A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins

Kleinlogel, Sonja; Terpitz, Ulrich; Legrum, Barbara; Gökbuget, Deniz; Boyden, Edward S.; Bamann, Christian; Wood, Paul; Bamberg, Ernst

doi:10.1038/nmeth.1766

Cited by 79 publications

(109 citation statements)

References 34 publications

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“…Kleinlogel and coworkers reported a gene fusion strategy for stoichiometric co-expression of two microbial rhodopsins as a single polypeptide chain 59 . We made fused constructs of several channelrhodopsins and Arch, but these showed poor trafficking.…”

Section: Methodsmentioning

confidence: 99%

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

et al. 2014

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All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and 2, which show improved brightness and voltage sensitivity, microsecond response times, and produce no photocurrent. We engineered a novel channelrhodopsin actuator, CheRiff, which shows improved light sensitivity and kinetics, and spectral orthogonality to the QuasArs. A co-expression vector, Optopatch, enabled crosstalk-free genetically targeted all-optical electrophysiology. In cultured neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials in dendritic spines, synaptic transmission, sub-cellular microsecond-timescale details of action potential propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In brain slice, Optopatch induced and reported action potentials and subthreshold events, with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without use of conventional electrodes.

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Section: Methodsmentioning

confidence: 99%

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

et al. 2014

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“…After MEA experiments, retinal flat mounts were stained with antibodies against channelrhodopsin 41 and Brn3a. Tissue from NHP1 was labeled with anti-GFP antibodies in green and anti-Brn3a antibodies in red ( Figure 4H).…”

Section: Electrophysiological Recordings Reveal Catch-driven Responsementioning

confidence: 99%

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina

et al. 2017

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The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. Second-and third-order neurons are spared in these diseases, making it possible to restore retinal light responses using optogenetics. Viral expression of channelrhodopsin in the third-order neurons under ubiquitous promoters was previously shown to restore visual function, albeit at light intensities above illumination safety thresholds. Here, we report (to our knowledge, for the first time) activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin-Ca 2+ -permeable channelrhodopsin (CatCh) at safe light intensities. High-level CatCh expression was achieved due to a new promoter based on the regulatory region of the gamma-synuclein gene (SNCG) allowing strong expression in ganglion cells across species. Our promoter, in combination with clinically proven adeno-associated virus 2 (AAV2), provides CatCh expression in peri-foveolar ganglion cells responding robustly to light under the illumination safety thresholds for the human eye. On the contrary, the threshold of activation and the proportion of unresponsive cells were much higher when a ubiquitous promoter (cytomegalovirus [CMV]) was used to express CatCh. The results of our study suggest that the inclusion of optimized promoters is key in the path to clinical translation of optogenetics.

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“…A method for optimized tandem expression of excitatory and inhibitory opsins has been recently developed (73). Genetic engineering is currently used to expand and optimize available opsins in three important aspects: light sensitivity, speed, and spectral response (Fig.…”

Section: The Optogenetics Toolboxmentioning

confidence: 99%

Cardiac optogenetics

Entcheva

2013

American Journal of Physiology-Heart and Circulatory Physiology

113

105

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Optogenetics is an emerging technology for optical interrogation and control of biological function with high specificity and high spatiotemporal resolution. Mammalian cells and tissues can be sensitized to respond to light by a relatively simple and well-tolerated genetic modification using microbial opsins (light-gated ion channels and pumps). These can achieve fast and specific excitatory or inhibitory response, offering distinct advantages over traditional pharmacological or electrical means of perturbation. Since the first demonstrations of utility in mammalian cells (neurons) in 2005, optogenetics has spurred immense research activity and has inspired numerous applications for dissection of neural circuitry and understanding of brain function in health and disease, applications ranging from in vitro to work in behaving animals. Only recently (since 2010), the field has extended to cardiac applications with less than a dozen publications to date. In consideration of the early phase of work on cardiac optogenetics and the impact of the technique in understanding another excitable tissue, the brain, this review is largely a perspective of possibilities in the heart. It covers the basic principles of operation of light-sensitive ion channels and pumps, the available tools and ongoing efforts in optimizing them, overview of neuroscience use, as well as cardiac-specific questions of implementation and ideas for best use of this emerging technology in the heart. channelrhodopsin; light-sensitive ion channels; optical mapping THIS ARTICLE is part of a collection on Physiological Basis of Cardiovascular Cell and Gene Therapies. Other articles appearing in this collection, as well as a full archive of all collections, can be found online at http://ajpheart.physiology.org/. Background

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A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins

Cited by 79 publications

References 34 publications

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina

Cardiac optogenetics

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