PGE2 upregulates the Na+/K+ ATPase in HepG2 cells via EP4 receptors and intracellular calcium

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP–Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

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EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

10.1101/224162 ◽

2017 ◽

Author(s):

Chi-Lun Chang ◽

Yu-Ju Chen ◽

Jen Liou

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Live Cell Imaging ◽

Cell Imaging ◽

Time Lapse ◽

Cellular Functions ◽

Binding Ability ◽

Spatiotemporal Regulation ◽

Time Lapse Imaging

AbstractThe endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.SummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

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Deep learning pipeline for cell edge segmentation of time-lapse live cell images

10.1101/191858 ◽

2017 ◽

Cited By ~ 2

Author(s):

Chuangqi Wang ◽

Xitong Zhang ◽

Hee June Choi ◽

Bolun Lin ◽

Yudong Yu ◽

...

Keyword(s):

Neural Networks ◽

Deep Learning ◽

Live Cell Imaging ◽

Phase Contrast ◽

Deep Neural Networks ◽

Cell Imaging ◽

Time Lapse ◽

Live Cell ◽

Live Cells ◽

Quantitative Analyses

AbstractQuantitative live cell imaging has been widely used to study various dynamical processes in cell biology. Phase contrast microscopy is a popular imaging modality for live cell imaging since it does not require labeling and cause any phototoxicity to live cells. However, phase contrast images have posed significant challenges for accurate image segmentation due to complex image features. Fluorescence live cell imaging has also been used to monitor the dynamics of specific molecules in live cells. But unlike immunofluorescence imaging, fluorescence live cell images are highly prone to noise, low contrast, and uneven illumination. These often lead to erroneous cell segmentation, hindering quantitative analyses of dynamical cellular processes. Although deep learning has been successfully applied in image segmentation by automatically learning hierarchical features directly from raw data, it typically requires large datasets and high computational cost to train deep neural networks. These make it challenging to apply deep learning in routine laboratory settings. In this paper, we evaluate a deep learning-based segmentation pipeline for time-lapse live cell movies, which uses small efforts to prepare the training set by leveraging the temporal coherence of time-lapse image sequences. We train deep neural networks using a small portion of images in the movies, and then predict cell edges for the entire image frames of the same movies. To further increase segmentation accuracy using small numbers of training frames, we integrate VGG16 pretrained model with the U-Net structure (VGG16-U-Net) for neural network training. Using live cell movies from phase contrast, Total Internal Reflection Fluorescence (TIRF), and spinning disk confocal microscopes, we demonstrate that the labeling of cell edges in small portions (5∼10%) can provide enough training data for the deep learning segmentation. Particularly, VGG16-U-Net produces significantly more accurate segmentation than U-Net by increasing the recall performance. We expect that our deep learning segmentation pipeline will facilitate quantitative analyses of challenging high-resolution live cell movies.

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A single probe to sense Al(iii) colorimetrically and Cd(ii) by turn-on fluorescence in physiological conditions and live cells, corroborated by X-ray crystallographic and theoretical studies

Dalton Transactions ◽

10.1039/c4dt01433b ◽

2015 ◽

Vol 44 (9) ◽

pp. 4123-4132 ◽

Cited By ~ 35

Author(s):

Chirantan Kar ◽

Soham Samanta ◽

Sudeep Goswami ◽

Aiyagari Ramesh ◽

Gopal Das

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Selective Recognition ◽

Live Cells ◽

Paper Strip ◽

Theoretical Studies ◽

X Ray ◽

Physiological Conditions ◽

Turn On ◽

Single Probe

Selective recognition of Al3+and Cd2+by UV-Vis and fluorescence based techniques using a cinnamaldehyde functionalized conjugated ligand, and its applications in paper strip and live cell imaging.

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Time-Lapse, Live-Cell Imaging of Potassium Efflux in Cell Exposed to Nanosecond Pulsed Electric Fields

Biophysical Journal ◽

10.1016/j.bpj.2020.11.1493 ◽

2021 ◽

Vol 120 (3) ◽

pp. 223a

Author(s):

Flavia Mazzarda ◽

Esin B. Sozer ◽

Julia L. Pittaluga ◽

Claudia Muratori ◽

P. Thomas Vernier

Keyword(s):

Electric Fields ◽

Live Cell Imaging ◽

Cell Imaging ◽

Pulsed Electric Fields ◽

Time Lapse ◽

Live Cell ◽

Potassium Efflux ◽

Nanosecond Pulsed Electric Fields

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Dye selection for live cell imaging of intact siRNA

Biological Chemistry ◽

10.1515/bc-2011-256 ◽

2012 ◽

Vol 393 (1-2) ◽

pp. 23-35 ◽

Cited By ~ 10

Author(s):

Markus Hirsch ◽

Dennis Strand ◽

Mark Helm

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Dyes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Lapse ◽

Live Cell ◽

High Yield ◽

Ph Variation ◽

Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

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Live-Cell Imaging of Caspase Activation for High-Content Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109343207 ◽

2009 ◽

Vol 14 (8) ◽

pp. 956-969 ◽

Cited By ~ 31

Author(s):

Christophe Antczak ◽

Toshimitsu Takagi ◽

Christina N. Ramirez ◽

Constantin Radu ◽

Hakim Djaballah

Keyword(s):

Cell Death ◽

Live Cell Imaging ◽

Cell Imaging ◽

Exposure Period ◽

Live Cells ◽

Assay Method ◽

Caspase Activation ◽

Fluorogenic Substrate ◽

Automated Imaging ◽

First Time

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488™ fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. ( Journal of Biomolecular Screening 2009:956-969)

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Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Journal of Visualized Experiments ◽

10.3791/3145 ◽

2011 ◽

Cited By ~ 35

Author(s):

Imke G. de Jong ◽

Katrin Beilharz ◽

Oscar P. Kuipers ◽

Jan- Willem Veening

Keyword(s):

Bacillus Subtilis ◽

Streptococcus Pneumoniae ◽

Live Cell Imaging ◽

Cell Imaging ◽

Time Lapse ◽

Live Cell ◽

Time Lapse Microscopy

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

10.1101/2020.03.03.974923 ◽

2020 ◽

Author(s):

Patricia A. Clow ◽

Nathaniel Jillette ◽

Jacqueline J. Zhu ◽

Albert W. Cheng

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Repetitive Sequences ◽

Binary Sequence ◽

Three Dimensional ◽

Live Cell ◽

Live Cells ◽

Imaging Method ◽

Genomic Locus ◽

Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

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