Eya1 protein phosphatase regulates tight junction formation in lung distal epithelium.
ABSTRACT: Little is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity, such as aPKC?. We recently reported that Eya1 phosphatase functions through aPKC?-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKC? phosphorylation levels, aPKC?-mediated TJ protein phosphorylation and Notch1-Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wild-type Eya1 or partially inhibiting aPKC? in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1(-/-) lung epithelial TJ defects. These findings uncover novel functions for the Eya1-aPKC?-Notch1-Cdc42 pathway as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area.
Project description:Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKC? phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in vivo or in vitro. In addition, in Eya1(-/-) lungs, perpendicular division is not maintained and Numb is segregated to both daughter cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1(-/-) lung phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching. Indeed, genetic activation of Notch partially rescues Eya1(-/-) lung epithelial defects. These findings uncover novel functions for Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.
Project description:In response to stress- or tissue-damage-induced apoptosis, unaffected epithelial cells undergo compensatory proliferation to maintain the integrity of the epithelium. Proximal signals regulating this response are not fully understood, but c-Jun N-terminal kinase (JNK) activity appears to be critical for both apoptosis and compensatory proliferation. Disruption of epithelial cell apical-basal polarity occurs in early cancer development and is often correlated with increased proliferation by means not fully characterized. We considered whether disruption of the various polarity complexes could provide signals identifying damaged epithelial cells and thus lead to apoptosis-induced compensatory proliferation.We identify the Cdc42/Par6/atypical protein kinase C (aPKC) Par polarity complex as uniquely and specifically regulating apoptosis-induced compensatory proliferation in Drosophila epithelia. Genetic depletion of individual components or disruption of formation and localization of this complex, but not other polarity complexes, induces JNK-dependent apoptosis and JNK-dependent compensatory proliferation following radiation injury. When apoptosis execution is blocked, by p35 expression, Cdc42/Par6/aPKC-depleted tissues uniquely hyperproliferate, leading to tissue and organ overgrowth. Disruption of Cdc42/Par6/aPKC leads to activation of JNK through increased Rho1 and Rok activity and Rok's capacity to activate myosin but not F-actin.We show that the Cdc42/Par6/aPKC polarity complex influences both a physiologic compensatory proliferation response after irradiation injury and a contrived compensatory non-cell-autonomous hyperproliferation response when cell-autonomous apoptosis, resulting from Cdc42/Par6/aPKC disruption, is inhibited. These results suggest the possibility that in cancer where apoptotic regulation is disrupted, loss of Cdc42/Par6/aPKC polarity complex organization or localization could contribute to tumor hyperproliferation and explain how polarity disruption contributes to tumor development.
Project description:Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6-aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. We used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by ?-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion.
Project description:Enteropathogenic Escherichia coli (EPEC) uses a type three secretion system to inject effector proteins into host intestinal epithelial cells, causing diarrhea. EPEC induces the formation of pedestals underlying attached bacteria, disrupts tight junction (TJ) structure and function, and alters apico-basal polarity by redistributing the polarity proteins Crb3 and Pals1, although the mechanisms are unknown. Here we investigate the temporal relationship of PAR polarity complex and TJ disruption following EPEC infection. EPEC recruits active aPKC?, a PAR polarity protein, to actin within pedestals and at the plasma membrane prior to disrupting TJ. The EPEC effector EspF binds the endocytic protein sorting nexin 9 (SNX9). This interaction impacts actin pedestal organization, recruitment of active aPKC? to actin at cell-cell borders, endocytosis of JAM-A S285 and occludin, and TJ barrier function. Collectively, data presented herein support the hypothesis that EPEC-induced perturbation of TJ is a downstream effect of disruption of the PAR complex and that EspF binding to SNX9 contributes to this phenotype. aPKC? phosphorylates polarity and TJ proteins and participates in actin dynamics. Therefore, the early recruitment of aPKC? to EPEC pedestals and increased interaction with actin at the membrane may destabilize polarity complexes ultimately resulting in perturbation of TJ.
Project description:Cdc42 regulates epithelial morphogenesis together with the Par complex (Baz/Par3-Par6-aPKC), Crumbs (Crb/CRB3) and Stardust (Sdt/PALS1). However, how these proteins work together and interact during epithelial morphogenesis is not well understood. To address this issue, we used the genetically amenable Drosophila pupal photoreceptor and follicular epithelium. We show that during epithelial morphogenesis active Cdc42 accumulates at the developing apical membrane and cell-cell contacts, independently of the Par complex and Crb. However, membrane localization of Baz, Par6-aPKC and Crb all depend on Cdc42. We find that although binding of Cdc42 to Par6 is not essential for the recruitment of Par6 and aPKC to the membrane, it is required for their apical localization and accumulation, which we find also depends on Par6 retention by Crb. In the pupal photoreceptor, membrane recruitment of Par6-aPKC also depends on Baz. Our work shows that Cdc42 is required for this recruitment and suggests that this factor promotes the handover of Par6-aPKC from Baz onto Crb. Altogether, we propose that Cdc42 drives morphogenesis by conferring apical identity, Par-complex assembly and apical accumulation of Crb.
Project description:Epithelial cells are polarized along their apical-basal axis by the action of the small GTPase Cdc42, which is known to activate the aPKC kinase at the apical domain. However, loss of aPKC kinase activity was reported to have only mild effects on epithelial cell polarity. Here, we show that Cdc42 also activates a second kinase, Pak1, to specify apical domain identity in Drosophila and mammalian epithelia. aPKC and Pak1 phosphorylate an overlapping set of polarity substrates in kinase assays. Inactivating both aPKC kinase activity and the Pak1 kinase leads to a complete loss of epithelial polarity and morphology, with cells losing markers of apical polarization such as Crumbs, Par3/Bazooka, or ZO-1. This function of Pak1 downstream of Cdc42 is distinct from its role in regulating integrins or E-cadherin. Our results define a conserved dual-kinase mechanism for the control of apical membrane identity in epithelia.
Project description:The polarity protein complex Par6/atypical protein kinase (aPKC)/Cdc42 regulates polarization processes during epithelial morphogenesis, astrocyte migration, and axon specification. We, as well as others, have shown that this complex is also required for disruption of apical-basal polarity during the oncogene ErbB2-induced transformation and transforming growth factor beta-induced epithelial-mesenchymal transition of mammary epithelial cells. Here, we report that expression of Par6 by itself in mammary epithelial cells induces epidermal growth factor-independent cell proliferation and development of hyperplastic three-dimensional acini without affecting apical-basal polarity. This is dependent on the ability of Par6 to interact with aPKC and Cdc42, but not Lgl and Par3, and its ability to promote sustained activation of MEK/ERK signaling. Down-regulation of Cdc42 or aPKC expression suppresses the ability of Par6 to induce proliferation, demonstrating that Par6 promotes cell proliferation by interacting with aPKC and Cdc42. We also show that Par6 is overexpressed in breast cancer-derived cell lines and in both precancerous breast lesions and advanced primary human breast cancers, suggesting that Par6 overexpression regulates tumor initiation and progression. Thus, in addition to regulating cell polarization processes, Par6 is an inducer of cell proliferation in breast epithelial cells.
Project description:The formation of highly branched epithelial structures is critical for the development of many essential organs, including lung, liver, pancreas, kidney and mammary glands. Elongation and branching of these structures require precise control of complex morphogenetic processes that are dependent upon coordinate regulation of cell shape, apical-basal polarity, proliferation, migration, and interactions among multiple cell types. Herein, we demonstrate that temporal-spatial regulation of epithelial cell polarity by the small GTPase, CDC42, is essential for branching morphogenesis of the developing lung. Epithelial cell-specific deletion of CDC42 in fetal mice disrupted epithelial cell polarity, the actin cytoskeleton, intercellular contacts, directional trafficking of proteins, proliferation and mitotic spindle orientation, impairing the organization and patterning of the developing respiratory epithelium and adjacent mesenchyme. Transition from a pseudostratified to a simple columnar epithelium was impaired, consistent with coordinate dysregulation of epithelial cell polarity, mitotic spindle orientation, and repositioning of mitotic cells within the epithelium during cell cycle progression. Expression of sonic hedgehog and its receptor, patched-1, was decreased, while fibroblast growth factor 10 expression in the mesenchyme was expanded, resulting in disruption of branching morphogenesis and bronchiolar smooth muscle formation in this model. CDC42 is required for spatial positioning of proliferating epithelial cells, as well as signaling interactions between the epithelium and mesenchyme and is, therefore, essential for formation and maintenance of the respiratory tract during morphogenesis of the fetal lung.
Project description:Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6-aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6-aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6-aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6-aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.
Project description:Cdc42 has been implicated in numerous biochemical pathways during epithelial morphogenesis, including the control of spindle orientation during mitosis, the establishment of apical-basal polarity, the formation of apical cell-cell junctions, and polarized secretion. To investigate the signaling pathways through which Cdc42 mediates these diverse effects, we have screened an siRNA library corresponding to the 36 known Cdc42 target proteins, in a human bronchial epithelial cell line. Two targets, PAK4 and Par6B, were identified as necessary for the formation of apical junctions. PAK4 is recruited to nascent cell-cell contacts in a Cdc42-dependent manner, where it is required for the maturation of primordial junctions into apical junctions. PAK4 kinase activity is essential for junction maturation, but overexpression of an activated PAK4 mutant disrupts this process. Par6B, together with its binding partner aPKC, is necessary both for junction maturation and for the retention of PAK4 at sites of cell-cell contact. This study demonstrates that controlled regulation of PAK4 is required for apical junction formation in lung epithelial cells and highlights potential cross-talk between two Cdc42 targets, PAK4 and Par6B.