A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

Journal: Nature chemical biology
Published Date: Apr 1, 2018

Abstract

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization 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 and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

Authors

  • Kiryl D Piatkevich
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Erica E Jung
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Christoph Straub
    Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
  • Changyang Linghu
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Demian Park
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Ho-Jun Suk
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Daniel R Hochbaum
    Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
  • Daniel Goodwin
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Eftychios Pnevmatikakis
    Simons Center Data Analysis, Simons Foundation, New York, NY, USA.
  • Nikita Pak
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Takashi Kawashima
    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
  • Chao-Tsung Yang
    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
  • Jeffrey L Rhoades
    Picower Institute for Learning & Memory and Department of Brain & Cognitive Sciences, MIT, Cambridge, MA, USA.
  • Or Shemesh
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Shoh Asano
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Young-Gyu Yoon
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Limor Freifeld
    Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
  • Jessica L Saulnier
    Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
  • Clemens Riegler
    Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA, USA.
  • Florian Engert
    Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA, USA.
  • Thom Hughes
    Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, USA.
  • Mikhail Drobizhev
    Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, USA.
  • Bálint Szabó
    Nanobiosensorics Group, Institute of Technical Physics and Materials Science, Centre for Energy Research, Hung. Acad. Sci., Budapest, Hungary.
  • Misha B Ahrens
    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
  • Steven W Flavell
    Picower Institute for Learning & Memory and Department of Brain & Cognitive Sciences, MIT, Cambridge, MA, USA.
  • Bernardo L Sabatini
    Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
  • Edward S Boyden
    Media Lab, Massachusetts Institute of Technology, Cambridge, United States.

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