A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro

AbstractDopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in finely controlling output functions is currently challenging, primarily due to a lack of techniques to visualize spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed ‘R-GenGAR-DA’. More specifically, a circular permutated red fluorescent protein (cpmApple) was inserted into the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated reasonable dynamic range (ΔF/F0 = –50%) and DA affinity (EC50 = 0.7 µM) as well as the highest selectivity for DA over NE (143-fold) amongst available DA biosensors. Due to its high selectivity, R-GenGAR-DA1.2 allowed dual-color fluorescence live imaging for monitoring DA and NE, combined with the existing green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (>350-fold) in HeLa cells and hippocampal neurons grown from primary culture. By enabling precise measurement of DA, as well as simultaneous visualization of DA and NE, the red-DA biosensor R-GenGAR-DA1.2 is promising in advancing our understanding of the interplay between DA and NE in organizing key brain functions.Significance StatementThe neuromodulators dopamine and norepinephrine modulate a broad range of brain functions, often in concert. One current challenge is to measure dopamine and norepinephrine dynamics simultaneously with high spatial and temporal resolution. We therefore developed a red-dopamine biosensor that has 143-fold higher selectivity for dopamine over norepinephrine. Taking advantage of its high selectivity for dopamine over norepinephrine, this red-dopamine biosensor allowed dual-color fluorescence live imaging for monitoring dopamine and norepinephrine in both HeLa cells and hippocampal neurons in vitro combined with the existing green-norepinephrine biosensor that has 350-fold selectivity for norepinephrine over dopamine. Thus, this approach can provide new opportunities to advance our understanding of high spatial and temporal dynamics of dopamine and norepinephrine in normal and abnormal brain functions.

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Stability of the Human Papillomavirus Type 18 E2 Protein Is Regulated by a Proteasome Degradation Pathway through Its Amino-Terminal Transactivation Domain

Journal of Virology ◽

10.1128/jvi.75.16.7244-7251.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7244-7251 ◽

Cited By ~ 39

Author(s):

Sophie Bellanger ◽

Caroline Demeret ◽

Sylvain Goyat ◽

Françoise Thierry

Keyword(s):

Human Papillomavirus ◽

Hela Cells ◽

Half Life ◽

Fluorescent Protein ◽

Growth Arrest ◽

Transactivation Domain ◽

Mcf7 Cells ◽

E2 Protein ◽

Amino Terminal

ABSTRACT The E2 proteins of papillomaviruses regulate both viral transcription and DNA replication. The human papillomavirus type 18 (HPV18) E2 protein has been shown to repress transcription of the oncogenic E6 and E7 genes, inducing growth arrest in HeLa cells. Using HPV18 E2 fused to the green fluorescent protein (GFP), we showed that this protein was short-lived in transfected HeLa cells. Real-time microscopy experiments indicated that the E2-dependent signal increased for roughly 24 h after transfection and then rapidly disappeared, indicating that E2 was unstable in HeLa cells and could confer instability to GFP. Similar studies done with a protein lacking the transactivation domain indicated that this truncation strongly stabilizes the E2 protein. In vitro, full-length E2 or the transactivation domain alone was efficiently ubiquitinated, whereas deletion of the transactivation domain strongly decreased the ubiquitination of the E2 protein. Proteasome inhibition in cells expressing E2 increased its half-life about sevenfold, which was comparable to the half-life of the amino-terminally truncated protein. These characteristics of E2 instability were independent of the E2-mediated G1 growth arrest in HeLa cells, as they were reproduced in MCF7 cells, where E2 does not affect the cell cycle. Altogether, these experiments showed that the HPV18 E2 protein was degraded by the ubiquitin-proteasome pathway through its amino-terminal transactivation domain. Tight regulation of the stability of the HPV 18 E2 protein may be essential to avoid accumulation of a potent transcriptional repressor and antiproliferative agent during the viral vegetative cycle.

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Substance P excites GABAergic neurons in the mouse central amygdala through neurokinin 1 receptor activation

Journal of Neurophysiology ◽

10.1152/jn.00883.2014 ◽

2015 ◽

Vol 114 (4) ◽

pp. 2500-2508 ◽

Cited By ~ 6

Author(s):

L. Sosulina ◽

C. Strippel ◽

H. Romo-Parra ◽

A. L. Walter ◽

T. Kanyshkova ◽

...

Keyword(s):

Substance P ◽

Fluorescent Protein ◽

Input Resistance ◽

Receptor Activation ◽

Gabaergic Neurons ◽

Acid Decarboxylase ◽

Central Amygdala ◽

Cellular Mechanisms ◽

Action Potential Firing

Substance P (SP) is implicated in stress regulation and affective and anxiety-related behavior. Particularly high expression has been found in the main output region of the amygdala complex, the central amygdala (CE). Here we investigated the cellular mechanisms of SP in CE in vitro, taking advantage of glutamic acid decarboxylase-green fluorescent protein (GAD67-GFP) knockin mice that yield a reliable labeling of GABAergic neurons, which comprise 95% of the neuronal population in the lateral section of CE (CEl). In GFP-positive neurons within CEl, SP caused a membrane depolarization and increase in input resistance, associated with an increase in action potential firing frequency. Under voltage-clamp conditions, the SP-specific membrane current reversed at −101.5 ± 2.8 mV and displayed inwardly rectifying properties indicative of a membrane K+ conductance. Moreover, SP responses were blocked by the neurokinin type 1 receptor (NK1R) antagonist L-822429 and mimicked by the NK1R agonist [Sar9,Met(O2)11]-SP. Immunofluorescence staining confirmed localization of NK1R in GFP-positive neurons in CEl, predominantly in PKCδ-negative neurons (80%) and in few PKCδ-positive neurons (17%). Differences in SP responses were not observed between the major types of CEl neurons (late firing, regular spiking, low-threshold bursting). In addition, SP increased the frequency and amplitude of GABAergic synaptic events in CEl neurons depending on upstream spike activity. These data indicate a NK1R-mediated increase in excitability and GABAergic activity in CEl neurons, which seems to mostly involve the PKCδ-negative subpopulation. This influence can be assumed to increase reciprocal interactions between CElon and CEloff pathways, thereby boosting the medial CE (CEm) output pathway and contributing to the anxiogenic-like action of SP in the amygdala.

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A Novel Cellular Model to Study Angiotensin II AT2 Receptor Function in Breast Cancer Cells

International Journal of Peptides ◽

10.1155/2012/745027 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 5

Author(s):

Sylvie Rodrigues-Ferreira ◽

Marina Morel ◽

Rosana I. Reis ◽

Françoise Cormier ◽

Véronique Baud ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Fluorescent Protein ◽

Receptor Activation ◽

At1 Receptor ◽

At2 Receptor ◽

Cellular Model ◽

Selective Antagonist

Recent studies have highlighted the AT1 receptor as a potential therapeutic target in breast cancer, while the role of the AT2 subtype in this disease has remained largely neglected. The present study describes the generation and characterization of a new cellular model of human invasive breast cancer cells (D3H2LN-AT2) stably expressing high levels of Flag-tagged human AT2 receptor (Flag-hAT2). These cells exhibit high-affinity binding sites for AngII, and total binding can be displaced by the AT2-selective antagonist PD123319 but not by the AT1-selective antagonist losartan. Of interest, high levels of expression of luciferase and green fluorescent protein make these cells suitable for bioluminescence and fluorescence studies in vitro and in vivo. We provide here a novel tool to investigate the AT2 receptor functions in breast cancer cells, independently of AT1 receptor activation.

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In vivo and in vitro effects of vortioxetine on molecules associated with neuroplasticity

Journal of Psychopharmacology ◽

10.1177/0269881116667710 ◽

2016 ◽

Vol 31 (3) ◽

pp. 365-376 ◽

Cited By ~ 6

Author(s):

Pirathiv Kugathasan ◽

Jessica Waller ◽

Ligia Westrich ◽

Aicha Abdourahman ◽

Joseph A Tamm ◽

...

Keyword(s):

Protein Level ◽

Hippocampal Neurons ◽

Transcript Level ◽

Middle Aged ◽

Α Subunit ◽

Aged Mice ◽

Positive Effects ◽

Brain Functions

Neuroplasticity is fundamental for brain functions, abnormal changes of which are associated with mood disorders and cognitive impairment. Neuroplasticity can be affected by neuroactive medications and by aging. Vortioxetine, a multimodal antidepressant, has shown positive effects on cognitive functions in both pre-clinical and clinical studies. In rodent studies, vortioxetine increases glutamate neurotransmission, promotes dendritic branching and spine maturation, and elevates hippocampal expression of the activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) at the transcript level. The present study aims to assess the effects of vortioxetine on several neuroplasticity-related molecules in different experimental systems. Chronic (1 month) vortioxetine increased Arc/Arg3.1 protein levels in the cortical synaptosomes of young and middle-aged mice. In young mice, this was accompanied by an increase in actin-depolymerizing factor (ADF)/cofilin serine 3 phosphorylation without altering the total ADF/cofilin protein level, and an increase in the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation at serine 845 (S845) without altering serine 831 (S831) GluA1 phosphorylation nor the total GluA1 protein level. Similar effects were detected in cultured rat hippocampal neurons: Acute vortioxetine increased S845 GluA1 phosphorylation without changing S831 GluA1 phosphorylation or the total GluA1 protein level. These changes were accompanied by an increase in α subunit of Ca2+/calmodulin-dependent kinase (CaMKIIα) phosphorylation (at threonine 286) without changing the total CaMKIIα protein level in cultured neurons. In addition, chronic (1 month) vortioxetine, but not fluoxetine, restored the age-associated reduction in Arc/Arg3.1 and c-Fos transcripts in the frontal cortex of middle-aged mice. Taken together, these results demonstrated that vortioxetine modulates molecular targets that are related to neuroplasticity.

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Imaging Molecular Targeting In Living Neural Cultures

Microscopy and Microanalysis ◽

10.1017/s1431927600019462 ◽

1999 ◽

Vol 5 (S2) ◽

pp. 1228-1229

Author(s):

Christopher S. Wallace ◽

Michael A. Silverman ◽

Michelle A. Burack ◽

Janis E. Lochner ◽

Richard G. Allen ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Hippocampal Neurons ◽

Protein Targeting ◽

Fluorescent Protein ◽

Complex Structure ◽

Time Lapse ◽

The Central Nervous System

Recent technical advances in the ability to attach an endogenously fluorescent protein sequence—i.e., green fluorescent protein or GFP and its derivatives--to any protein of experimental interest promises to mark a new era of progress in the study of protein targeting. Bringing these new tools to bear on neurons of the central nervous system has been challenging, however, because they have a very complex structure and are relatively difficult to transfect because they are post-mitotic.We use two cell culture approaches to characterize protein trafficking within neurons of the central nervous system in vitro. The first is a dissociated culture of hippocampal neurons from embryonic (El8) rats which is especially suited to analysis by conventional light microscopy because these neurons are grown on glass coverslips at low density. Neurons cultured in this way develop a morphology comparable to that seen in vivo and permit the establishment of axons and dendrites to be analyzed by time-lapse microscopy.

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Propofol Neurotoxicity Is Mediated by p75 Neurotrophin Receptor Activation

Anesthesiology ◽

10.1097/aln.0b013e318242a48c ◽

2012 ◽

Vol 116 (2) ◽

pp. 352-361 ◽

Cited By ~ 93

Author(s):

Matthew L. Pearn ◽

Yue Hu ◽

Ingrid R. Niesman ◽

Hemal H. Patel ◽

John C. Drummond ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Receptor Activation ◽

Neurotrophin Receptor ◽

P75 Neurotrophin Receptor ◽

Wild Type ◽

P75 Ntr ◽

Green Fluorescent ◽

Developing Neurons

Background Propofol exposure to neurons during synaptogenesis results in apoptosis, leading to cognitive dysfunction in adulthood. Previous work from our laboratory showed that isoflurane neurotoxicity occurs through p75 neurotrophin receptor (p75(NTR)) and subsequent cytoskeleton depolymerization. Given that isoflurane and propofol both suppress neuronal activity, we hypothesized that propofol also induces apoptosis in developing neurons through p75(NTR). Methods Days in vitro 5-7 neurons were exposed to propofol (3 μM) for 6 h and apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Primary neurons from p75(NTR-/-) mice or wild-type neurons were treated with propofol, with or without pretreatment with TAT-Pep5 (10 μM, 15 min), a specific p75(NTR) inhibitor. P75(NTR-/-) neurons were transfected for 72 h with a lentiviral vector containing the synapsin-driven p75(NTR) gene (Syn-p75(NTR)) or control vector (Syn-green fluorescent protein) before propofol. To confirm our in vitro findings, wild-type mice and p75(NTR-/-) mice (PND5) were pretreated with either TAT-Pep5 or TAT-ctrl followed by propofol for 6 h. Results Neurons exposed to propofol showed a significant increase in Cl-Csp3, an effect attenuated by TAT-Pep5 and hydroxyfasudil. Apoptosis was significantly attenuated in p75(NTR-/-) neurons. In p75(NTR-/-) neurons transfected with Syn-p75(NTR), propofol significantly increased Cl-Csp3 in comparison with Syn-green fluorescent protein-transfected p75(NTR-/-) neurons. Wild-type mice exposed to propofol exhibited increased Cl-Csp3 in the hippocampus, an effect attenuated by TAT-Pep5. By contrast, propofol did not induce apoptosis in p75(NTR-/-) mice. Conclusion These results demonstrate that propofol induces apoptosis in developing neurons in vivo and in vitro and implicate a role for p75(NTR) and the downstream effector RhoA kinase.

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Highly active neurons emerging in vitro

Journal of Neurophysiology ◽

10.1152/jn.00663.2020 ◽

2021 ◽

Author(s):

Mami Okada ◽

Rena Kono ◽

Yu Sato ◽

Chiaki Kobayashi ◽

Ryuta Koyama ◽

...

Keyword(s):

Neuronal Network ◽

Hippocampal Neurons ◽

Fluorescent Protein ◽

Primary Cultures ◽

Early Gene ◽

High Frequencies ◽

Firing Rates ◽

Highly Active ◽

High Firing

Mean firing rates vary across neurons in a neuronal network. Whereas most neurons infrequently emit spikes, a small fraction of neurons exhibit extremely high frequencies of spikes; this fraction of neurons plays a pivotal role in information processing; however, little is known about how these outliers emerge and whether they are maintained over time. In primary cultures of mouse hippocampal neurons, we traced highly active neurons every 24 h for 7 weeks by optically observing the fluorescent protein dVenus; the expression of dVenus was controlled by the promoter of Arc, an immediate early gene that is induced by neuronal activity. Under default-mode conditions, 0.3-0.4% of neurons were spontaneously Arc-dVenus positive, exhibiting high firing rates. These neurons were spatially clustered, exhibited intermittently repeated dVenus expression, and often continued to express Arc-dVenus for approximately two weeks. Thus, highly active neurons constitute a few select functional subpopulations in the neuronal network.

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Fluorescent Indicators for Live-Cell and in Vitro Detection of Inorganic Cadmium Dynamics

10.21203/rs.3.rs-1103117/v1 ◽

2021 ◽

Author(s):

Shulin Hu ◽

Jun Yang ◽

Anqi Liao ◽

Ying Lin ◽

Shuli Liang

Keyword(s):

Fluorescent Protein ◽

Dynamic Range ◽

Cadmium Concentration ◽

High Dynamic Range ◽

Ion Concentration ◽

Cadmium Ion ◽

Fluorescent Indicators ◽

Cadmium Detection ◽

Time Readout

Abstract Cadmium contamination is a severe threat to the environment and food safety. Thus, there is an urgent need to develop highly sensitive and selective cadmium detection tools. The engineered fluorescent indicator is a powerful tool for the rapid detection of inorganic cadmium in the environment. In this study, the development of yellow fluorescent indicators of cadmium chloride by inserting a fluorescent protein at different positions of the high cadmium-specific repressor and optimizing the flexible linker between the connection points is reported. These indicators provide a fast, sensitive, specific, high dynamic range, and real-time readout of cadmium ion dynamics in solution. Under optimal conditions, the fluorescent indicators N0C0/N1C1 showed a linear response to cadmium concentration within the range from 10/30 to 50/100 nM and with a detection limit of 10/33 nM. Escherichia coli cells containing the indicator were used to further study the response of cadmium ion concentration in living cells. E. coli N1C1 could respond to different concentrations of cadmium ions. This study provides a rapid and straightforward method for cadmium ion detection in vitro and the potential for biological imaging.

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Ca2+ Homeostasis in the Endoplasmic Reticulum: Coexistence of High and Low [Ca2+] Subcompartments in Intact HeLa Cells

The Journal of Cell Biology ◽

10.1083/jcb.139.3.601 ◽

1997 ◽

Vol 139 (3) ◽

pp. 601-611 ◽

Cited By ~ 85

Author(s):

Mayte Montero ◽

Javier Alvarez ◽

Wilhelm J.J. Scheenen ◽

Rosario Rizzuto ◽

Jacopo Meldolesi ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Steady State ◽

Hela Cells ◽

Fluorescent Protein ◽

Divalent Cations ◽

Calibration Procedure ◽

Continuous Release ◽

Recombinant Aequorin ◽

Green Fluorescent

Two recombinant aequorin isoforms with different Ca2+ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca2+ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca2+]er or [Sr2+]er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca2+] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca2+ concentration, the [Ca2+]er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca2+]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr2+ was used as a Ca2+ surrogate), the bulk of the organelle was shown to accumulate free [cation2+]er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca2+], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca2+ or Sr2+, is presented. Moreover, a few other key problems concerning the ER Ca2+ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP3 vary depending on their initial [Ca2+]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca2+] the cation is simultaneously accumulated; (b) stimulation of Ca2+ release by receptor-generated InsP3 is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation2+]er of the InsP3 receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca2+], observed when the cells are incubated in Ca2+-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).

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