scholarly journals In vivo imaging of calcium dynamics in zebrafish hepatocytes

2022 ◽  
Author(s):  
Macarena Pozo-Morales ◽  
Ines Garteizgogeascoa ◽  
Camille Perazzolo ◽  
Sumeet Pal Singh
Keyword(s):  
Calcium Signaling ◽  
In Vivo Imaging ◽  
Molecular Mechanisms ◽  
Calcium Oscillations ◽  
Calcium Flux ◽  
Calcium Dynamics ◽  
Cell Ablation ◽  
Mammalian Liver ◽  
Intracellular Calcium Dynamics

Hepatocytes were the first cell-type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a non-invasive manner due to the optical inaccessibility of the mammalian liver. Here we take advantage of the transparency of the zebrafish larvae to develop a setup that allows in vivo imaging of calcium flux in zebrafish hepatocytes at cellular resolution. Using this, we provide quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo, which are lost upon starvation. Feeding recommences calcium waves in the liver, but in a spatially restricted manner. Further, ethanol treatment as well as cell ablation induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, while the latter leads to a single calcium spike. Overall, we demonstrate the presence of oscillations, waves and spikes in vivo. Thus, our study introduces a platform for observing diverse calcium dynamics while maintaining the native environment of the liver, which will help investigations into the dissection of molecular mechanisms supporting the intra- and intercellular calcium signaling in the liver.

scholarly journals Orange Fluorescent Proteins: Filling the Spectral Gap

2014 ◽  
Vol 70 (a1) ◽  
pp. C1670-C1670
Author(s):  
Sergei Pletnev ◽  
Daria Shcherbakova ◽  
Oksana Subach ◽  
Vladimir Malashkevich ◽  
Steven Almo ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Spectral Properties ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Emission Wavelength ◽  
Microscopy Imaging ◽  
Large Stokes Shift

Fluorescent proteins (FPs) have become valuable tools for molecular biology, biochemistry, medicine, and cancer research. Starting from parent green fluorescent protein (GFP), most challenging task of the FPs studies was the development of FPs with longer excitation/emission wavelength. This pursuit was motivated by advantages of so-called red-shifted FPs, namely, lower background of cellular autofluorescence in microscopy, lower light scattering and reduced tissue absorbance of longer wavelengths for in vivo imaging. In addition to FPs with regular spectral properties, there are proteins of other types available, including FPs with a large Stokes shift and photoconvertible FPs. These special kinds of FPs have become useful in super-resolution microscopy, imaging of enzyme activities, protein-protein interactions, photolabeling, and in vivo imaging. According to their emission wavelength, red-shifted FPs could be divided in the following groups: 520-540 nm yellow FPs (YFPs), 540-570 nm orange FPs (OFPs), 570-620 nm red FPs (RFPs), and > 620 nm far-RFPs. Red shift of the excitation/emission bands of these FPs is predominantly achieved by extension of the conjugated system of the chromophore and its protonation/deprotonation. The variety of spectral properties of FPs (excitation and emission wavelength, quantum yield, brightness, photo- and pH- stability, photoconversion, large Stokes shift, etc) results from the different chromophore structures and its interactions with surrounding amino acid residues. In this work we focus on structural studies and molecular mechanisms of FPs with orange emission.

scholarly journals TRPM4 is required for calcium oscillations downstream from the stretch-activated TRPV4 channel

2021 ◽  
Author(s):  
Oleg Yarishkin ◽  
Tam T. Phuong ◽  
Felix Vazquez-Chona ◽  
Jacques A Bertrand ◽  
Sarah Redmon ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Molecular Mechanisms ◽  
Transient Receptor Potential ◽  
Membrane Surface ◽  
Receptor Potential ◽  
Calcium Oscillations ◽  
Transient Receptor Potential Vanilloid ◽  
Transient Receptor Potential Melastatin ◽  
Transient Receptor ◽  
Insight Into

Transduction of mechanical information is influenced by physical, chemical and thermal cues but the molecular mechanisms through which transducer activation shapes temporal signaling remain underexplored. In the present study, electrophysiology, histochemistry and functional imaging were combined with gene silencing and heterologous expression to gain insight into calcium signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated nonselective cation channel. We show that trabecular meshwork (TM) cells, which employ mechanotransduction to actively regulate intraocular pressure, respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with a monovalent cation current that was suppressed by BAPTA, Ruthenium Red and 9-phenanthrol, an inhibitor of TRPM4 (Transient Receptor Potential Melastatin 4) channels. Accordingly, TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings indicated that TRPM4-dependent oscillations required release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. These findings provide insight into the complexity of membrane-cytosolic interactions during TRPV4 signaling and may foster strategies to promote homeostatic regulation and counter pathological remodeling within the conventional outflow pathway of the mammalian eye.

scholarly journals Spatiotemporal restriction of endothelial cell calcium signaling is required during leukocyte transmigration

2020 ◽  
Vol 218 (1) ◽  
Author(s):  
Prarthana J. Dalal ◽  
David P. Sullivan ◽  
Evan W. Weber ◽  
David B. Sacks ◽  
Matthias Gunzer ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Calcium Signaling ◽  
Dominant Negative ◽  
Calcium Flux ◽  
Inhibitory Peptide ◽  
Leukocyte Transmigration ◽  
Cell Calcium ◽  
Leukocyte Transendothelial Migration ◽  
Calmodulin Kinase

Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.

scholarly journals Independently paced calcium oscillations in progenitor and differentiated cells in an ex vivo epithelial organ

2021 ◽  
Author(s):  
Anna Kim ◽  
Amanda Nguyen ◽  
Marco Marchetti ◽  
Denise Montell ◽  
Beth Pruitt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Cell Types ◽  
Cytosolic Calcium ◽  
Calcium Oscillations ◽  
Enteroendocrine Cells ◽  
Calcium Dynamics ◽  
Differentiated Cells ◽  
Different Cell Types

Cytosolic calcium is a highly dynamic, tightly regulated, and broadly conserved cellular signal. Calcium dynamics have been studied widely in cellular monocultures, yet in vivo most organs comprise heterogeneous populations of stem and differentiated cells. We examined calcium dynamics in each cell type of the adult Drosophila intestine, a self-renewing epithelial organ where multipotent stem cells give rise to mature absorptive enterocytes and secretory enteroendocrine cells. Here we perform live imaging of whole organs ex vivo, and we employ orthogonal expression of red and green calcium sensors to determine whether calcium oscillations between different cell types are coupled. We show that stem cell daughters adopt strikingly distinct patterns of calcium oscillations when they acquire their terminal fates: enteroendocrine cells exhibit single-cell calcium oscillations, while long-range calcium waves propagate rhythmically across large fields of enterocytes. These multicellular waves do not propagate through progenitor cells (stem cells and undifferentiated enterocyte precursors), whose oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates calcium oscillations in all three cell types, even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. Our findings establish that cells adopt fate-specific modes of calcium dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in the same epithelium are paced independently.

scholarly journals In vivo imaging of calcium dynamics within identified target cells during synaptogenesis

2003 ◽  
Vol 43 (supplement) ◽  
pp. S233
Author(s):  
H. Kazama ◽  
T. Morimoto-Tanifuji ◽  
A. Nose
Keyword(s):  
In Vivo Imaging ◽  
Target Cells ◽  
Calcium Dynamics

scholarly journals Long-Term Oral Administration of Theaphenon-E Improves Cardiomyocyte Mechanics and Calcium Dynamics by Affecting Phospholamban Phosphorylation and ATP Production

2018 ◽  
Vol 47 (3) ◽  
pp. 1230-1243 ◽  
Author(s):  
Leonardo Bocchi ◽  
Monia Savi ◽  
Valeria Naponelli ◽  
Rocchina Vilella ◽  
Gianluca Sgarbi ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Intracellular Calcium ◽  
Green Tea ◽  
Mitochondrial Respiration ◽  
Calcium Dynamics ◽  
Dietary Polyphenols ◽  
Intracellular Calcium Dynamics ◽  
In Vivo Administration

Background/Aims: Dietary polyphenols from green tea have been shown to possess cardio-protective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms. Methods: The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2. Results: GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB. Conclusions: Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2.

scholarly journals Non-Invasive In Vivo Imaging of Calcium Signaling in Mice

PLoS ONE ◽  
2007 ◽  
Vol 2 (10) ◽  
pp. e974 ◽  
Author(s):  
Kelly L. Rogers ◽  
Sandrine Picaud ◽  
Emilie Roncali ◽  
Raphaël Boisgard ◽  
Cesare Colasante ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
In Vivo Imaging ◽  
Non Invasive

scholarly journals Effects of Standardized Green Tea Extract and Its Main Component, EGCG, on Mitochondrial Function and Contractile Performance of Healthy Rat Cardiomyocytes

Nutrients ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2949
Author(s):  
Rocchina Vilella ◽  
Gianluca Sgarbi ◽  
Valeria Naponelli ◽  
Monia Savi ◽  
Leonardo Bocchi ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Green Tea ◽  
Mitochondrial Function ◽  
Tap Water ◽  
Water Solution ◽  
Calcium Dynamics ◽  
Rat Cardiomyocytes ◽  
Endoplasmic Reticulum Calcium ◽  
Intracellular Calcium Dynamics

We recently showed that the long-term in vivo administration of green tea catechin extract (GTE) resulted in hyperdynamic cardiomyocyte contractility. The present study investigates the mechanisms underlying GTE action in comparison to its major component, epigallocatechin-3-gallate (EGCG), given at the equivalent amount that would be in the entirety of GTE. Twenty-six male Wistar rats were given 40 mL/day of a tap water solution with either standardized GTE or pure EGCG for 4 weeks. Cardiomyocytes were then isolated for the study. Cellular bioenergetics was found to be significantly improved in both GTE- and EGCG-fed rats compared to that in controls as shown by measuring the maximal mitochondrial respiration rate and the cellular ATP level. Notably, the improvement of mitochondrial function was associated with increased levels of oxidative phosphorylation complexes, whereas the cellular mitochondrial mass was unchanged. However, only the GTE supplement improved cardiomyocyte mechanics and intracellular calcium dynamics, by lowering the expression of total phospholamban (PLB), which led to an increase of both the phosphorylated-PLB/PLB and the sarco-endoplasmic reticulum calcium ATPase/PLB ratios. Our findings suggest that GTE might be a valuable adjuvant tool for counteracting the occurrence and/or the progression of cardiomyopathies in which mitochondrial dysfunction and alteration of intracellular calcium dynamics constitute early pathogenic factors.

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