Recent progress in the development of genetically encoded Ca&lt;sup&gt;2+ &lt;/sup&gt;indicators

Horikawa, Kazuki

doi:10.2152/jmi.62.24

Cited by 24 publications

(15 citation statements)

References 32 publications

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“…In contrast, much larger fluctuations of GCaMP3 fluorescence are seen in neonatal SCN cultured under similar conditions. Further studies using larger sample size and more sensitive reporters such as aequorin [ 69 ] or newer genetically encoded Ca 2+ indicators [ 76 ] may provide more secure evidence of circadian Ca 2+ oscillations in the heart.…”

Section: Discussionmentioning

confidence: 99%

Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by β-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue

2016

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Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a β-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+.

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Section: Discussionmentioning

confidence: 99%

Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by β-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue

2016

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“…For example RV caused an increase in Ca 2+ transients but RSV elevated [Ca 2+ ] c with few Ca 2+ transients until just prior to cell death, and PV infection disrupted the normal Ca 2+ oscillations of HeLa cells. Future studies using these GECI-expressing cell lines, as well as additional lines developed with other GECI varieties, will be able to further dissect the mechanisms by which viruses exploit ER Ca 2+ signaling (10, 22, 23). …”

Section: Discussionmentioning

confidence: 99%

“…In order to study both aspects of Ca 2+ signaling, a wide range of fluorescent Ca 2+ indicators have been developed to enable real time observations of changes in Ca 2+ levels [reviewed in (10)]. All Ca 2+ indicators are based upon fluorescent molecules that bind Ca 2+ ions and have different spectral properties between the Ca 2+ -free and the Ca 2+ -bound states.…”

Section: Introductionmentioning

confidence: 99%

“…There are three classes of GECIs: [1] bioluminescent sensors based on the aequorin protein, [2] Forester resonance energy transfer (FRET) based two-color fluorescent protein indicators, and [3] single fluorescent protein-based indicators. While there are many varieties of these GECIs [reviewed in (10, 17)], the function of both single color and the FRET based sensors are based on a similar mechanism, which uses a Ca 2+ responsive element [typically calmodulin (CaM) and the M13 CaM-binding peptide] inserted into a fluorescent protein. In the case of the FRET based sensors, the CaM-M13 motif is inserted between CFP/YFP FRET pairs, such that upon Ca 2+ binding the CaM conformational change brings CFP and YFP closer together resulting in increased FRET efficiency.…”

Section: Introductionmentioning

confidence: 99%

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Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells

Perry

Ramachandran

Utama

et al. 2015

Methods

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Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections.

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“…However, recent developments of exogenous and in particular genetically encoded fluorescent indicators of neuronal activity such as GCaMP and YC-Nano have revolutionized the targeted expression of fluorescence with much higher signal levels. And even transgenetic lines exist [ 14 ]. Furthermore, recent work has shown that the dynamics of flavoprotein fluorescence can be optically mapped in wild-type CNS tissue as an indicator of oxidative metabolism [ 15 , 16 ], which might be also a useful tool in experimental stroke research.…”

Section: State-of-the-art Techniques To Study Neuronal Reorganizatmentioning

confidence: 99%

State-of-the-Art Techniques to Causally Link Neural Plasticity to Functional Recovery in Experimental Stroke Research

Wahl

2018

Neural Plasticity

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Current experimental stroke research faces the same challenge as neuroscience: to transform correlative findings in causative ones. Research of recent years has shown the tremendous potential of the central nervous system to react to noxious stimuli such as a stroke: Increased plastic changes leading to reorganization in form of neuronal rewiring, neurogenesis, and synaptogenesis, accompanied by transcriptional and translational turnover in the affected cells, have been described both clinically and in experimental stroke research. However, only minor attempts have been made to connect distinct plastic remodeling processes as causative features for specific behavioral phenotypes. Here, we review current state-of the art techniques for the examination of cortical reorganization and for the manipulation of neuronal circuits as well as techniques which combine anatomical changes with molecular profiling. We provide the principles of the techniques together with studies in experimental stroke research which have already applied the described methodology. The tools discussed are useful to close the loop from our understanding of stroke pathology to the behavioral outcome and may allow discovering new targets for therapeutic approaches. The here presented methods open up new possibilities to assess the efficiency of rehabilitative strategies by understanding their external influence for intrinsic repair mechanisms on a neurobiological basis.

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Recent progress in the development of genetically encoded Ca<sup>2+ </sup>indicators

Abstract: GECIs are categorized into two groups based on the molecular design, i.e., two -FP based and single -FP based one as is discussed in many reviews (Table1, 2)(7, 8). REVIEW Recent progress in the development

Cited by 24 publications

References 32 publications

Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by β-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue

Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by β-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue

Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells

State-of-the-Art Techniques to Causally Link Neural Plasticity to Functional Recovery in Experimental Stroke Research

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