Engineering genetically encoded indicators for neurotransmission

• Author / Creator

  Dalangin, Rochelin R. S.

• Information is transmitted between neurons through the flow of different ions and the release of neurotransmitters at speeds on the order of milliseconds. The advent of genetically encoded fluorescent protein (FP)-based optical indicators for neuronal activity, which can be precisely targeted towards desired populations of cells and enabled non-invasive imaging of both single neurons and populations of neurons, revolutionized our understanding of neurotransmission. In this thesis, we detail our efforts to expand the repertoire of genetically encoded indicators for neurotransmission by combining directed evolution and rational design.First, we expand the pool of analytes for which genetically encoded indicators are available by engineering a new series of green FP-based aspartate indicators, which we call “ODIN1”, from a new aspartate/glutamate periplasmic binding protein. By analyzing the protein’s solvent accessibility, we were able to identify the optimal insertion point for a circularly permutated (cp) green FP and engineer bright prototypes with large dynamic ranges. Further optimization efforts to introduce specificity for aspartate over glutamate led to variants with different and well-separated affinities for aspartate and glutamate. By tethering the ODIN1 variants on the surface of cells using the SpyTag and SpyCatcher system, we were able to show that ODIN1 had good response towards aspartate that is comparable to other first-generation indicators for amino acids. Second, we capitalized on our efforts from engineering ODIN1 by engineering the first single FP-based glutamine indicator using a glutamine binding protein that is homologous to our aspartate/glutamate binding protein. By careful optimization of the linkers, we engineered a highly specific variant, which we refer to as “Qigon1”, with relatively high affinity and modest response. Using SpyTag and SpyCatcher, we demonstrated that Qigon1 is functional on the cell surface.Next, we describe the development of a new red genetically encoded calcium ion (Ca2+) indicator (GECI), RCaMP3.0, using mRuby3, one of the brightest red FPs to date. RCaMP3.0 shows a significantly larger dynamic range than its predecessor but still has low brightness and affinity for Ca2+. As a result, its performance in cells is limited.Last, we expand the spectral palette of available GECIs by engineering a new series of far-red Ca2+ indicators, the FR-GECO series, based on a new monomeric far-red FP. The FR-GECOs have high affinity and large dynamic ranges, are bright under one-photon and two-photon illumination in vitro and offer fast and sensitive detection of single action potentials in neurons.

• Subjects / Keywords

  • fluorescent protein
  • biosensors
  • indicator
  • neurotransmitter
  • neurotransmission
  • imaging

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-xm0e-c663

• 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.