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      Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging

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          Abstract

          Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca 2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca 2+ receptor moiety. We demonstrate super-resolution imaging of Ca 2+ concentration in cultured cells and optoacoustic Ca 2+ imaging in implanted tumor cells in mice under controlled Ca 2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

          Abstract

          Calcium and other analytes can be imaged at super-resolution and in vivo with photo-switchable sensors.

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          Most cited references71

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          Optimization of a GCaMP calcium indicator for neural activity imaging.

          Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
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            Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

            Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.
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              Engineering and characterization of a superfolder green fluorescent protein.

              Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.
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                Author and article information

                Contributors
                andre.stiel@helmholtz-muenchen.de
                Journal
                Nat Biotechnol
                Nat Biotechnol
                Nature Biotechnology
                Nature Publishing Group US (New York )
                1087-0156
                1546-1696
                29 November 2021
                29 November 2021
                2022
                : 40
                : 4
                : 598-605
                Affiliations
                [1 ]GRID grid.4567.0, ISNI 0000 0004 0483 2525, Institute of Biological and Medical Imaging, Helmholtz Zentrum München, ; Neuherberg, Germany
                [2 ]GRID grid.6936.a, ISNI 0000000123222966, Chair of Biogenic Functional Materials, Technische Universität München, ; Straubing, Germany
                [3 ]GRID grid.5037.1, ISNI 0000000121581746, Science for Life Laboratory, KTH Royal Institute of Technology, ; Stockholm, Sweden
                [4 ]GRID grid.4567.0, ISNI 0000 0004 0483 2525, Intracellular Transport and RNA Biology Group, Institute of Structural Biology, Helmholtz Zentrum München, ; Neuherberg, Germany
                [5 ]GRID grid.6936.a, ISNI 0000000123222966, Center for Translational Cancer Research, School of Medicine, Technical University of Munich, ; Munich, Germany
                [6 ]GRID grid.6582.9, ISNI 0000 0004 1936 9748, Institute of Pharmaceutical Biotechnology, Ulm University, ; Ulm, Germany
                [7 ]GRID grid.6936.a, ISNI 0000000123222966, Munich Institute of Robotics and Machine Intelligence, Technical University of Munich, ; Munich, Germany
                Author information
                http://orcid.org/0000-0002-2462-8834
                http://orcid.org/0000-0001-6651-3851
                http://orcid.org/0000-0002-9940-2143
                http://orcid.org/0000-0003-0265-019X
                http://orcid.org/0000-0002-4774-7449
                http://orcid.org/0000-0003-4005-4997
                http://orcid.org/0000-0002-9988-0233
                http://orcid.org/0000-0001-8675-6797
                Article
                1100
                10.1038/s41587-021-01100-5
                9005348
                34845372
                ab421f34-a5dc-4370-8ef1-5a6a86c26ead
                © The Author(s) 2021

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 13 May 2020
                : 17 September 2021
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft (German Research Foundation);
                Award ID: STI656/1-1
                Award ID: STI656/1-1
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100010663, EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council);
                Award ID: 638314
                Award ID: 638314
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100010902, National Science Foundation of China | Joint Research Fund for Overseas Chinese Scholars and Scholars in Hong Kong and Macao;
                Award ID: 201306960006
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100010661, EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020);
                Award ID: 732720
                Award Recipient :
                Categories
                Article
                Custom metadata
                © The Author(s), under exclusive licence to Springer Nature America, Inc. 2022

                Biotechnology
                fluorescence imaging,optical imaging,x-ray crystallography,molecular engineering,super-resolution microscopy

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