scholarly journals Localization of Phosphatidylinositol (3,4,5)-Trisphosphate to Phagosomes in Entamoeba histolytica Achieved Using Glutathione S-Transferase- and Green Fluorescent Protein-Tagged Lipid Biosensors

2009 ◽  
Vol 78 (1) ◽  
pp. 125-137 ◽  
Author(s):  
Yevgeniya A. Byekova ◽  
Rhonda R. Powell ◽  
Brenda H. Welter ◽  
Lesly A. Temesvari
Keyword(s):  
Green Fluorescent Protein ◽  
Entamoeba Histolytica ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Major Product ◽  
Pi3 Kinase ◽  
Ph Domain ◽  
Intestinal Protozoan ◽  
Ph Domains ◽  
Green Fluorescent

ABSTRACT Entamoeba histolytica is an intestinal protozoan parasite that causes amoebic dysentery and liver abscess. Phagocytosis by the parasite is a critical virulence process, since it is a prerequisite for tissue invasion and establishment of chronic infection. While the roles of many of the proteins that regulate phagocytosis-related signaling events in E. histolytica have been characterized, the functions of lipids in this cellular process remain largely unknown in this parasite. In other systems, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a major product of phosphoinositide 3 kinase (PI3-kinase) activity, is essential for phagocytosis. Pleckstrin homology (PH) domains are protein domains that specifically bind to PIP3. In this study, we utilized glutathione S-transferase (GST)- and green fluorescent protein (GFP)-labeled PH domains as lipid biosensors to characterize the spatiotemporal aspects of PIP3 distribution during various endocytic processes in E. histolytica. PIP3-specific biosensors accumulated at extending pseudopodia and in phagosomal cups in trophozoites exposed to erythrocytes but did not localize to pinocytic compartments during the uptake of a fluid-phase marker, dextran. Our results suggest that PIP3 is involved in the early stages of phagosome formation in E. histolytica. In addition, we demonstrated that PIP3 exists at high steady-state levels in the plasma membrane of E. histolytica and that these levels, unlike those in mammalian cells, are not abolished by serum withdrawal. Finally, expression of a PH domain in trophozoites inhibited erythrophagocytosis and enhanced motility, providing genetic evidence supporting the role of PI3-kinase signaling in these processes in E. histolytica.

scholarly journals Dual Targeting of Osh1p, a Yeast Homologue of Oxysterol-binding Protein, to both the Golgi and the Nucleus-Vacuole Junction

2001 ◽  
Vol 12 (6) ◽  
pp. 1633-1644 ◽  
Author(s):  
Timothy P. Levine ◽  
Sean Munro
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Cholesterol Homeostasis ◽  
Deletion Mapping ◽  
Ph Domain ◽  
Oxysterol Binding Protein ◽  
Wide Range ◽  
Ph Domains ◽  
Green Fluorescent

Oxysterol binding protein (OSBP) is the only protein known to bind specifically to the group of oxysterols with potent effects on cholesterol homeostasis. Although the function of OSBP is currently unknown, an important role is implicated by the existence of multiple homologues in all eukaryotes so far examined. OSBP and a subset of homologues contain pleckstrin homology (PH) domains. Such domains are responsible for the targeting of a wide range of proteins to the plasma membrane. In contrast, OSBP is a peripheral protein of Golgi membranes, and its PH domain targets to the trans-Golgi network of mammalian cells. In this article, we have characterized Osh1p, Osh2p, and Osh3p, the three homologues of OSBP inSaccharomyces cerevisiae that contain PH domains. Examination of a green fluorescent protein (GFP) fusion to Osh1p revealed a striking dual localization with the protein present on both the late Golgi, and in the recently described nucleus-vacuole (NV) junction. Deletion mapping revealed that the PH domain of Osh1p specified targeting to the late Golgi, and an ankyrin repeat domain targeting to the NV junction, the first such targeting domain identified for this structure. GFP fusions to Osh2p and Osh3p showed intracellular distributions distinct from that of Osh1p, and their PH domains appear to contribute to their differing localizations.

Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

1999 ◽  
Vol 112 (16) ◽  
pp. 2705-2714
Author(s):  
E.M. Burns ◽  
L. Christopoulou ◽  
P. Corish ◽  
C. Tyler-Smith
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Quantitative Measurement ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Chromosome Loss ◽  
Facs Analysis ◽  
Fluorescent Cells ◽  
Green Fluorescent ◽  
Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo

2018 ◽  
Vol 194 ◽  
pp. 29-39 ◽  
Author(s):  
Fatemeh Motevalli ◽  
Azam Bolhassani ◽  
Shilan Hesami ◽  
Sepideh Shahbazi
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants

2003 ◽  
Vol 373 (2) ◽  
pp. 403-408 ◽  
Author(s):  
Nadya G. GURSKAYA ◽  
Arkady F. FRADKOV ◽  
Natalia I. POUNKOVA ◽  
Dmitry B. STAROVEROV ◽  
Maria E. BULINA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Random Mutagenesis ◽  
Wild Type ◽  
Green Luminescence ◽  
Mammalian Cell Lines ◽  
Biological Tool ◽  
Fluorescent Pattern ◽  
Green Fluorescent

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu222→Gly (E222G) substitution, which removed the evolutionarily invariant Glu222. Re-introduction of Glu222 into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of ‘humanized’ aceGFP and β-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

scholarly journals Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods

2008 ◽  
Vol 411 (2) ◽  
pp. 441-448 ◽  
Author(s):  
Shu-Chin Yip ◽  
Robert J. Eddy ◽  
Angie M. Branch ◽  
Huan Pang ◽  
Haiyan Wu ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Leading Edge ◽  
Carcinoma Cells ◽  
Immunological Methods ◽  
Ph Domain ◽  
Membrane Recruitment ◽  
Secondary Messenger ◽  
Ph Domains ◽  
Green Fluorescent ◽  
Kinetics Of

Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P3, which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P3 in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P3 synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P3 production using a specific monoclonal anti-PtdIns(3,4,5)P3 antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P3 staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P3 levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P3 production, measured by the membrane translocation of an epitope-tagged BTKPH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4–5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P3 hydrolysis by measuring the decay of the PtdIns(3,4,5)P3 signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P3 membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P3 turnover occurs within seconds of synthesis. In contrast, BTKPH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P3 by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P3 accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P3] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P3in vitro. These data suggest that anti-PtdIns(3,4,5)P3 antibodies are a useful tool to detect localized PtdIns(3,4,5)P3, and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.

scholarly journals Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools

1998 ◽  
Vol 143 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Péter Várnai ◽  
Tamás Balla
Keyword(s):  
Fluorescent Probe ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Protein Fusion ◽  
Fusion Construct ◽  
Cellular Expression ◽  
Ph Domains ◽  
Green Fluorescent

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol– labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

scholarly journals High-Titer Retroviral Vectors Containing the Enhanced Green Fluorescent Protein Gene for Efficient Expression in Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3316-3321 ◽  
Author(s):  
Ana Limón ◽  
Javier Briones ◽  
Teresa Puig ◽  
Mercé Carmona ◽  
Oscar Fornas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hematopoietic Cells ◽  
Retroviral Vectors ◽  
Green Fluorescent Protein Gene ◽  
Efficient System ◽  
Green Fluorescent

Abstract Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34+ cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.

scholarly journals Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

1999 ◽  
Vol 10 (6) ◽  
pp. 2033-2050 ◽  
Author(s):  
Karen Jordan ◽  
Joell L. Solan ◽  
Michel Dominguez ◽  
Michael Sia ◽  
Art Hand ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Dye Transfer ◽  
Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

Sign in / Sign up

Export Citation Format

Share Document