A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters
| Autor: | Nikita Pak, Mikhail Drobizhev, Jeffrey L. Rhoades, Jessica L. Saulnier, Demian Park, Shoh Asano, Clemens Riegler, Edward S. Boyden, Misha B. Ahrens, Florian Engert, Young-Gyu Yoon, Ho-Jun Suk, Daniel Hochbaum, Changyang Linghu, Takashi Kawashima, Eftychios A. Pnevmatikakis, Steven W. Flavell, Kiryl D. Piatkevich, Daniel Goodwin, Bálint Szabó, Erica E. Jung, Chao-Tsung Yang, Limor Freifeld, Bernardo L. Sabatini, Or A. Shemesh, Thomas E. Hughes, Christoph Straub |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Male Opsin Computer science Cell Separation Optogenetics Protein Engineering Hippocampus Article Fluorescence 03 medical and health sciences Mice Postsynaptic potential Genes Reporter synaptic transmission Animals Humans Caenorhabditis elegans Molecular Biology Zebrafish mouse Gene Library Neurons biology Subthreshold conduction HEK 293 cells Brain Cell Biology Robotics biology.organism_classification Directed evolution subthreshold activity Flow Cytometry Luminescent Proteins 030104 developmental biology HEK293 Cells Microscopy Fluorescence C. elegans Female Directed Molecular Evolution Neuroscience voltage imaging |
| Zdroj: | Nature chemical biology |
| ISSN: | 1552-4469 |
| Popis: | We here present a new way to engineer complex proteins toward multidimensional specifications, through a simple yet scalable directed evolution strategy. By robotically picking mammalian cells that are identified, under a microscope, to express proteins that simultaneously exhibit several specific properties, we can screen through hundreds of thousands of proteins in a library in a matter of a few hours, evaluating each along multiple performance axes. We demonstrate the power of this approach by identifying a novel genetically encoded fluorescent voltage indicator, simultaneously optimizing brightness and membrane localization of the protein using our microscopy-guided cell picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1, and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices as well as in larval zebrafish in vivo. We also demonstrate measurement of postsynaptic responses downstream of optogenetically controlled neurons in C. elegans. |
| Databáze: | OpenAIRE |
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