Development of fluorescent protein-based indicators for neuronal activity imaging

• Author / Creator

  Abdelfattah, Ahmed

• Fluorescence microscopy is currently the most powerful imaging technique for interrogation of neural circuits. Accordingly, tremendous efforts have been invested in engineering fluorescent proteins (FPs) to act as indicators for various biochemical processes in neural circuits, resulting in a plethora of genetically encoded FP-based indicators for neural imaging. In this thesis, we report the development of various FP-based indicators for neuronal imaging. Specifically, we describe our efforts to expand the current repertoire of neuronal activity indicators to include a voltage indicator with red-shifted fluorescence, a highlightable voltage indicator, and a green fluorescent indicator for the neurotransmitter γ-aminobutyric acid (GABA). First, we added to the spectral palette of practically useful voltage indicators by developing a bright red-shifted FP-based voltage indicator (FlicR1). Using protein engineering and directed evolution, we engineered FlicR1 to have sufficient speed and sensitivity to report single action potentials in single-trial recordings. Because it is excitable with yellow light, FlicR1 is the first FP-based voltage indicator that can be used in conjunction with blue-light-activated optogenetic actuators to report optically driven action potentials. Using FlicR1 as a template, we further engineered voltage indicators that utilize two copies of voltage sensitive domains linked in tandem by a circularly permutated FP. This design allowed us to create two new voltage indicators: a red intensiometric voltage indicator (tdFlicR Δ110AR) and a green/red ratiometric voltage indicator (tdFlicR-VK-ASAP). We demonstrate their utility by imaging membrane potential changes in cultured human cell lines and primary neuron cultures. Furthermore, we engineered a highlightable voltage indicator (FlicGR0.7). FlicGR0.7 can be photoconverted from a green to a red fluorescent form using blue light illumination. We demonstrate that both the green and red fluorescent forms of FlicGR0.7 are sensitive to membrane potential changes in mammalian cells. Finally, we developed a green FP-based GABA indicator (GABA-G1). Upon binding to GABA, GABA-G1 decreases its green fluorescence. Through rational design and directed protein evolution we engineered GABA-G1 to have a 1-fold dynamic range in vitro. Although we attempted several strategies to target GABA-G1 to the surface of mammalian cells, it remained trapped in the endoplasmic reticulum.

• Subjects / Keywords

  • Protein engineering
  • Fluorescent proteins
  • Fluorescence imaging
  • Voltage imaging
  • Genetically encoded voltage indicator

• Graduation date

  Fall 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R37P8TT90

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.