Project description:Columnar epithelia establish their luminal domains and their mitotic spindles parallel to the basal surface and undergo symmetric cell divisions in which the cleavage furrow bisects the apical domain. Hepatocyte lumina interrupt the lateral domain of neighboring cells perpendicular to two basal domains and their cleavage furrow rarely bifurcates the luminal domains. We determine that the serine/threonine kinase Par1b defines lumen position in concert with the position of the astral microtubule anchoring complex LGN-NuMA to yield the distinct epithelial division phenotypes. Par1b signaling via the extracellular matrix (ECM) in polarizing cells determined RhoA/Rho-kinase activity at cell-cell contact sites. Columnar MDCK and Par1b-depleted hepatocytic HepG2 cells featured high RhoA activity that correlated with robust LGN-NuMA recruitment to the metaphase cortex, spindle alignment with the substratum, and columnar organization. Reduced RhoA activity at the metaphase cortex in HepG2 cells and Par1b-overexpressing MDCK cells correlated with a single or no LGN-NuMA crescent, tilted spindles, and the development of lateral lumen polarity.
Project description:The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.
Project description:A polarized architecture is central to both epithelial structure and function. In many cells, polarity involves mutual antagonism between the Par complex and the Scribble (Scrib) module. While molecular mechanisms underlying Par-mediated apical determination are well-understood, how Scrib module proteins specify the basolateral domain remains unknown. Here, we demonstrate dependent and independent activities of Scrib, Discs-large (Dlg), and Lethal giant larvae (Lgl) using the Drosophila follicle epithelium. Our data support a linear hierarchy for localization, but rule out previously proposed protein-protein interactions as essential for polarization. Cortical recruitment of Scrib does not require palmitoylation or polar phospholipid binding but instead an independent cortically stabilizing activity of Dlg. Scrib and Dlg do not directly antagonize atypical protein kinase C (aPKC), but may instead restrict aPKC localization by enabling the aPKC-inhibiting activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each required, all three together are not sufficient to antagonize the Par complex. Our data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg.
Project description:Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell-matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical-basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity.
Project description:Defining the three body axes is a central event of vertebrate morphogenesis. Establishment of left-right (L-R) asymmetry in development follows the determination of dorsal-ventral and anterior-posterior (A-P) body axes, although the molecular mechanism underlying precise L-R symmetry breaking in reference to the other two axes is still poorly understood. Here, by removing both Vangl1 and Vangl2, the two mouse homologues of a Drosophila core planar cell polarity (PCP) gene Van Gogh (Vang), we reveal a previously unrecognized function of PCP in the initial breaking of lateral symmetry. The leftward nodal flow across the posterior notochord (PNC) has been identified as the earliest event in the de novo formation of L-R asymmetry. We show that PCP is essential in interpreting the A-P patterning information and linking it to L-R asymmetry. In the absence of Vangl1 and Vangl2, cilia are positioned randomly around the centre of the PNC cells and nodal flow is turbulent, which results in disrupted L-R asymmetry. PCP in mouse, unlike what has been implicated in other vertebrate species, is not required for ciliogenesis, cilium motility, Sonic hedgehog (Shh) signalling or apical docking of basal bodies in ciliated tracheal epithelial cells. Our data suggest that PCP acts earlier than the unidirectional nodal flow during bilateral symmetry breaking in vertebrates and provide insight into the functional mechanism of PCP in organizing the vertebrate tissues in development.
Project description:Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. Here we report instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. We show that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals.
Project description:Background: Interleukin-22 (IL-22) impacts the integrity of intestinal epithelia and has been associated with the development of colitis-associated cancer and inflammatory bowel diseases (IBD). Previous data suggest that IL-22 protects the mucosal barrier and promotes wound healing and barrier defect. We hypothesized, that IL-22 modulates cell polarity of intestinal epithelial cells (IECs) acting on tight junction assembly. The aim of the study was to investigate IL-22-dependent mechanisms in the reprogramming of intestinal epithelia. Methods: IECs were exposed to IL-22 at various concentrations. IECs in Matrigel® were grown to 3-dimensional cysts in the presence or absence of IL-22 and morphology and expression of polarity proteins were analyzed by confocal microscopy. Epithelial cell barrier (TER and sandwich assay) and TJ assembly analysis (calcium-switch assay) were performed. TJ and cell polarity protein expression were assessed by western blotting and confocal microscopy. Cell migration and invasion assays were performed. Induction of epithelial-mesenchymal transition (EMT) was assessed by RT-qPCR analysis and western blotting. Signaling pathway analyses were performed by phosphoblotting and functional assays after blocking STAT3 and ERK signaling pathways. Using the toxoplasma-model of terminal ileitis, IL-22-knock-out mice were compared to wild-type littermates, analyzed for barrier function using one-path-impedance-analysis and macromolecular flux (H3-mannitol, Ussing-chambers). Results: IECs exhibited a barrier defect after IL-22 exposure. TJ protein distribution and expression were severely impaired. Delayed recovery in the calcium-switch assay was observed suggesting a defect in TJ assembly. Analyzing the 3D-cyst model, IL-22 induced multi-lumen and aberrant cysts, and altered the localization of cell polarity proteins. Cell migration and invasion was caused by IL-22 as well as induction of EMT. Interestingly, only inhibition of the MAPK pathway, rescued the TJal barrier defect, while blocking STAT3 was relevant for cell survival. In addition, ileal mucosa of IL-22 deficient mice was protected from the barrier defect seen in Toxoplasma gondii-induced ileitis in wild type mice shown by significantly higher Re values and correspondingly lower macromolecule fluxes. Conclusion: IL-22 impairs intestinal epithelial cell barrier by inducing EMT, causing defects in epithelial cell polarity and increasing cell motility and cell invasion. IL-22 modulates TJ protein expression and mediates tight junctional (TJal) barrier defects via ERK pathway.
Project description:Peri-centrosomal positioning of the mammalian Golgi apparatus is known to involve microtubule-based motility, but its importance for cellular physiology is a major unanswered question. Here, we identify golgin-160 and GMAP210 as proteins required for centripetal motility of Golgi membranes. In the absence of either golgin, peri-centrosomal positioning of the Golgi apparatus was disrupted while the cytoskeleton remained intact. Although secretion persisted with normal kinetics, it was evenly distributed in response to wounding rather than directed to the wound edge. Strikingly, these cells also completely failed to polarize. Further, directionally persistent cell migration was inhibited such that wound closure was impaired. These findings not only reveal novel roles for golgin-160 and GMAP210 in conferring membrane motility but also indicate that Golgi positioning has an active role in directed secretion, cell polarity, and wound healing.
Project description:In contrast to our significant understanding of signaling cascades that determine cell polarity in lower eukaryotic or immortalized cells, little is known about the transcriptional program that governs mammalian epithelial polarization in vivo. Here we show, using conditional gene ablation and three-dimensional tissue culture, that the homeobox transcription factor Cdx2 controls apical-basolateral polarity in mouse enterocytes and human colonic epithelial cells. Cdx2 regulates a comprehensive gene network involved in endo-lysosomal maturation and protein transport. In the absence of Cdx2, defective protein trafficking impairs apical-basal transport and induces ectopic lumen formation. These defects are partially recapitulated by suppression of key apical transport components, Rab11a and Kif3b, which are regulated by Cdx2. Furthermore, Cdx2 deficiency affects components that control the organization of microvillus actin cytoskeleton, leading to severe microvillus atrophy. These results demonstrate that Cdx2 regulates epithelial cell polarity and morphogenesis through control of apical protein transport and endo-lysosomal function.
Project description:Cell polarity is regulated by evolutionarily conserved polarity factors whose precise higher-order organization at the cell cortex is largely unknown. Here we image frontally the cortex of live fission yeast cells using time-lapse and super-resolution microscopy. Interestingly, we find that polarity factors are organized in discrete cortical clusters resolvable to ~50-100 nm in size, which can form and become cortically enriched by oligomerization. We show that forced co-localization of the polarity factors Tea1 and Tea3 results in polarity defects, suggesting that the maintenance of both factors in distinct clusters is required for polarity. However, during mitosis, their co-localization increases, and Tea3 helps to retain the cortical localization of the Tea1 growth landmark in preparation for growth reactivation following mitosis. Thus, regulated spatial segregation of polarity factor clusters provides a means to spatio-temporally control cell polarity at the cell cortex. We observe similar clusters in Saccharomyces cerevisiae and Caenorhabditis elegans cells, indicating this could be a universal regulatory feature.