Alteration of Epithelial Structure and Function Associated with PtdIns(4,5)P2 Degradation by a Bacterial Phosphatase

Elucidation of the role of PtdIns(4,5)P2 in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P2, generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P2 in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cδ, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na+/H+ exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P2. Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P2 was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P2-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP5) into inositol 1,4,5,6-tetrakisphosphate (IP4), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP4. In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P2 with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na+/H+ exchange caused by PtdIns(4,5)P2 hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.

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Uniform cAMP Stimulation of Dictyostelium Cells Induces Localized Patches of Signal Transduction and Pseudopodia

Molecular Biology of the Cell ◽

10.1091/mbc.e03-08-0566 ◽

2003 ◽

Vol 14 (12) ◽

pp. 5019-5027 ◽

Cited By ~ 76

Author(s):

Marten Postma ◽

Jeroen Roelofs ◽

Joachim Goedhart ◽

Theodorus W.J. Gadella ◽

Antonie J.W.G. Visser ◽

...

Keyword(s):

Fluorescent Protein ◽

Relative Concentration ◽

Surface Membrane ◽

Leading Edge ◽

Ph Domain ◽

Cytosolic Ph ◽

Entire Boundary ◽

A Cell ◽

Green Fluorescent ◽

Camp Stimulation

The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1μM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 s after stimulation and disappears after ∼20 s. A second translocation of PHCrac-GFP starts after ∼30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of ∼4-5 μm, each covering ∼10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at ∼10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.

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Impact of Heterogeneity within Cultured Cells on Bacterial Invasion: Analysis of Pseudomonas aeruginosa andSalmonella enterica Serovar Typhi Entry into MDCK cells by Using a Green Fluorescent Protein-Labelled Cystic Fibrosis Transmembrane Conductance Regulator Receptor

Infection and Immunity ◽

10.1128/iai.68.2.861-870.2000 ◽

2000 ◽

Vol 68 (2) ◽

pp. 861-870 ◽

Cited By ~ 18

Author(s):

A. Alev Gerçeker ◽

Tanweer Zaidi ◽

Peter Marks ◽

David E. Golan ◽

Gerald B. Pier

Keyword(s):

Epithelial Cells ◽

Protein Expression ◽

Fluorescent Protein ◽

Cultured Cells ◽

Mdck Cells ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Bacterial Uptake ◽

Cftr Protein ◽

Green Fluorescent

ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel that also serves as a receptor for entry ofPseudomonas aeruginosa and Salmonella entericaserovar Typhi into epithelial cells. To evaluate heterogeneity in CFTR protein expression in cultured cells and the effect of heterogeneity on internalization of different P. aeruginosa and serovar Typhi strains, we used two-color flow cytometry and confocal laser microscopy to study bacterial uptake by Madin-Darby canine kidney (MDCK) type I epithelial cells stably expressing a green fluorescent protein (GFP)-CFTR fusion construct (MDCK–GFP-CFTR cells). We found a strong correlation between cell size and GFP-CFTR protein expression, with 60 to 70% of cells expressing low levels of GFP-CFTR protein, 20 to 30% expressing intermediate levels, and <10% expressing high levels. The cells were sorted into low-, intermediate-, or high-level producers of CFTR protein; in vitro growth of each sorted population yielded the same distribution of CFTR protein expression as that in the original population. Cells expressing either low or high levels of CFTR protein internalized bacteria poorly; maximal bacterial uptake occurred in the cells expressing intermediate levels of CFTR protein. Treatment of MDCK cells with sodium butyrate markedly enhanced the production of CFTR protein without increasing cell size; butyrate treatment also increased the proportion of cells with internalized bacteria. However, there were fewer bacteria per butyrate-treated cell and, for P. aeruginosa, there was an overall decrease in the total level of bacterial uptake. The most highly ingested bacterial strains were internalized by fewer total MDCK–GFP-CFTR cells, indicating preferential bacterial uptake by a minority of epithelial cells within a given culture. Confocal fluorescence microscopy showed that P. aeruginosa and serovar Typhi induced cytoplasmic accumulation of CFTR protein close to the plasma membrane where the bacteria were adherent. These results show that within a population of MDCK–GFP-CFTR cells, there are cells with markedly different abilities to ingest bacteria via CFTR, the majority of the P. aeruginosa and serovar Typhi cells are ingested by the one-fourth to one-third of the cells that exhibit an intermediate size and level of CFTR protein expression, and overexpression of the CFTR receptor does not increase total bacterial uptake but rather allows more epithelial cells to ingest fewer total bacteria.

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Developmental derivation of vocal fold mucosa from human induced pluripotent stem cells

10.21203/rs.2.12611/v1 ◽

2019 ◽

Author(s):

Vlasta Lungova ◽

Susan Thibeault

Keyword(s):

Epithelial Cells ◽

Pluripotent Stem Cells ◽

Vocal Fold ◽

Fluorescent Protein ◽

Three Dimensional ◽

Stratified Squamous Epithelium ◽

Three Dimensional Models ◽

Induced Pluripotent ◽

Green Fluorescent

Abstract Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. We report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa.

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Inhibitory receptor FcγRIIb mediates the effects of IgG on a phagosome acidification and a sequential dephosphorylation system comprising SHIPs and Inpp4a

Innate Immunity ◽

10.1177/1753425917701553 ◽

2017 ◽

Vol 23 (4) ◽

pp. 401-409 ◽

Cited By ~ 5

Author(s):

Tomohiro Segawa ◽

Kaoru Hazeki ◽

Kiyomi Nigorikawa ◽

Atsuko Nukuda ◽

Tomoki Tanizawa ◽

...

Keyword(s):

Fluorescent Protein ◽

Fluorescence Probe ◽

Pleckstrin Homology Domain ◽

Raw 264.7 Macrophages ◽

Lipid Phosphatases ◽

Src Homology 2 ◽

Lipid Phosphatase ◽

Src Homology ◽

Src Homology 2 Domain ◽

Green Fluorescent

The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in Raw 264.7 macrophages. The clearance of PIs was much faster when the phagocytosed particles were coated with IgG. The effect of IgG was not observed in the macrophages deficient in FcγRIIb, an inhibitory IgG receptor. To identify the lipid phosphatases responsible for the FcγRIIb-accelerated PI clearance, we prepared a panel of lipid phosphatase-deficient cells. The lack of a PI 5-phosphatase Src homology 2 domain-containing inositol-5-phosphatase (SHIP)1 or SHIP2 impaired the FcγRIIb-accelerated clearance of PIs. The lack of a PI 4-phosphatase Inpp4a also impaired the accelerated PIs clearance. In the FcγRIIb- and Inpp4a-deficient cells, acidification of the formed phagosome was slowed. These results suggested that FcγRIIb drives the sequential dephosphorylation system comprising SHIPs and Inpp4a, and accelerates phagosome acidification.

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Myo6 Facilitates the Translocation of Endocytic Vesicles from Cell Peripheries

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0767 ◽

2003 ◽

Vol 14 (7) ◽

pp. 2728-2743 ◽

Cited By ~ 134

Author(s):

Laura Aschenbrenner ◽

TinThu Lee ◽

Tama Hasson

Keyword(s):

Epithelial Cells ◽

Fluorescent Protein ◽

Early Endosome ◽

Sufficient Time ◽

Significant Delay ◽

Transfected Cells ◽

Early Endosomes ◽

Green Fluorescent ◽

Endocytic Vesicles ◽

Transferrin Uptake

Immunolocalization studies in epithelial cells revealed myo6 was associated with peripherally located vesicles that contained the transferrin receptor. Pulse-chase experiments after transferrin uptake showed that these vesicles were newly uncoated endocytic vesicles and that myo6 was recruited to these vesicles immediately after uncoating. GIPC, a putative myo6 tail binding protein, was also present. Myo6 was not present on early endosomes, suggesting that myo6 has a transient association with endocytic vesicles and is released upon early endosome fusion. Green fluorescent protein (GFP) fused to myo6 as well as the cargo-binding tail (M6tail) alone targeted to the nascent endocytic vesicles. Overexpression of GFP-M6tail had no effect on a variety of organelle markers; however, GFP-M6tail displaced the endogenous myo6 from nascent vesicles and resulted in a significant delay in transferrin uptake. Pulse-chase experiments revealed that transferrin accumulated in uncoated vesicles within the peripheries of transfected cells and that Rab5 was recruited to the surface of these vesicles. Given sufficient time, the transferrin did traffic to the perinuclear sorting endosome. These data suggest that myo6 is an accessory protein required for the efficient transportation of nascent endocytic vesicles from the actin-rich peripheries of epithelial cells, allowing for timely fusion of endocytic vesicles with the early endosome.

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Targeting Mucosal Sites by Polymeric Immunoglobulin Receptor-directed Peptides

Journal of Experimental Medicine ◽

10.1084/jem.20020581 ◽

2002 ◽

Vol 196 (4) ◽

pp. 551-555 ◽

Cited By ~ 24

Author(s):

Kendra D. White ◽

J. Donald Capra

Keyword(s):

Epithelial Cells ◽

Fluorescent Protein ◽

Secretory Component ◽

In Vivo Studies ◽

Polymeric Immunoglobulin Receptor ◽

Immunoglobulin Receptor ◽

Potential Binding Site ◽

Green Fluorescent

Polymeric immunoglobulins provide first line humoral defense at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR) on mucosal and glandular epithelial cells. Previous studies from our laboratory suggested that amino acids 402–410 of the Cα3 domain of dimeric IgA (dIgA) represented a potential binding site for the pIgR. Here by binding human secretory component to overlapping decapeptides of Cα3, we confirm these residues and also uncover an additional site. Furthermore, we show that the Cα3 motif appears to be sufficient to direct transport of green fluorescent protein through the pIgR-specific cellular transcytosis system. An alternative approach identified phage peptides, selected from a library by the in vitro Madin Darby Canine Kidney transcytosis assay, for pIgR-mediated transport through epithelial cells. Some transcytosis-selected peptides map to the same 402–410 pIgR-binding Cα3 site. Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport. In addition to identifying small peptides that are both bound and transported by the pIgR, this study provides evidence that the pIgR-mediated mucosal secretion system may represent a means of targeting small molecule therapeutics and genes to mucosal epithelial cells.

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Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00034.2003 ◽

2003 ◽

Vol 285 (3) ◽

pp. L691-L700 ◽

Cited By ~ 31

Author(s):

Jason M. Roper ◽

Rhonda J. Staversky ◽

Jacob N. Finkelstein ◽

Peter C. Keng ◽

Michael A. O'Reilly

Keyword(s):

Epithelial Cells ◽

Fluorescent Protein ◽

Airway Epithelial Cells ◽

Clara Cell ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Green Fluorescence ◽

Experimental Conditions ◽

Green Fluorescent

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1α and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.

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Phosphatidylinositol-4,5-Bisphosphate-Rich Plasma Membrane Patches Organize Active Zones of Endocytosis and Ruffling in Cultured Adipocytes

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.9102-9123.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 9102-9123 ◽

Cited By ~ 54

Author(s):

Shaohui Huang ◽

Larry Lifshitz ◽

Varsha Patki-Kamath ◽

Richard Tuft ◽

Kevin Fogarty ◽

...

Keyword(s):

Plasma Membrane ◽

Large Scale ◽

Plasma Membranes ◽

Fluorescent Protein ◽

Dimensional Space ◽

Three Dimensional ◽

Actin Dynamics ◽

Ph Domain ◽

Membrane Ruffling ◽

Green Fluorescent

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

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Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.2.c486 ◽

2001 ◽

Vol 281 (2) ◽

pp. C486-C495 ◽

Cited By ~ 14

Author(s):

Melissa H. Little ◽

Lorine Wilkinson ◽

Darren L. Brown ◽

Michael Piper ◽

Toshiya Yamada ◽

...

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Fluorescent Protein ◽

Protein Localization ◽

Proteolytic Processing ◽

Proximal Tubule Cells ◽

Epithelial Monolayers ◽

Vertebrate Orthologs ◽

Localization Signal ◽

Green Fluorescent

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

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