Imaging local estrogen production in single living cells with recombinant fluorescent indicators

Picazo, Fernando; Domingo, Beatriz; Pérez‐Ortiz, José Manuel; Llopis, Juan

doi:10.1016/j.bios.2010.09.024

Cited by 3 publications

(5 citation statements)

References 37 publications

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“…In spite of this, until recently (Zhao et al, 2011a) the only available color of single-wavelength GECI was green [camgaroo (Yu et al, 2003), pericam (Nagai et al, 2001), and the Case sensors (Leder et al, 2010) were engineered from EYFP but have GFP-like green fluorescence]. For analytes other than Ca 2+ , sensors with several different colors have been constructed: Hyper, a yellow peroxide sensor (Belousov et al, 2006); blue, cyan, green, and yellow sensors for maltose (Marvin et al, 2011); a yellow sensor of molecular strain (Ichimura et al, 2012); Frex, a yellow sensor of NADH (Zhao et al, 2011b); PermELI, a yellow estrogen sensor (Picazo et al, 2011); and a yellow indicator of ATP:ADP ratio (Berg et al, 2009). …”

Section: Introductionmentioning

confidence: 99%

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Akerboom

Calderón

Tian

et al. 2013

Front. Mol. Neurosci.

667

663

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Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

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Section: Introductionmentioning

confidence: 99%

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Akerboom

Calderón

Tian

et al. 2013

Front. Mol. Neurosci.

667

663

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“…ImageJ software [47] with customized macros was used to subtract the background from raw images and to create ratio images in intensity-modulated display mode (Venus images were used as intensity modulator). We also used the so called 3-Cube method to estimate the absolute FRET efficiency ( E ) and the relative concentration of donor and acceptor fluorochromes ([D]/[A]) [48], [49].…”

Section: Methodsmentioning

confidence: 99%

Visualization of Phosphatidic Acid Fluctuations in the Plasma Membrane of Living Cells

2014

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We developed genetically-encoded fluorescent sensors based on Förster Resonance Energy Transfer to monitor phosphatidic acid (PA) fluctuations in the plasma membrane using Spo20 as PA-binding motif. Basal PA levels and phospholipase D activity varied in different cell types. In addition, stimuli that activate PA phosphatases, leading to lower PA levels, increased lamellipodia and filopodia formation. Lower PA levels were observed in the leading edge than in the trailing edge of migrating HeLa cells. In MSC80 and OLN93 cells, which are stable cell lines derived from Schwann cells and oligodendrocytes, respectively, a higher ratio of diacylglycerol to PA levels was demonstrated in the membrane processes involved in myelination, compared to the cell body. We propose that the PA sensors reported here are valuable tools to unveil the role of PA in a variety of intracellular signaling pathways.

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“…84 Estrogens altered the fluorescence signal of cells transfected with the indicators in a dose-dependent manner. They imaged local estrogen production in adrenocortical H295 cells expressing aromatase and transfected with the fluorescent sensors.…”

Section: Single Cell Analysis By Direct Fluorescencementioning

confidence: 99%

“…Picazo et al developed biosensors based on the estrogen receptor R-ligand binding domain and fluorescent proteins to detect estrogenic compounds. 84 Estrogens altered the fluorescence signal of cells transfected with the indicators in a dosedependent manner. They imaged local estrogen production in adrenocortical H295 cells expressing aromatase and transfected with the fluorescent sensors.…”

Section: ' Single Cell Analysis By Direct Fluorescencementioning

confidence: 99%

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Chemical Analysis of Single Cells

et al. 2011

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Models and Data Analysis for Monitoring Single Cell Exocytosis 4379 Microfabricated Devices in the Measurement of Exocytosis at Single Cells 4380 Electrode Arrays 4380 Field Effect Transistors Used to Monitor Exocytosis at Cells 4380 Impedance Measurements 4380 Scanning Electrochemical Microscopy (SECM) Measurements at Single Cells 4380 Morphology 4380 Metabolism 4381 Release 4381 Uptake 4381 Ion Selective Electrodes 4381

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Imaging local estrogen production in single living cells with recombinant fluorescent indicators

Cited by 3 publications

References 37 publications

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Visualization of Phosphatidic Acid Fluctuations in the Plasma Membrane of Living Cells

Chemical Analysis of Single Cells

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