scholarly journals FLUORESCENT-PROTEIN STAINING OF NUCLEOLI IN MONOLAYER CELL CULTURES: DETERMINANTS AND SIGNIFICANCE

1970 ◽  
Vol 18 (10) ◽  
pp. 746-755 ◽  
Author(s):  
JOHN C. MAISEL
Keyword(s):  
Fluorescent Protein ◽  
Human Tumor ◽  
Logarithmic Growth Phase ◽  
Hela Cell Lines ◽  
Ionic Bonds ◽  
Γ Globulins ◽  
Nucleolar Component ◽  
Protein Staining ◽  
Ionic Binding ◽  
Fixed Cell

In order to distinguish whether fluorescent-protein staining of nucleoli in acetone-fixed cell culture monolayers is a useless artifact or whether nucleolus-globulin affinity reflects a physiologic interaction between cells and serum, the phenomenon was investigated by further characterizing the interacting components and the conditions required. Staining proved to be a time-dependent and temperature-dependent, two-step procedure: initial modification of nucleoli by smaller serum γ-globulins is required for later ionic binding to nucleoli of fluorescein-labeled, nonimmune α-, β- or larger α-globulins. The initial modification is essential only in cells grown in minimal serum and harvested toward the end of the logarithmic growth phase of the culture. The nucleolar component binding the conjugate is both heat- and pH-stable, is maintained in situ apparently by ionic bonds and most likely includes a saline-insoluble, nonhistone basic protein. Previous serum modification of nucleoli in living primarily explanted monkey kidney cells, detected with this technique, could not be demonstrated with established H-Ep 2 or HeLa cell lines of human tumor origin. These data suggest that serum-dependent nucleolar fluorescent protein staining might be a useful artifact, signifying something about nucleolar organization or content of unexplained relation to cell growth.

scholarly journals Degradation of SAMHD1 by Vpx Is Independent of Uncoating

2015 ◽  
Vol 89 (10) ◽  
pp. 5701-5713 ◽  
Author(s):  
Paula Jáuregui ◽  
Eric C. Logue ◽  
Megan L. Schultz ◽  
Stephanie Fung ◽  
Nathaniel R. Landau
Keyword(s):  
T Cells ◽  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Hela Cell ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Nuclear Localization Sequence ◽  
Hela Cell Lines ◽  
Localization Sequence ◽  
Green Fluorescent

ABSTRACTSterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm.IMPORTANCESAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.

scholarly journals A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0241161
Author(s):  
Timothy S. Jarvela ◽  
Kriti Chaplot ◽  
Iris Lindberg
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Alpha Synuclein ◽  
Cellular Processes ◽  
Tev Protease ◽  
Protease Cleavage ◽  
Green Fluorescent ◽  
Fixed Cell ◽  
Cell Microscopy ◽  
Extracellular Environment

Alpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of alpha-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized alpha-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between alpha-synuclein and green fluorescent protein and subjected cells to brief treatment with TEV protease after incubation with tagged PFFs. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, protection from TEV cleavage can be used to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.

scholarly journals Minimal genetically encoded tags for fluorescent protein labeling in living neurons

2021 ◽  
Author(s):  
Aleksandra Arsić ◽  
Cathleen Hagemann ◽  
Nevena Stajković ◽  
Timm Schubert ◽  
Ivana Nikić-Spiegel
Keyword(s):  
Click Chemistry ◽  
Unnatural Amino Acids ◽  
Genome Engineering ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Live Cell ◽  
Cell Labeling ◽  
Neurofilament Light Chain ◽  
Neurofilament Light ◽  
Fixed Cell

AbstractModern light microscopy, including super-resolution techniques, brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with click chemistry. UAAs are site-specifically incorporated into a protein of interest by genetic code expansion. If the UAA carries a strained alkene or alkyne moiety it can be conjugated to a tetrazine-bearing fluorophore via a strain-promoted inverse-electron-demand Diels–Alder cycloaddition (SPIEDAC), a variant of bioorthogonal click chemistry. The minimal size of the incorporated tag and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision make SPIEDAC live-cell labeling unique. However, until now, this type of labeling has not been used in complex, non-dividing cells, such as neurons. Using neurofilament light chain as a target protein, we established SPIEDAC labeling in living primary neurons and applied it for fixed-cell, live-cell, dual-color pulse—chase and super-resolution microscopy. We also show that SPIEDAC labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced microscopy techniques, we anticipate that SPIEDAC labeling will contribute to novel discoveries in neurobiology.

scholarly journals Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
M. Neefjes ◽  
B. A. C. Housmans ◽  
G. G. H. van den Akker ◽  
L. W. van Rhijn ◽  
T. J. M. Welting ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Fluorescent Protein ◽  
Cellular Signaling ◽  
Reporter Genes ◽  
Firefly Luciferase ◽  
Body Fluids ◽  
Signal Interference ◽  
Red Fluorescent Protein ◽  
Hela Cell Lines ◽  
Slow Kinetics

AbstractReporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we compared the characteristics of five reporter genes (Firefly luciferase, stable- and unstable Nano luciferase, secretable Gaussia luciferase and Red Fluorescent Protein) to study complex body fluids. For this comparison, the NFκB Response Element (NFκB-RE) and Smad Binding Element (SBE) were identically cloned into the five different reporter vectors. Reporter characteristics were evaluated by kinetic and concentration–response measurements in SW1353 and HeLa cell lines. Finally, reporter compatibility with complex body fluids (fetal calf serum, knee joint synovial fluid and human serum) and inter-donor variation were evaluated. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases. Intracellularly measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. Secreted Gaussia luciferase appeared to be incompatible with complex body fluids, due to variability in inter-donor signal interference. Unstable Nano luciferase demonstrated clear inducibility, high sensitivity and compatibility with complex body fluids and therefore can be recommended for cellular signaling studies using complex body fluids.

scholarly journals High-Content Screening: A New Approach to Easing Key Bottlenecks in the Drug Discovery Process

1997 ◽  
Vol 2 (4) ◽  
pp. 249-259 ◽  
Author(s):  
Kenneth A. Giuliano ◽  
Robbin L. DeBiasio ◽  
R. Terry Dunlay ◽  
Albert Gough ◽  
Joanne M. Volosky ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput Screening ◽  
Nuclear Dna ◽  
Spatial Information ◽  
Fluorescent Protein ◽  
Human Tumor ◽  
Nuclear Dna Content ◽  
High Content Screening ◽  
Drug Induced ◽  
Cell Functions

Recent improvements in target discovery and high throughput screening (HTS) have increased the pressure at key points along the drug discovery pipeline. High-content screening (HCS) was developed to ease bottlenecks that have formed at target validation and lead optimization points in the pipeline. HCS defines the role of targets in cell functions by combining fluorescence-based reagents with the ArrayScan™ System to automatically extract temporal and spatial information about target activities within cells. The ArrayScan System is a tabletop instrument that includes optics for subcellular resolution of fluorescence signals from many cells in a field within a well of a microtiter plate. One demonstrated application is a high-content screen designed to measure the drug-induced transport of a green fluorescent protein-human glucocorticoid receptor chimeric protein from the cytoplasm to the nucleus of human tumor cells. A high-content screen is also described for the multiparametric measurement of apoptosis. This single screen provides measurements of nuclear size and shape changes, nuclear DNA content, mitochondrial potential, and actin-cytoskeletal rearrangements during drug-induced programmed cell death. The next generation HCS system is a miniaturized screening platform, the CellChip™ System, that will increase the throughput of HCS, while integrating HCS with HTS on the same platform.

scholarly journals Nestin-Linked Green Fluorescent Protein Transgenic Nude Mouse for Imaging Human Tumor Angiogenesis

2005 ◽  
Vol 65 (12) ◽  
pp. 5352-5357 ◽  
Author(s):  
Yasuyuki Amoh ◽  
Meng Yang ◽  
Lingna Li ◽  
Jose Reynoso ◽  
Michael Bouvet ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Nude Mouse ◽  
Tumor Angiogenesis ◽  
Fluorescent Protein ◽  
Human Tumor ◽  
Green Fluorescent
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