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Optimization of genetically encoded indicators for neuronal imaging

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posted on 2024-10-14, 16:29 authored by Glenn TurnerGlenn Turner, Abhi Aggarwal, Benjamin Arthur, Daniel BusheyDaniel Bushey, Helen FarrantsHelen Farrants, Jeremy HassemanJeremy Hasseman, Vivek JayaramanVivek Jayaraman, John Koberstein, Ilya Kolb, Wyatt KorffWyatt Korff, Loren Looger, Abigail Stuart, Adrian Negrean, Kaspar PodgoroskiKaspar Podgoroski, Daniel ReepDaniel Reep, Eric SchreiterEric Schreiter, Hiroshi ShiozakiHiroshi Shiozaki, Nelson SprustonNelson Spruston, Alison G. Tebo, Arthur Tsang, Getahun Tsegaye, Amelia Waring, Jihong ZhengJihong Zheng, GENIE Project Team

GENIE = Genetically ENcoded Indicators and Effectors

Protein sensors are routinely used to image neuronal activity and they underlie many recent advances in cellular functional imaging. However, many sensors still have major limitations when used in vivo. The GENIE Project Team engineers and optimizes fluorescent sensors for imaging of cellular activity in the intact nervous system of experimental animals. We have developed sensors for calcium, voltage and different neurotransmitters.

This poster describes the GENIE screening process and updates to four sensors. Presented at the Society for Neuroscience meeting, October 7, 2024.

SCaMP: Red Calcium Sensor

  • New red calcium indicator based on mScarlet - the brightest of the mRFPs
  • Initial design work lead to a sensor with sensitivity and kinetics comparable to jRGECO.
  • Optimization in progress with 47 sites targeted for mutagenesis and ~5000 variants screened.
  • Next: top performing single mutations will be combined to test for further performance improvements
  • Anticipated release Summer 2025.

iGluSnFR4: Glutamate sensor

  • Built off iGluSnFR3 (Aggarwal…Podgorski Nat. Methods 2023) by site-directed mutagenesis and neuronal screening
  • 5x increase in signal:noise over predecessor in culture
  • Improved binding kinetics
  • Anticipated release Spring 2025

RubyACR: Far-Red Opto-Inhibitors in Drosophila

  • RubyACRs (Govorunova…Spudich PNAS 2020) are optogenetic anion channels with a far-red action spectrum
  • We show that they are effective optogenetic inhibitors in freely behaving Drosophila
  • Anticipated release early 2025

WHaloCaMP2: Chemigenetic Calcium Sensor

  • WHaloCaMP is composed of a calcium-binding protein domain that interacts with a HaloTag-bound synthetic dye (Farrants…Schreiter Nature Methods 2024)
  • Users can set sensor Em/Ex by picking from a suite of Janelia Fluor dyes.
  • Bioavailability described in Grimm…Lavis Nature Methods 2020
  • Far-red calcium imaging using JF669
  • Sensor supports fluorescence lifetime imaging (FLIM) for absolute calcium concentration
  • Screened 3388 total variants - top hits exhibited 5x improved SNR


Funding

Howard Hughes Medical Institute

History