HCO3- transport through anoctamin/transmembrane protein ANO1/TMEM16A, in pancreatic acinar cells, regulates luminal pH

The identification of ANO1/TMEM16A as the likely calcium-dependent chloride channel of exocrine glands has led to a more detailed understanding of its biophysical properties. This includes a calcium-dependent change in channel selectivity and evidence that HCO3-permeability can be significant. Here we use freshly isolated pancreatic acini that preserve the luminal structure to measure intraluminal pH and test the idea that ANO1/TMEM16A contributes to luminal pH balance. Our data show that, under physiologically relevant stimulation with 10 pm cholesystokinin, the luminal acid load that results from the exocytic fusion of zymogen granules is significantly blunted by HCO3-buffer in comparison with HEPES, and that this is blocked by the specific TMEM16A inhibitor T16inh-A01. Furthermore, in a model of acute pancreatitis, we observed substantive luminal acidification and provide evidence that ANO1/TMEM16A acts to attenuate this pH shift. We conclude that ANO1/TMEM16A is a significant pathway in pancreatic acinar cells for HCO3-secretion into the lumen

Phosphoinositide 3-kinase p110δ promotes lumen formation through the enhancement of apico-basal polarity and basal membrane organization

Signalling triggered by adhesion to the extracellular matrix plays a key role in the spatial orientation of epithelial polarity and formation of lumens in glandular tissues. Phosphoinositide 3-kinase signalling in particular is known to influence the polarization process during epithelial cell morphogenesis. Here, using Madin–Darby canine kidney epithelial cells grown in 3D culture, we show that the p110d isoform of phosphoinositide 3-kinase co-localizes with focal adhesion proteins at the basal surface of polarized cells. Pharmacological, siRNA- or kinase-dead-mediated inhibition of p110d impair the early stages of lumen formation, resulting in inverted polarized cysts, with no laminin or type IV collagen assembly at cell/extracellular matrix contacts. p110d also regulates the organization of focal adhesions and membrane localization of dystroglycan. Thus, we uncover a previously unrecognized role for p110d in epithelial cells in the orientation of the apico-basal axis and lumen formation

Myosin VI and vinculin cooperate during the morphogenesis of cadherin cell–cell contacts in mammalian epithelial cells

Cooperation between cadherins and the actin cytoskeleton controls many aspects of epithelial biogenesis. We report here that myosin VI critically regulates the morphogenesis of epithelial cell–cell contacts. As epithelial monolayers mature in culture, discontinuous cell–cell contacts are initially replaced by continuous (cohesive) contacts. Myosin VI is recruited to cell contacts as they become linear and cohesive, where it forms a biochemical complex with epithelial cadherin (E-cadherin). Myosin VI is necessary for strong cadherin adhesion, for cells to form cohesive linear contacts, and for the integrity of the apical junctional complex. We find that vinculin mediates this effect of myosin VI. Myosin VI is necessary for vinculin and E-cadherin to interact. A combination of gain and loss of function approaches identifies vinculin as a downstream effector of myosin VI that is necessary for the integrity of intercellular contacts. We propose that myosin VI and vinculin form a molecular apparatus that generates cohesive cell–cell contacts in cultured mammalian epithelia

Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures

The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell-cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell-cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesi

Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherin-based bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts

TorsinA folding and N-linked glycosylation are sensitive to redox homeostasis

The Endoplasmic Reticulum (ER) is responsible for the folding and post-translational modification of secretory proteins, as well as for triaging misfolded proteins. During folding, there is a complex yet only partially understood interplay between disulfide bond formation, which is an enzyme catalyzed event in the oxidizing environment of the ER, along with other post-translational modifications (PTMs) and chaperone-supported protein folding. Here, we used the glycoprotein torsinA as a model substrate to explore the impact of ER redox homeostasis on PTMs and protein biogenesis. TorsinA is a AAA+ ATPase with unusual oligomeric properties and controversial functions. The deletion of a C-terminal glutamic acid residue (∆E) is associated with the development of Early-Onset Torsion Dystonia, a severe movement disorder. TorsinA differs from other AAA+ ATPases since it is an ER resident, and as a result of its entry into the ER torsinA contains two N-linked glycans and at least one disulfide bond. The role of these PTMs on torsinA biogenesis and function and the identity of the enzymes that catalyze them are poorly defined. Using a yeast torsinA expression system, we demonstrate that a specific protein disulfide isomerase, Pdi1, affects the folding and N-linked glycosylation of torsinA and torsinA∆E in a redox-dependent manner, suggesting that the acquisition of early torsinA folding intermediates is sensitive to perturbed interactions between Cys residues and the quality control machinery. We also highlight the role of specific Cys residues during torsinA biogenesis and demonstrate that torsinA∆E is more sensitive than torsinA when these Cys residues are mutated.Fil: Honer, Jonas. University of Pittsburgh; Estados UnidosFil: Niemeyer, Katie M.. University of Pittsburgh; Estados UnidosFil: Fercher, Christian. The University of Queensland; AustraliaFil: Diez Tissera, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Jaberolansar, Noushin. The University of Queensland; AustraliaFil: Jafrani, Yohaann M.A.. The University of Queensland; AustraliaFil: Zhou, Chun. The University of Queensland; AustraliaFil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Shewan, Annette M.. The University of Queensland; AustraliaFil: Schulz, Benjamin L.. The University of Queensland; AustraliaFil: Brodsky, Jeffrey L.. University of Pittsburgh; Estados UnidosFil: Zacchi, Lucia Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. University of Pittsburgh; Estados Unidos. The University of Queensland; Australi

Phosphoinositides in cell architecture

Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity.We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues

Phosphoinositides as Determinants of Membrane Identity, Apicobasal Polarity, and Lumen Formation

Epithelial cells sense their surrounding environment via mechanical and chemical stimuli, responding to multiple sources of tension and force, including those generated by cell-cell and cell-extracellular matrix (ECM) interaction: they respond to these cues by developing an apicobasal axis of polarity. Phosphoinositides (PIs) are structural components of biological membranes that control a diverse array of signaling pathways through spatiotemporal recruitment of effectors containing PI-specific binding domain(s). Thus they have been shown to modulate a plethora of cellular processes including actin polymerization, cell migration, proliferation, differentiation, and vesicular trafficking. PIs are enriched in different membranes and their levels are tightly regulated by specific PI kinases and phosphatases. During the past decade, PIs have come to the fore as specific markers that define membrane identity, acting as critical regulators of the cell polarization process. In this review, we have examined how PIs are able to assign identity to polarized epithelial cell plasma membrane domains and integrate in space and time complex signaling pathways to trigger appropriate cellular responses to environmental cues. PIs are implicated in a vast array of cellular responses that are central for morphogenesis such as, but not limited to, cytoskeletal changes, cytokinesis, and recruitment of downstream effectors to govern mechanisms involved in polarization and lumen formation. Subversion by pathogens of PI metabolism and plasma membrane identity in polarized cells and the clinical relevance of research on PIs were also discussed