scholarly journals Akt kinases are required for efficient feeding by macropinocytosis in Dictyostelium

2018 ◽  
Author(s):  
Thomas D. Williams ◽  
Sew-Yeu Peak-Chew ◽  
Peggy Paschke ◽  
Robert R. Kay
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Biology ◽  
Large Scale ◽  
Model Organism ◽  
Dependent Manner ◽  
Fluid Uptake ◽  
Small G Protein ◽  
Knockout Mutants ◽  
Summary Statement

AbstractMacropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum. In Dictyostelium, macropinocytic cups are organised by ‘macropinocytic patches’ in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and direct actin polymerisation to their periphery. Here, we show that a classical (PkbA) and a variant (PkbR1) Akt protein kinase acting downstream of PI(3,4,5)P3 are together are near-essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Akt targets identified by phosphoproteomics were highly enriched in small G-protein regulators, including the RhoGAP GacG. GacG knockout mutants make few macropinosomes but instead redeploy their cytoskeleton from macropinocytosis to motility, moving rapidly but taking up little fluid. The function of Akt in cell feeding through control of macropinosome size has implications for cancer cell biology.Summary statementDictyostelium amoebae feed by macropinocytosis in a PIP3 dependent manner. In the absence of PI3-kinases or the downstream Akt protein kinases, cells have smaller macropinosomes and nearly abolished fluid uptake.

scholarly journals Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+mobilization and degranulation in mast cells

2011 ◽  
Vol 22 (24) ◽  
pp. 4908-4917 ◽  
Author(s):  
Deepti Gadi ◽  
Alice Wagenknecht-Wiesner ◽  
David Holowka ◽  
Barbara Baird
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Time Course ◽  
Fluorescent Protein ◽  
Immunoglobulin E ◽  
Dependent Manner ◽  
Effector Domain ◽  
Granule Exocytosis ◽  
Kinase C

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca2+concentrations and oscillatory association of PKCβ–enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca2+mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca2+entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

scholarly journals Simvastatin Efficiently Reduces Levels of Alzheimer’s Amyloid Beta in Yeast

2019 ◽  
Vol 20 (14) ◽  
pp. 3531 ◽  
Author(s):  
Sudip Dhakal ◽  
Mishal Subhan ◽  
Joshua M. Fraser ◽  
Kenneth Gardiner ◽  
Ian Macreadie
Keyword(s):  
Fusion Protein ◽  
Amyloid Beta ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Dependent Manner ◽  
Green Fluorescence ◽  
Convenient Means ◽  
Ergosterol Synthesis ◽  
Green Fluorescent

A large-scale epidemiology study on statins previously showed that simvastatin was unique among statins in reducing the incidence of dementia. Since amyloid beta (Aβ42) is the protein that is most associated with Alzheimer’s disease, this study has focused on how simvastatin influences the turnover of native Aβ42 and Aβ42 fused with green fluorescent protein (GFP), in the simplest eukaryotic model organism, Saccharomyces cerevisiae. Previous studies have established that yeast constitutively producing Aβ42 fused to GFP offer a convenient means of analyzing yeast cellular responses to Aβ42. Young cells clear the GFP fusion protein and do not have green fluorescence while the older population of cells retains the fusion protein and exhibits green fluorescence, offering a fast and convenient means of studying factors that affect Aβ42 turnover. In this study the proportion of cells having GFP fused to Aβ after exposure to simvastatin, atorvastatin and lovastatin was analyzed by flow cytometry. Simvastatin effectively reduced levels of the cellular Aβ42 protein in a dose-dependent manner. Simvastatin promoted the greatest reduction as compared to the other two statins. A comparison with fluconazole, which targets that same pathway of ergosterol synthesis, suggests that effects on ergosterol synthesis do not account for the reduced amounts of Aβ42 fused to GFP. The levels of native Aβ42 following treated with simvastatin were also examined using a more laborious approach, quantitative MALDI TOF mass spectrometry. Simvastatin efficiently reduced levels of native Aβ42 from the population. This work indicates a novel action of simvastatin in reducing levels of Aβ42 providing new insights into how simvastatin exerts its neuroprotective role. We hypothesize that this reduction may be due to protein clearance.

The C2 domains of classical/conventional PKCs are specific PtdIns(4,5)P2-sensing domains

2007 ◽  
Vol 35 (5) ◽  
pp. 1046-1048 ◽  
Author(s):  
S. Corbalán-García ◽  
M. Guerrero-Valero ◽  
C. Marín-Vicente ◽  
J.C. Gómez-Fernández
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Living Cells ◽  
Model Membranes ◽  
Membrane Localization ◽  
Dependent Manner ◽  
C2 Domain ◽  
C2 Domains ◽  
Local Densities

The C2 domains of cPKCs [classical/conventional PKCs (protein kinase Cs)] bind to membranes in a Ca2+-dependent manner and thereby act as cellular Ca2+ effectors. Recent findings have demonstrated that the C2 domain of cPKCs interacts specifically with PtdIns(4,5)P2 through its polybasic cluster located in the β3–β4-strands, this interaction being critical for the membrane localization of these enzymes in living cells. In addition, these C2 domains exhibit higher affinity to bind PtdIns(4,5)P2 than any other polyphosphate phosphatidylinositols. It has also been shown that the presence of PtdIns(4,5)P2 in model membranes decreases the Ca2+ concentration required for classical C2 domains to bind them. Overall, the studies reviewed here suggest a new mechanism of membrane docking by the C2 domains of cPKCs in which the local densities of phosphatidylserine and PtdIns(4,5)P2 on the inner leaflet of the plasma membrane are sufficient to drive Ca2+-activated membrane docking during a physiological Ca2+ signal.

scholarly journals Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

2018 ◽  
Author(s):  
Sanduo Zheng ◽  
Lok-To Sham ◽  
Frederick A. Rubino ◽  
Kelly Brock ◽  
William P. Robins ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Biology ◽  
Model Organism ◽  
Structural Data ◽  
Outer Face ◽  
Antibiotic Development ◽  
Cell Wall Assembly ◽  
Lipid Ii

AbstractThe peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein prior to its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data are only available for MurJ from the extremophile Thermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the Gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Finally, through the use of sequence co-evolution analysis we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

Microcompartments within the yeast plasma membrane

2013 ◽  
Vol 394 (2) ◽  
pp. 189-202 ◽  
Author(s):  
Hans Merzendorfer ◽  
Jürgen J. Heinisch
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Model Organism ◽  
Dynamic Interaction ◽  
Eukaryotic Cells ◽  
Classical Concept ◽  
Yeast Plasma Membrane ◽  
Bud Neck ◽  
Microscopic Studies ◽  
Membrane Compartments

Abstract Recent research in cell biology makes it increasingly clear that the classical concept of compartmentation of eukaryotic cells into different organelles performing distinct functions has to be extended by microcompartmentation, i.e., the dynamic interaction of proteins, sugars, and lipids at a suborganellar level, which contributes significantly to a proper physiology. As different membrane compartments (MCs) have been described in the yeast plasma membrane, such as those defined by Can1 and Pma1 (MCCs and MCPs), Saccharomyces cerevisiae can serve as a model organism, which is amenable to genetic, biochemical, and microscopic studies. In this review, we compare the specialized microcompartment of the yeast bud neck with other plasma membrane substructures, focusing on eisosomes, cell wall integrity-sensing units, and chitin-synthesizing complexes. Together, they ensure a proper cell division at the end of mitosis, an intricately regulated process, which is essential for the survival and proliferation not only of fungal, but of all eukaryotic cells.

scholarly journals Real-time measurement of E2:ERα transcriptional activity in living cells

2019 ◽  
Author(s):  
Manuela Cipolletti ◽  
Stefano Leone ◽  
Stefania Bartoloni ◽  
Claudia Busonero ◽  
Filippo Acconcia
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Cell Biology ◽  
Transcriptional Activity ◽  
Receptor Activation ◽  
Living Cells ◽  
Membrane Localization ◽  
Dependent Manner ◽  
Plasma Membrane Localization ◽  
The Impact

AbstractKinetic analyses of diverse physiological processes have the potential to unveil new aspects of the molecular regulation of cell biology at temporal levels. 17β-estradiol (E2) regulates diverse physiological effects by binding to the estrogen receptor α (ERα), which primarily works as a transcription factor. Although many molecular details of the modulation of ERα transcriptional activity have been discovered including the impact of receptor plasma membrane localization and its relative E2-evoked signalling, the knowledge of real-time ERα transcriptional dynamics in living cells is lacking. Here, we report the generation of MCF-7 and HeLa cells stably expressing a modified luciferase under the control of an E2-sensitive promoter, which activity can be continuously monitored in living cells and show that E2 induces a linear increase in ERα transcriptional activity. Ligand-independent (e.g., epidermal growth factor) receptor activation was also detected in a time-dependent manner. Kinetic profiles of ERα transcriptional activity measured in the presence of both receptor antagonists and inhibitors of ERα plasma membrane localization reveals a biphasic dynamic of receptor behaviour underlying novel aspects of receptor-regulated transcriptional effects. Finally, analysis of the rate of the dose-dependent E2 induction of ERα transcriptional activity demonstrates that low doses of E2 induce an effect identical to that determined by high concentrations of E2 as a function of the duration of hormone administration. Overall, we present the characterization of sensitive stable cell lines where to study the kinetic of E2 transcriptional signaling and to identify new aspects of ERα function in different physiological or pathophysiological conditions.

Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO3− secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

2011 ◽  
Vol 301 (1) ◽  
pp. C137-C149 ◽  
Author(s):  
Suparerk Laohapitakworn ◽  
Jirawan Thongbunchoo ◽  
La-iad Nakkrasae ◽  
Nateetip Krishnamra ◽  
Narattaphol Charoenphandhu
Keyword(s):  
Plasma Membrane ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Intestinal Epithelium ◽  
Epithelial Transport ◽  
Ussing Chamber ◽  
Membrane Resistance ◽  
Regulatory Action ◽  
Dependent Manner ◽  
Gf 109203X

Besides being a Ca2+-regulating hormone, parathyroid hormone (PTH) has also been shown to regulate epithelial transport of certain ions, such as Cl−, HCO3−, and Na+, particularly in the kidney. Although the intestinal epithelium also expressed PTH receptors, little was known regarding its mechanism in the regulation of intestinal ion transport. We investigated the ion regulatory role of PTH in intestinal epithelium-like Caco-2 monolayer by Ussing chamber technique and alternating current impedance spectroscopy. It was found that Caco-2 cells rapidly responded to PTH within 1 min by increasing apical HCO3− secretion. CFTR served as the principal route for PTH-stimulated apical HCO3− efflux, which was abolished by various CFTR inhibitors, namely, NPPB, glycine hydrazide-101 (GlyH-101), and CFTRinh-172, as well as by small interfering RNA against CFTR. Concurrently, the plasma membrane resistance was decreased with no changes in the plasma membrane capacitance or paracellular permeability. HCO3− was probably supplied by basolateral uptake via the electrogenic Na+-HCO3− cotransporter and by methazolamide-sensitive carbonic anhydrase, while the resulting intracellular H+ might be extruded by both apical and basolateral Na+/H+ exchangers. Furthermore, the PTH-stimulated HCO3− secretion was markedly reduced by protein kinase A (PKA) inhibitor (PKI 14–22 amide) and phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY-294002), but not by intracellular Ca2+ chelator (BAPTA-AM) or protein kinase C inhibitor (GF-109203X). In conclusion, the present study provided evidence that PTH directly and rapidly stimulated apical HCO3− secretion through CFTR in PKA- and PI3K-dependent manner, which was a novel noncalciotropic, ion regulatory action of PTH in the intestinal epithelium.

Erythrocyte Dematin Regulates Glucose Transport Via Akt Phosphorylation and 14-3-3zeta Association.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1990-1990
Author(s):  
Morvarid Mohseni ◽  
Anwar Khan ◽  
Athar H. Chishti
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Cell Biology ◽  
Glucose Transporter ◽  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Degradation Pathway ◽  
Actin Binding ◽  
Glucose Transporter 1

Abstract Abstract 1990 Poster Board I-1012 Erythrocyte dematin is a widely expressed actin-binding and bundling protein, and functions as a suppressor of RhoA signaling in fibroblasts (Mohseni and Chishti, Molecular Cell Biology 28: 4712-4718, 2008). Dematin is a substrate of multiple protein kinases, and its actin bundling activity is regulated by cAMP dependent protein kinase. Recently, we identified a novel interaction between dematin and glucose transporter-1 (GLUT1) that is critically important for erythrocyte shape and membrane mechanical properties (Khan et al., Journal of Biological Chemistry 283:14600-14609, 2008). Since homologues of dematin and GLUT1 exist in many non-erythroid cells, we proposed that a conserved mechanism might couple related sugar transporters, such as the insulin-responsive glucose transporter-4 (GLUT4), to the actin cytoskeleton via dematin. Immunocytochemistry established the presence of dematin in 3T3-L1 adipocytes, and a small pool of dematin and GLUT4-containing vesicles co-localized in 3T3-L1 cells under both basal and insulin-stimulated conditions. Plasma membrane sheet assays indicate that upon insulin stimulation, dematin translocates to the plasma membrane along with GLUT4, resulting in partial co-localization at the plasma membrane. Furthermore, dematin RNAi treated 3T3-L1 cells show reduced GLUT4 protein expression, suggesting that dematin may regulate a sub-population of GLUT4 via the lysosomal degradation pathway in adipocytes. Importantly, glucose transport was reduced by ∼28% in 3T3-L1 adipocytes depleted of dematin, and by ∼15% in the dematin headpiece knockout (HPKO) mouse primary adipocytes. Since a significant amount of dematin did not co-localize with GLUT4 in the cytosol and plasma membrane, biochemical interaction between dematin and GLUT4 could not be verified using immunoprecipitation and transfection assays. Although dematin does not bind directly to GLUT4 under these conditions, a possibility existed that this interaction may be transient and mediated through an adaptor protein. Interestingly, dematin contains seven 14-3-3 binding sites, and 14-3-3 adaptor has been shown to be functionally involved in GLUT4 trafficking. We demonstrate that phosphorylated dematin binds to 14-3-3 in 3T3-L1 adipocytes under both basal and insulin stimulated conditions. Mutagenesis studies identify serine-85 on dematin as the primary phospho-binding site for 14-3-3zeta. Furthermore, using pharmacological inhibitors, Akt is identified as the likely protein kinase that phosphorylates dematin to mediate the biochemical interactions between dematin and 14-3-3zeta. Together, our results identify erythrocyte dematin as a potential regulator of glucose transporter trafficking and degradation pathways in adipocytes with functional implications for glucose homeostasis, diabetes, and obesity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Shuttling and sorting lipid-modified cargo into the cilia

2016 ◽  
Vol 44 (5) ◽  
pp. 1273-1280 ◽  
Author(s):  
Louise A. Stephen ◽  
Shehab Ismail
Keyword(s):  
Plasma Membrane ◽  
Human Cell ◽  
Primary Cilia ◽  
Cell Types ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Small G Protein ◽  
Molecular Machinery ◽  
Modified Proteins ◽  
Almost All

Primary cilia are hair-like microtubule-based organelles that can be found on almost all human cell types. Although the cilium is not separated from the cell by membranes, their content is different from that of the cell body and their membrane composition is distinct from that of the plasma membrane. Here, we will introduce a molecular machinery that shuttles and sorts lipid-modified proteins to the cilium, thus contributing in maintaining its distinct composition. The mechanism involves the binding of the GDI-like solubilising factors, uncoordinated (UNC)119a, UNC119b and PDE6D, to the lipid-modified ciliary cargo and the specific release of the cargo in the cilia by the ciliary small G-protein Arl3 in a GTP-dependent manner.

scholarly journals Involvement of Pca1 in ROS-mediated apoptotic cell death induced by alpha-thujone in the fission yeast (Schizosaccharomyces pombe)

2020 ◽  
Vol 20 (4) ◽  
Author(s):  
Hizlan Hincal Agus ◽  
Gizem Kok ◽  
Ezgi Derinoz ◽  
Didem Oncel ◽  
Sedanur Yilmaz
Keyword(s):  
Cell Death ◽  
Schizosaccharomyces Pombe ◽  
Cell Biology ◽  
Apoptotic Cell ◽  
Survival Rates ◽  
Model Organism ◽  
Apoptotic Cell Death ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Apoptotic Factors

ABSTRACT Alpha-thujone, widely used in beverages (1–5 mg/kg), is known to have cytotoxic effects, but the mode of action and the role of potential apoptotic proteins in yeast cell death should be unraveled. In this study, we used Schizosaccharomyces pombe, which is a promising unicellular model organism in mechanistic toxicology and cell biology, to investigate the involvement of pro-apoptotic factors in alpha-thujone-induced cell death. We showed alpha-thujone-induced ROS accumulation-dependent cytotoxicity and apoptosis. In addition, we used superoxide dismutase-deficient cells (sod1 and sod2 mutants) to understand the effect of oxidative stress. Alpha-thujone caused significant cytotoxicity and apoptotic cell death, particularly in sod mutants. Moreover, two potential apoptotic factors, pca1 and pnu1 (pombe caspase-1 and pombe nuc1) were investigated to understand which factor mediates alpha-thujone-induced cell death. Pca1-deficient cells showed increased survival rates and reduced apoptosis in comparison to parental cells after chemical treatment while pnu1 mutation did not cause any significant change and the response was found identical as of parental cells. Yeast responded to alpha-thujone in caspase-dependent manner which was very similar to that for acetic acid. In conclusion, alfa-thujone-induced apoptosis and accounting mechanisms, which were mediated by ROS and driven by Pca1, were clarified in the unicellular model, S. pombe.

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