scholarly journals CD2AP links actin to PI3 kinase activity to extend epithelial cell height and constrain cell area

2019 ◽  
Vol 219 (1) ◽  
Author(s):  
Yuou Wang ◽  
William M. Brieher
Keyword(s):  
Actin Polymerization ◽  
Mdck Cells ◽  
Cell Area ◽  
Critical Determinant ◽  
Sh3 Domains ◽  
Lateral Membrane ◽  
Cell Height ◽  
Pi3k Activation ◽  
Phosphoinositide 3 Kinase

Maintaining the correct ratio of apical, basal, and lateral membrane domains is important for epithelial physiology. Here, we show that CD2AP is a critical determinant of epithelial membrane proportions. Depletion of CD2AP or phosphoinositide 3-kinase (PI3K) inhibition results in loss of F-actin and expansion of apical–basal domains, which comes at the expense of lateral membrane height in MDCK cells. We demonstrate that the SH3 domains of CD2AP bind to PI3K and are necessary for PI3K activity along lateral membranes and constraining cell area. Tethering the SH3 domains of CD2AP or p110γ to the membrane is sufficient to rescue CD2AP-knockdown phenotypes. CD2AP and PI3K are both upstream and downstream of actin polymerization. Since CD2AP binds to both actin filaments and PI3K, CD2AP might bridge actin assembly to PI3K activation to form a positive feedback loop to support lateral membrane extension. Our results provide insight into the squamous to cuboidal to columnar epithelial transitions seen in complex epithelial tissues in vivo.

scholarly journals RacG Regulates Morphology, Phagocytosis, and Chemotaxis

2006 ◽  
Vol 5 (10) ◽  
pp. 1648-1663 ◽  
Author(s):  
Baggavalli P. Somesh ◽  
Georgia Vlahou ◽  
Miho Iijima ◽  
Robert H. Insall ◽  
Peter Devreotes ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Free System ◽  
Mild Phenotype ◽  
Particle Uptake ◽  
Phosphoinositide 3 Kinase

ABSTRACTRacG is an unusual member of the complex family of Rho GTPases inDictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 μM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPγS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or moreDictyosteliumRho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

BCR activation of PI3K is Vav-independent in murine B cells

2004 ◽  
Vol 32 (5) ◽  
pp. 781-784 ◽  
Author(s):  
E. Vigorito ◽  
E. Clayton ◽  
M. Turner
Keyword(s):  
B Cells ◽  
Tyrosine Kinases ◽  
Adaptor Proteins ◽  
Pleckstrin Homology Domain ◽  
Pi3k Activation ◽  
Lipid Product ◽  
Lipid Kinases ◽  
Phosphoinositide 3 Kinase

BCR (B-cell antigen receptor)-induced Ca2+ signalling is initiated by activation of tyrosine kinases, which in concert with adaptor proteins and lipid kinases regulate PLC (phospholipase C) γ2 activation. Vav and PI3K (phosphoinositide 3-kinase) are required for optimal Ca2+ responses, although it has not been established, in primary B-cells, if both proteins are components of the same pathway. In vitro evidence suggests that binding of the PI3K lipid product PIP3 to Vav pleckstrin homology domain contributes to Vav activation. However, pharmacological inhibition of PI3K by wortmannin or deletion of the p110δ catalytic subunit has no effect on Vav activation in response to BCR engagement, suggesting that this mechanism does not operate in vivo. We also show that PI3K recruitment to phosphorylated-tyrosine-containing complexes is Vav-independent. Taken together with our previous observation that protein kinase B phosphorylation is normal in Vav-deficient B-cells, we suggest that PI3K activation is Vav-independent in response to strong signals delivered by multivalent cross-linking.

scholarly journals Phosphoinositide 3-Kinase Catalytic Subunit Deletion and Regulatory Subunit Deletion Have Opposite Effects on Insulin Sensitivity in Mice

2005 ◽  
Vol 25 (5) ◽  
pp. 1596-1607 ◽  
Author(s):  
Saskia M. Brachmann ◽  
Kohjiro Ueki ◽  
Jeffrey A. Engelman ◽  
Ronald C. Kahn ◽  
Lewis C. Cantley
Keyword(s):  
Insulin Sensitivity ◽  
Glucose Uptake ◽  
Mammalian Cells ◽  
Ex Vivo ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Pi3k Activation ◽  
Insulin Dependent ◽  
Phosphoinositide 3 Kinase

ABSTRACT Studies ex vivo have shown that phosphoinositide 3-kinase (PI3K) activity is necessary but not sufficient for insulin-stimulated glucose uptake. Unexpectedly, mice lacking either of the PI3K regulatory subunits p85α or p85β exhibit increased insulin sensitivity. The insulin hypersensitivity is particularly unexpected in p85α−/− p55α−/− p50α−/− mice, where a decrease in p110α and p110β catalytic subunits was observed in insulin-sensitive tissues. These results raised the possibility that decreasing total PI3K available for stimulation by insulin might circumvent negative feedback loops that ultimately shut off insulin-dependent glucose uptake in vivo. Here we present results arguing against this explanation. We show that p110α+/− p110β+/− mice exhibit mild glucose intolerance and hyperinsulinemia in the fasted state. Unexpectedly, p110α+/− p110β+/− mice showed a ∼50% decrease in p85 expression in liver and muscle. Consistent with this in vivo observation, knockdown of p110 by RNA interference in mammalian cells resulted in loss of p85 proteins due to decreased protein stability. We propose that insulin sensitivity is regulated by a delicate balance between p85 and p110 subunits and that p85 subunits mediate a negative role in insulin signaling independent of their role as mediators of PI3K activation.

scholarly journals Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 3081-3093 ◽  
Author(s):  
Cunxi Li ◽  
Mingming Hao ◽  
Zheng Cao ◽  
Wei Ding ◽  
Ramona Graves-Deal ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Distinct Region ◽  
Lateral Membrane ◽  
Polarized Epithelial Cells

Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-α and that TGF-α is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of μ1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α–containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.

Abstract 41: Platelet Dream Plays a Critical Role During Thrombogenesis in Mice

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Kyungho Kim ◽  
Alan Tseng ◽  
Andrew Barazia ◽  
Joseph E Italiano ◽  
Jaehyung Cho
Keyword(s):  
Thrombus Formation ◽  
Regulatory Element ◽  
Critical Role ◽  
Flow Chamber ◽  
Regulatory Function ◽  
Pi3k Activation ◽  
Granule Secretion ◽  
Phosphoinositide 3 Kinase

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain. Using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that hematopoietic and endothelial cell DREAM are required for platelet thrombus formation following arteriolar injury. DREAM deletion also prolonged tail bleeding times. In vitro flow chamber assays and in vivo adoptive transfer experiments indicated the importance of platelet DREAM in thrombogenesis. We found that deletion of platelet DREAM does not alter ultrastructural features but significantly impaired aggregation and ATP secretion induced by collagen-related peptide (CRP), ADP, or A23187, but not thrombin or U46619. Biochemical studies showed that platelet DREAM is required for phosphoinositide 3-kinase (PI3K) activation induced by GPVI-, A23187-, or integrin-mediated signaling. Studies using DREAM-null platelets and isoform-specific PI3K inhibitors revealed that platelet DREAM positively regulates granule secretion, Ca2+ mobilization, and aggregation induced by CRP or A23187 through PI3K class Iβ (PI3K-Iβ) activity. Genetic and pharmacological studies in megakaryoblastic MEG-01 cells showed that DREAM regulates A23187-induced Ca2+ mobilization and that the regulatory function of DREAM requires Ca2+ binding and PI3K-Iβ activity. Taken together, we have identified platelet DREAM as a novel regulator of PI3K-Iβ activity during thrombus formation.

scholarly journals Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

2017 ◽  
Vol 28 (8) ◽  
pp. 1088-1100 ◽  
Author(s):  
Lynne A. Lapierre ◽  
Elizabeth H. Manning ◽  
Kenya M. Mitchell ◽  
Cathy M. Caldwell ◽  
James R. Goldenring
Keyword(s):  
Mdck Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Cyst Formation ◽  
Epithelial Polarity ◽  
Lateral Membrane ◽  
Low Calcium ◽  
Single Lumen ◽  
Apical Junctions ◽  
Temporal Localization

MARK2 regulates the establishment of polarity in Madin–Darby canine kidney (MDCK) cells in part through phosphorylation of serine 227 of Rab11-FIP2. We identified Eps15 as an interacting partner of phospho-S227-Rab11-FIP2 (pS227-FIP2). During recovery from low calcium, Eps15 localized to the lateral membrane before pS227-FIP2 arrival. Later in recovery, Eps15 and pS227-FIP2 colocalized at the lateral membrane. In MDCK cells expressing the pseudophosphorylated FIP2 mutant FIP2(S227E), during recovery from low calcium, Eps15 was trapped and never localized to the lateral membrane. Mutation of any of the three NPF domains within GFP-FIP2(S227E) rescued Eps15 localization at the lateral membrane and reestablished single-lumen cyst formation in GFP-FIP2(S227E)–expressing cells in three-dimensional (3D) culture. Whereas expression of GFP-FIP2(S227E) induced the loss of E-cadherin and occludin, mutation of any of the NPF domains of GFP-FIP2(S227E) reestablished both proteins at the apical junctions. Knockdown of Eps15 altered the spatial and temporal localization of pS227-FIP2 and also elicited formation of multiple lumens in MDCK 3D cysts. Thus an interaction of Eps15 and pS227-FIP2 at the appropriate time and location in polarizing cells is necessary for proper establishment of epithelial polarity.

Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy

1992 ◽  
Vol 20 (2) ◽  
pp. 218-221
Author(s):  
Henning F. Bjerregaard
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
South African ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Electrophysiological Measurements ◽  
Renal Epithelial Cell Line ◽  
Darby Canine Kidney

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

scholarly journals Actin Polymerization in Macrophages in Response to Oxidized LDL and Apoptotic Cells: Role of 12/15-Lipoxygenase and Phosphoinositide 3-Kinase

2003 ◽  
Vol 14 (10) ◽  
pp. 4196-4206 ◽  
Author(s):  
Yury I. Miller ◽  
Dorothy S. Worrall ◽  
Colin D. Funk ◽  
James R. Feramisco ◽  
Joseph L. Witztum
Keyword(s):  
Cell Membrane ◽  
Actin Polymerization ◽  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Oxidation Products ◽  
Apoptotic Cells ◽  
Lipid Oxidation Products ◽  
Phosphoinositide 3 Kinase

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO–transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages.

scholarly journals Effect of Epsilon Toxin–GFP on MDCK Cells and Renal Tubules In Vivo

2004 ◽  
Vol 52 (7) ◽  
pp. 931-942 ◽  
Author(s):  
Alex Soler-Jover ◽  
Juan Blasi ◽  
Inma Gómez de Aranda ◽  
Piedad Navarro ◽  
Maryse Gibert ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Renal Tubules ◽  
Epsilon Toxin

scholarly journals Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity.

1992 ◽  
Vol 116 (4) ◽  
pp. 889-899 ◽  
Author(s):  
D A Wollner ◽  
K A Krzeminski ◽  
W J Nelson
Keyword(s):  
Epithelial Cell ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Cell ◽  
Cell Contact ◽  
Surface Polarity ◽  
Lateral Membrane ◽  
E Cadherin ◽  
Cell Cell

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.

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