Abstract:Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensor… Show more
“…The faster kinetics of the ASAP indicators compared to ArcLight enabled a shorter time to peak when reporting cardiac action potentials ( Figure 2E–F , Figure 2—figure supplement 1 ). These results demonstrate that ASAP2s can image cardiac action potentials, as previously shown with other voltage indicators in vitro ( Kaestner et al, 2015 ; Tian et al, 2011 ; Leyton-Mange et al, 2014 ; Chang Liao et al, 2015 ; Werley et al, 2017 ) and in vivo ( Chang Liao et al, 2015 ; Tsutsui et al, 2010 ; Hou et al, 2014 ). 10.7554/eLife.25690.007 Figure 2.…”
Section: Resultssupporting
confidence: 85%
“…We next sought to compare the ability of ASAP2s and other indicators to report voltage dynamics in excitable cells. Given the interest in using indicators to image cardiac electrical activity ( Kaestner et al, 2015 ), we first expressed our indicators in cardiomyocytes differentiated from human embryonic stem cells. Consistent with our results in HEK293A cells, the response amplitude of ASAP2s to cardiac potentials was greater than that of the other indicators, reaching –45.1 ± 1.5% compared with –24.0 ± 1.8% for ASAP1 and –32.9 ± 1.8% for ArcLight ( Figure 2A–D , Video 1 ).…”
Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.DOI:
http://dx.doi.org/10.7554/eLife.25690.001
“…The faster kinetics of the ASAP indicators compared to ArcLight enabled a shorter time to peak when reporting cardiac action potentials ( Figure 2E–F , Figure 2—figure supplement 1 ). These results demonstrate that ASAP2s can image cardiac action potentials, as previously shown with other voltage indicators in vitro ( Kaestner et al, 2015 ; Tian et al, 2011 ; Leyton-Mange et al, 2014 ; Chang Liao et al, 2015 ; Werley et al, 2017 ) and in vivo ( Chang Liao et al, 2015 ; Tsutsui et al, 2010 ; Hou et al, 2014 ). 10.7554/eLife.25690.007 Figure 2.…”
Section: Resultssupporting
confidence: 85%
“…We next sought to compare the ability of ASAP2s and other indicators to report voltage dynamics in excitable cells. Given the interest in using indicators to image cardiac electrical activity ( Kaestner et al, 2015 ), we first expressed our indicators in cardiomyocytes differentiated from human embryonic stem cells. Consistent with our results in HEK293A cells, the response amplitude of ASAP2s to cardiac potentials was greater than that of the other indicators, reaching –45.1 ± 1.5% compared with –24.0 ± 1.8% for ASAP1 and –32.9 ± 1.8% for ArcLight ( Figure 2A–D , Video 1 ).…”
Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.DOI:
http://dx.doi.org/10.7554/eLife.25690.001
“…30 Using genetically encoded voltage sensors, APs were imaged in rat primary CMs, 31 whole mouse hearts, 32 and human embryonic stem cell-derived CMs. 33 Optical AP imaging was recently conducted successfully in hiPSC-CMs using the genetically encoded voltage indicator ArcLight.…”
Translational perspectiveCardiomyocytes (CMs) generated from human induced pluripotent stem cells are an evolving platform to understand molecular disease mechanism and evaluate cardiovascular drugs. A major limitation of this system is that they represent a heterogeneous mix of ventricular-, atrial-, and nodal-like CMs. By expressing a voltage-sensitive fluorescent protein under the control of lineage-specific promoters, we developed a convenient system allowing high-throughput subtype-specific optical action potential (AP) imaging in these cells. This enables not only quantification of electrical phenotypes in patient-specific CMs but also subtype-specific investigation of drug effects, which may aid both drug development and safety pharmacology in the cardiovascular field.
“…This study has confirmed the essential roles of Muller cells in visual perception and in normal retinal structure formation (Byrne et al, 2013). In this regard some technical strategies for gene delivery (Kaestner et al, 2015a; El-Shamayleh et al, 2016) and past works on tissue-specific expression of fluorescent protein sensors may be considered (Akemann et al, 2013; Kaestner et al, 2015b). …”
Section: Protein Photosensitizer For Nanoscopically-confined Photodynmentioning
Singlet oxygen generated in a type II photodynamic action, due to its limited lifetime (1 μs) and reactive distance (<10 nm), could regulate live cell function nanoscopically. The genetically-encoded protein photosensitizers (engineered fluorescent proteins such as KillerRed, TagRFP, and flavin-binding proteins such as miniSOG, Pp2FbFPL30M) could be expressed in a cell type- and/or subcellular organelle-specific manner for targeted protein photo-oxidative activation/desensitization. The newly emerged active illumination technique provides an additional level of specificity. Typical examples of photodynamic activation include permanent activation of G protein-coupled receptor CCK1 and photodynamic activation of ionic channel TRPA1. Protein photosensitizers have been used to photodynamically modulate major cellular functions (such as neurotransmitter release and gene transcription) and animal behavior. Protein photosensitizers are increasingly used in photon-driven nanomanipulation in cell physiology research.
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