Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein

2011 ◽  
Vol 435 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Ekaterina O. Serebrovskaya ◽  
Tatiana V. Gorodnicheva ◽  
Galina V. Ermakova ◽  
Elena A. Solovieva ◽  
George V. Sharonov ◽  
...  
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Green Light ◽  
Specific Cell ◽  
Light Illumination ◽  
Division Rate ◽  
Green Fluorescent ◽  
Mitosis And Meiosis

Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)–tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B–tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B–tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B–tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B–tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B–tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.

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 Light-Regulated Transcription of a Mitochondrial-Targeted K+ Channel

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2507
Author(s):  
Anja J. Engel ◽  
Laura-Marie Winterstein ◽  
Marina Kithil ◽  
Markus Langhans ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
K Channel ◽  
Physiological Changes ◽  
Channel Activity ◽  
Cryptochrome 2 ◽  
Green Fluorescent ◽  
Gfp Tag

The inner membranes of mitochondria contain several types of K+ channels, which modulate the membrane potential of the organelle and contribute in this way to cytoprotection and the regulation of cell death. To better study the causal relationship between K+ channel activity and physiological changes, we developed an optogenetic platform for a light-triggered modulation of K+ conductance in mitochondria. By using the light-sensitive interaction between cryptochrome 2 and the regulatory protein CIB1, we can trigger the transcription of a small and highly selective K+ channel, which is in mammalian cells targeted into the inner membrane of mitochondria. After exposing cells to very low intensities (≤0.16 mW/mm2) of blue light, the channel protein is detectable as an accumulation of its green fluorescent protein (GFP) tag in the mitochondria less than 1 h after stimulation. This system allows for an in vivo monitoring of crucial physiological parameters of mitochondria, showing that the presence of an active K+ channel causes a substantial depolarization compatible with the effect of an uncoupler. Elevated K+ conductance also results in a decrease in the Ca2+ concentration in the mitochondria but has no impact on apoptosis.

scholarly journals Functional Display of an α2 Integrin-Specific Motif (RKK) on the Surface of Baculovirus Particles

2005 ◽  
Vol 4 (4) ◽  
pp. 437-445 ◽  
Author(s):  
Reetta Riikonen ◽  
Heli Matilainen ◽  
Nina Rajala ◽  
Olli Pentikäinen ◽  
Mark Johnson ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Recombinant Baculovirus ◽  
Autographa Californica ◽  
Wild Type ◽  
Receptor Binding Site ◽  
Mammalian Cell Lines ◽  
Green Fluorescent

The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an α2 integrin, the α2I-domain. However, the interaction was not strong enough to overcome binding of wild type gp64 to the unknown cellular receptor(s) on the surface of α2 integrin-expressing cells (CHO-α2β1) or enhance the viral uptake. After treatment of these cells with phospholipase C, internalization of all viruses was blocked or decreased significantly. However, one of the RKK displaying viruses, AcGFP(K)gp64, was still able to internalize into CHO-α2β1 cells, although at a lower level as compared to non-treated cells. This may indicate the possible utilization of a PLC independent alternative route via, in this case, the α2β1 integrin.

scholarly journals An endogenous green fluorescent protein–photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer

Open Biology ◽  
2014 ◽  
Vol 4 (4) ◽  
pp. 130206 ◽  
Author(s):  
Cécile Fourrage ◽  
Karl Swann ◽  
Jose Raul Gonzalez Garcia ◽  
Anthony K. Campbell ◽  
Evelyn Houliston
Keyword(s):  
Energy Transfer ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Parallel Acquisition ◽  
Green Fluorescent

Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria . It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica , we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis

1999 ◽  
Vol 112 (12) ◽  
pp. 1835-1842 ◽  
Author(s):  
G.N. Naumov ◽  
S.M. Wilson ◽  
I.C. MacDonald ◽  
E.E. Schmidt ◽  
V.L. Morris ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Resolution ◽  
Fluorescent Protein ◽  
Specific Cell ◽  
Fixed Tissue ◽  
Cellular Expression ◽  
Liver Surface ◽  
Metastatic Process ◽  
Green Fluorescent

High resolution intravital videomicroscopy has provided a powerful tool for directly observing steps in the metastatic process, and for clarifying molecular mechanisms of metastasis and modes of action of anti-metastasis therapeutics. Cells previously have been identified in vivo using exogenously added fluorescent labels, limiting observations to a few cell divisions, or by natural markers (e.g. melanin) expressed only by specific cell types. Here we tested the utility of stable green fluorescent protein (GFP)-transfected cells for monitoring and quantifying sequential steps in the metastatic process. Using CHO-K1 cells that stably express GFP, we document the visualization and quantification by intravital videomicroscopy of sequential steps in metastasis within mouse liver, from initial arrest of cells in the microvasculature to the growth and angiogenesis of metastases. Individual, non-dividing cells, as well as micro- and macrometastases could clearly be detected and quantified, as could fine cellular details such as pseudopodial projections, even after extended periods of in vivo growth. We quantified the size distribution of micrometastases and their locations relative to the liver surface using 50 micrometer thick formalin-fixed tissue sections. The data suggest preferential growth and survival of micrometastases near the liver surface. Furthermore, we observed a small population of single cells that persisted over the 11 day observation period, which may represent dormant cells with potential for subsequent proliferation. This study demonstrates the advantages of GFP-expressing cells, coupled with real-time high resolution videomicroscopy, for long-term in vivo studies to visualize and quantify sequential steps of the metastatic process.

Organization of transport from endoplasmic reticulum to Golgi in higher plants

2000 ◽  
Vol 28 (4) ◽  
pp. 505-512 ◽  
Author(s):  
A. V. Andreeva ◽  
H. Zheng ◽  
C. M. Saint-Jore ◽  
M. A. Kutuzov ◽  
D. E. Evans ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Plant Cells ◽  
Yeast Cells ◽  
Green Fluorescent

In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endo-membrane trafficking in plant cells.

scholarly journals Two-Step Assembly Dynamics of the Bacillussubtilis Divisome

2009 ◽  
Vol 191 (13) ◽  
pp. 4186-4194 ◽  
Author(s):  
Pamela Gamba ◽  
Jan-Willem Veening ◽  
Nigel J. Saunders ◽  
Leendert W. Hamoen ◽  
Richard A. Daniel
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Localization Pattern ◽  
Ftsz Ring ◽  
Time Lapse Microscopy ◽  
Green Fluorescent ◽  
Posttranscriptional Level

ABSTRACT Cell division in bacteria is carried out by about a dozen proteins which assemble at midcell and form a complex known as the divisome. To study the dynamics and temporal hierarchy of divisome assembly in Bacillus subtilis, we have examined the in vivo localization pattern of a set of division proteins fused to green fluorescent protein in germinating spores and vegetative cells. Using time series and time-lapse microscopy, we show that the FtsZ ring assembles early and concomitantly with FtsA, ZapA, and EzrA. After a time delay of at least 20% of the cell cycle, a second set of division proteins, including GpsB, FtsL, DivIB, FtsW, Pbp2B, and DivIVA, are recruited to midcell. Together, our data provide in vivo evidence for two-step assembly of the divisome. Interestingly, overproduction of FtsZ advances the temporal assembly of EzrA but not of DivIVA, suggesting that a signal different from that of FtsZ polymerization drives the assembly of late divisome proteins. Microarray analysis shows that FtsZ depletion or overexpression does not significantly alter the transcription of division genes, supporting the hypothesis that cell division in B. subtilis is mainly regulated at the posttranscriptional level.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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