Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

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Real-Time Optical Monitoring of Ligand-Mediated Internalization of α1b-Adrenoceptor with Green Fluorescent Protein

Molecular Endocrinology ◽

10.1210/mend.12.8.0149 ◽

1998 ◽

Vol 12 (8) ◽

pp. 1099-1111 ◽

Cited By ~ 2

Author(s):

Takeo Awaji ◽

Akira Hirasawa ◽

Masakazu Kataoka ◽

Hitomi Shinoura ◽

Yasuhisa Nakayama ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Real Time ◽

Fluorescent Protein ◽

Optical Monitoring ◽

Green Fluorescent

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Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.97.13.7237 ◽

2000 ◽

Vol 97 (13) ◽

pp. 7237-7242 ◽

Cited By ~ 126

Author(s):

M. F. Garcia-Parajo ◽

G. M. J. Segers-Nolten ◽

J.-A. Veerman ◽

J. Greve ◽

N. F. van Hulst

Keyword(s):

Green Fluorescent Protein ◽

Real Time ◽

Fluorescent Protein ◽

Fluorescence Emission ◽

Protein Molecules ◽

Green Fluorescent

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Quantification of green fluorescent protein fluorescence using real-time PCR thermal cycler

BioTechniques ◽

10.2144/000112221 ◽

2006 ◽

Vol 41 (2) ◽

pp. 150-154 ◽

Cited By ~ 5

Author(s):

Jan Utermark ◽

Petr Karlovsky

Keyword(s):

Green Fluorescent Protein ◽

Real Time ◽

Real Time Pcr ◽

Fluorescent Protein ◽

Green Fluorescent Protein Fluorescence ◽

Protein Fluorescence ◽

Thermal Cycler ◽

Green Fluorescent

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Construction of Self-Transmissible Green Fluorescent Protein-Based Biosensor Plasmids and Their Use for Identification of N-Acyl Homoserine-Producing Bacteria in Lake Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.00677-10 ◽

2010 ◽

Vol 76 (18) ◽

pp. 6119-6127 ◽

Cited By ~ 11

Author(s):

Putthapoom Lumjiaktase ◽

Claudio Aguilar ◽

Tom Battin ◽

Kathrin Riedel ◽

Leo Eberl

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Bacterial Species ◽

Natural Habitat ◽

Homoserine Lactone ◽

Signal Molecules ◽

Broad Host Range ◽

Construction Of Self ◽

Green Fluorescent ◽

The Impact

ABSTRACT Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

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Green Fluorescent Protein as a Real Time Quantitative Reporter of Heterologous Protein Production

Biotechnology Progress ◽

10.1021/bp970121b ◽

1998 ◽

Vol 14 (2) ◽

pp. 351-354 ◽

Cited By ~ 76

Author(s):

C.R. Albano ◽

L. Randers-Eichhorn ◽

W.E. Bentley ◽

G. Rao

Keyword(s):

Green Fluorescent Protein ◽

Real Time ◽

Protein Production ◽

Heterologous Protein ◽

Fluorescent Protein ◽

Heterologous Protein Production ◽

Green Fluorescent

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Impact of signaling microcompartment geometry on GPCR dynamics in live retinal photoreceptors

The Journal of General Physiology ◽

10.1085/jgp.201210818 ◽

2012 ◽

Vol 140 (3) ◽

pp. 249-266 ◽

Cited By ~ 19

Author(s):

Mehdi Najafi ◽

Mohammad Haeri ◽

Barry E. Knox ◽

William E. Schiesser ◽

Peter D. Calvert

Keyword(s):

Green Fluorescent Protein ◽

Molecular Diffusion ◽

Fluorescent Protein ◽

Lateral Diffusion ◽

Live Cell ◽

Confocal Imaging ◽

Slow Phase ◽

Protein Diffusion ◽

Green Fluorescent ◽

The Impact

G protein–coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell–imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti–sapphire laser focused to the diffraction limit, which produced small sub–cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient Dlat = 0.130 ± 0.012 µm2s−1. Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.

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