex process that involves coordinated changes in plasma membrane composition, endocytic transport, and cytoskeleton organization. These changes are accomplished, at least in part, by the targeting and fusion of Rab11/FIP5-containing apical endosomes with the apical membrane initiation site (AMIS). Although AMIS formation and polarized transport of Rab11/FIP5-containing endosomes are crucial for the formation of a single apical lumen, the spatiotemporal regulation of this process remains poorly understood. Here, we demonstrate that the formation of the midbody during cytokinesis is a symmetry-breaking event that establishes the location of the AMIS. The interaction of FIP5 with SNX18, which is required for the formation of apical endocytic carriers, is inhibited by GSK-3 phosphorylation at FIP5-T276. Importantly, we show that FIP5-T276 phosphorylation occurs specifically during metaphase and anaphase, to ensure the fidelity and timing of FIP5-endosome targeting to the AMIS during apical lumen formation.

Snail destabilizes cell surface Crumbs3a.

During epithelial to mesenchymal transition (EMT), cells modulate expression of proteins resulting in loss of apical-basal polarity. Effectors of this EMT switch target the polarity protein Crumbs3a, a small transmembrane protein that is essential for generation of the apical membrane and tight junctions of mammalian epithelial cells. We previously showed that the Crumbs3 gene is a direct target of transcriptional regulation by Snail, a potent inducer of EMT. However, Snail has also been shown to have multiple non-transcriptional roles, including regulation of cell adhesion, proliferation and survival. Using SNAP-tag labeling, we determined that cell surface Crumbs3a has a half-life of approximately 3 h and that this cell surface half-life is significantly reduced when EMT is induced by Snail. We further observe that Snail induces differential glycosylation of Crumbs3a, including sialylation, suggesting a mechanism by which Crumbs3a may be destabilized. These results indicate that Crumbs3a is a post-translational target of Snail, in addition to being a transcriptional target. We conclude that Snail's ability to post-translationally modify and destabilize Crumbs3a augments the depolarizing process of EMT.

Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis.

The cellular mechanisms that drive growth and remodeling of the early intestinal epithelium are poorly understood. Current dogma suggests that the murine fetal intestinal epithelium is stratified, that villi are formed by an epithelial remodeling process involving the de novo formation of apical surface at secondary lumina, and that radial intercalation of the stratified cells constitutes a major intestinal lengthening mechanism. Here, we investigate cell polarity, cell cycle dynamics and cell shape in the fetal murine intestine between E12.5 and E14.5. We show that, contrary to previous assumptions, this epithelium is pseudostratified. Furthermore, epithelial nuclei exhibit interkinetic nuclear migration, a process wherein nuclei move in concert with the cell cycle, from the basal side (where DNA is synthesized) to the apical surface (where mitosis takes place); such nuclear movements were previously misinterpreted as the radial intercalation of cells. We further demonstrate that growth of epithelial girth between E12.5 and E14.5 is driven by microtubule- and actinomyosin-dependent apicobasal elongation, rather than by progressive epithelial stratification as was previously thought. Finally, we show that the actin-binding protein Shroom3 is crucial for the maintenance of the single-layered pseudostratified epithelium. In mice lacking Shroom3, the epithelium is disorganized and temporarily stratified during villus emergence. These results favor an alternative model of intestinal morphogenesis in which the epithelium remains single layered and apicobasally polarized throughout early intestinal development.

Localization of retinitis pigmentosa 2 to cilia is regulated by Importin beta2.

Ciliopathies represent a newly emerging group of human diseases that share a common etiology resulting from dysfunction of the cilium or centrosome. The gene encoding the retinitis pigmentosa 2 protein (RP2) is mutated in X-linked retinitis pigmentosa. RP2 localizes to the ciliary base and this requires the dual acylation of the N-terminus, but the precise mechanism by which RP2 is trafficked to the cilia is unknown. Here we have characterized an interaction between RP2 and Importin ß2 (transportin-1), a member of the Importin-ß family that regulates nuclear-cytoplasmic shuttling. We demonstrate that Importin ß2 is necessary for localization of RP2 to the primary cilium because ablation of Importin ß2 by shRNA blocks entry both of endogenous and exogenous RP2 to the cilium. Furthermore, we identify two distinct binding sites of RP2, which interact independently with Importin ß2. One binding site is a nuclear localization signal (NLS)-like sequence that is located at the N-terminus of RP2 and the other is an M9-like sequence within the tubulin folding cofactor C (TBCC) domain. Mutation of the NLS-like consensus sequence did not abolish localization of RP2 to cilia, suggesting that the sequence is not essential for RP2 ciliary targeting. Interestingly, we found that several missense mutations that cause human disease fall within the M9-like sequence of RP2 and these mutations block entry of RP2 into the cilium, as well as its interaction with Importin ß2. Together, this work further highlights a role of Importin ß2 in regulation of the entry of RP2 and other proteins into the ciliary compartment.

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.

The retinitis pigmentosa protein RP2 interacts with polycystin 2 and regulates cilia-mediated vertebrate development.

Ciliopathies represent a growing group of human genetic diseases whose etiology lies in defects in ciliogenesis or ciliary function. Given the established entity of renal-retinal ciliopathies, we have been examining the role of cilia-localized proteins mutated in retinitis pigmentosa (RP) in regulating renal ciliogenesis or cilia-dependent signaling cascades. Specifically, this study examines the role of the RP2 gene product with an emphasis on renal and vertebrate development. We demonstrate that in renal epithelia, RP2 localizes to the primary cilium through dual acylation of the amino-terminus. We also show that RP2 forms a calcium-sensitive complex with the autosomal dominant polycystic kidney disease protein polycystin 2. Ablation of RP2 by shRNA promotes swelling of the cilia tip that may be a result of aberrant trafficking of polycystin 2 and other ciliary proteins. Morpholino-mediated repression of RP2 expression in zebrafish results in multiple developmental defects that have been previously associated with ciliary dysfunction, such as hydrocephalus, kidney cysts and situs inversus. Finally, we demonstrate that, in addition to our observed physical interaction between RP2 and polycystin 2, dual morpholino-mediated knockdown of polycystin 2 and RP2 results in enhanced situs inversus, indicating that these two genes also regulate a common developmental process. This work suggests that RP2 may be an important regulator of ciliary function through its association with polycystin 2 and provides evidence of a further link between retinal and renal cilia function.

A novel Crumbs3 isoform regulates cell division and ciliogenesis via importin beta interactions.

The Crumbs family of apical transmembrane proteins regulates apicobasal polarity via protein interactions with a conserved C-terminal sequence, ERLI. However, one of the mammalian Crumbs proteins, Crumbs3 (CRB3) has an alternate splice form with a novel C-terminal sequence ending in CLPI (CRB3-CLPI). We report that CRB3-CLPI localizes to the cilia membrane and a membrane compartment at the mitotic spindle poles. Knockdown of CRB3-CLPI leads to both a loss of cilia and a multinuclear phenotype associated with centrosomal and spindle abnormalities. Using protein purification, we find that CRB3-CLPI interacts with importin beta-1 in a Ran-regulated fashion. Importin beta-1 colocalizes with CRB3-CLPI during mitosis, and a dominant-negative form of importin beta-1 closely phenocopies CRB3-CLPI knockdown. Knockdown of importin beta-1 blocks targeting of CRB3-CLPI to the spindle poles. Our data suggest an expanded role for Crumbs proteins in polarized membrane targeting and cell division via unique interactions with importin proteins.

Protein complexes that control renal epithelial polarity.

Establishment of epithelial apicobasal polarity is crucial for proper kidney development and function. In recent years, there have been important advances in our understanding of the factors that mediate the initiation of apicobasal polarization. Key among these are the polarity complexes that are evolutionarily conserved from simple organisms to humans. Three of these complexes are discussed in this review: the Crumbs complex, the Par complex, and the Scribble complex. The apical Crumbs complex consists of three proteins, Crumbs, PALS1, and PATJ, whereas the apical Par complex consists of Par-3, Par-6, and atypical protein kinase C. The lateral Scribble complex consists of Scribble, discs large, and lethal giant larvae. These complexes modulate kinase and small G protein activity such that the apical and basolateral complexes signal antagonistically, leading to the segregation of the apical and basolateral membranes. The polarity complexes also serve as scaffolds to direct and retain proteins at the apical membrane, the basolateral membrane, or the intervening tight junction. There is plasticity in apicobasal polarity, and this is best seen in the processes of epithelial-to-mesenchymal transition and the converse mesenchymal-to-epithelial transition. These transitions are important in kidney disease as well as kidney development, and modulation of the polarity complexes are critical for these transitions.

Direct interaction of two polarity complexes implicated in epithelial tight junction assembly.

Tight junctions help establish polarity in mammalian epithelia by forming a physical barrier that separates apical and basolateral membranes. Two evolutionarily conserved multi-protein complexes, Crumbs (Crb)-PALS1 (Stardust)-PATJ (DiscsLost) and Cdc42-Par6-Par3-atypical protein kinase C (aPKC), have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. Here we identify a biochemical and functional link between these two complexes that is mediated by Par6 and PALS1 (proteins associated with Lin7). The interaction between Par6 and PALS1 is direct, requires the amino terminus of PALS1 and the PDZ domain of Par6, and is regulated by Cdc42-GTP. The transmembrane protein Crb can recruit wild-type Par6, but not Par6 with a mutated PDZ domain, to the cell surface. Expression of dominant-negative PALS1-associated tight junction protein (PATJ) in MDCK cells results in mis-localization of PALS1, members of the Par3-Par6-aPKC complex and the tight junction marker, ZO-1. Similarly, overexpression of Par6 in MDCK cells inhibits localization of PALS1 to the tight junction. Our data highlight a previously unrecognized link between protein complexes that are essential for epithelial polarity and formation of tight junctions.

PATJ regulates tight junction formation and polarity in mammalian epithelial cells.

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.

Apical lumen formation in renal epithelia.

The ability to form epithelial lumina is a central architectural characteristic of virtually all organs and indispensable for their function. Ontogenetically, the kidney is one of the best-characterized organs, but concepts of the regulated formation of its hollow epithelial structures are still emerging. Epithelial cell lines provide the opportunity to study molecular mechanisms in simplified assays modeling cyst and tube formation. In these systems, several groups have identified molecules implicated in lumen formation, and their downregulation results in either multiple-lumen or no-lumen phenotypes. On the basis of these phenotypes, we propose a working model, assigning proteins to groups with similar functions. Defects within these specific protein groups lead to distinct epithelial phenotypes. Studies of mesenchymal-to-epithelial transition underline the importance of these protein groups, but converting these basic models of lumen formation to an understanding of the mesenchymal to tubule formation during kidney development is still challenging.

PALS1 regulates E-cadherin trafficking in mammalian epithelial cells.

Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.

Ciliary targeting of olfactory CNG channels requires the CNGB1b subunit and the kinesin-2 motor protein, KIF17.

Nonmotile cilia on olfactory sensory neurons (OSNs) compartmentalize signaling molecules, including odorant receptors and cyclic nucleotide-gated (CNG) channels, allowing for efficient, spatially confined responses to sensory stimuli . Little is known about the mechanisms of the ciliary targeting of olfactory CNG channels, composed of three subunits: CNGA2, CNGA4, and CNGB1b . Recent reports suggest that subunit composition of the retinal CNG channel influences localization, leading to disease . However, the mechanistic role of subunits in properly targeting native olfactory CNG channels remains unclear. Here, we show that heteromeric assembly with CNGB1b, containing a critical carboxy-terminal motif (RVxP), is required for ciliary trafficking of olfactory CNG channels. Movement of proteins within the cilia is governed by intraflagellar transport (IFT), a process that facilitates bidirectional movement of cargo along microtubules. Work in C. elegans has established that heterotrimeric and homodimeric kinesin-2 family members play a critical role in anterograde transport . In mammalian systems, the heterotrimeric KIF3a/KIF3b/KAP-3 complex plays a clear role in IFT; however, no role has been established for KIF17, the mammalian homolog of OSM-3 . Here, we demonstrate that KIF17 is required for olfactory CNG channel targeting, providing novel insights into mechanisms of mammalian ciliary transport.

Septate and paranodal junctions: kissing cousins.

A series of recent publications significantly advances our knowledge about the evolution and composition of septate junctions in arthropod and chordate species. These papers indicate that insect septate junctions share several structural and functional components with paranodal junctions, which join myelinating glial cells to axons in the vertebrate nervous system, and that both probably evolved from a common ancestral precursor.

The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost.

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95-Discs Large-zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. Here, we demonstrate that the L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homologue of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homologue, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ albeit indirectly via binding the Pals1 PDZ domain. In agreement, we find that a Drosophila homologue of Pals1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila Pals1 homologue has been demonstrated recently to represent Stardust, a crucial polarity gene in Drosophila. Thus, our data identifies a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity.

Akt1 regulates a JNK scaffold during excitotoxic apoptosis.

Cell survival is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. Here we describe a novel interaction between Akt1 and JNK interacting protein 1 (JIP1), a JNK pathway scaffold. Direct association between Akt1 and JIP1 was observed in primary neurons. Neuronal exposure to an excitotoxic stimulus decreased the Akt1-JIP1 interaction and concomitantly increased association between JIP1 and JNK. Akt1 interaction with JIP1 inhibited JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Consistent with this view, neurons from Akt1-deficient mice exhibited higher susceptibility to kainate than wild-type littermates. Overexpression of Akt1 mutants that bind JIP1 reduced excitotoxic apoptosis. These results suggest that Akt1 binding to JIP1 acts as a regulatory gate preventing JNK activation, which is released under conditions of excitotoxic injury.

Retinitis Pigmentosa GTPase Regulator (RPGR) protein isoforms in mammalian retina: insights into X-linked Retinitis Pigmentosa and associated ciliopathies.

Mutations in the cilia-centrosomal protein Retinitis Pigmentosa GTPase Regulator (RPGR) are a frequent cause of retinal degeneration. The RPGR gene undergoes complex alternative splicing and encodes multiple protein isoforms. To elucidate the function of major RPGR isoforms (RPGR 1-19 and RPGR ORF15), we have generated isoform-specific antibodies and examined their expression and localization in the retina. Using sucrose-gradient centrifugation, immunofluorescence and co-immunoprecipitation methods, we show that RPGR isoforms localize to distinct sub-cellular compartments in mammalian photoreceptors and associate with a number of cilia-centrosomal proteins. The RCC1-like domain of RPGR, which is present in all major RPGR isoforms, is sufficient to target it to the cilia and centrosomes in cultured cells. Our findings indicate that multiple isotypes of RPGR may perform overlapping yet somewhat distinct transport-related functions in photoreceptors.