Development of red fluorescent protein-based calcium ion and glutamate indicators

• Author / Creator

  Wu, Jiahui

• The discovery and subsequent applications of fluorescent proteins (FPs) launched a new era for live cell fluorescence imaging. The design and developments of FP-based indicators have further solidified the versatility of FPs and rendered them as indispensable tools in life science research now more than ever. Despite the tremendous developments and efforts invested in the field of FP-based indicators, there remain numerous opportunities in engineering indicators with improved or novel properties for studying biological processes in vivo. In this thesis we describe our efforts in developing a series of FP-based calcium ion (Ca2+) and glutamate indicators with various colors and useful spectral properties as versatile tools for interrogating cell signaling in cell biology. In this thesis, we first describe our efforts in employing protein engineering to expand the color palette of genetically encoded Ca2+ indicators to include intensiometric orange, improved red and ratiometric red fluorescent variants. We demonstrate these new indicators' utility by performing Ca2+ imaging in cultured human cell lines, slice culture of developing mouse neocortex, organotypic hippocampal slice cultures and the visual system of albino tadpoles. Using our intensiometric red Ca2+ indicators, R-GECO1 and R-GECO1.2, as templates, we further engineered a series of low affinity R-GECOs with dissociation constants (Kds) ranging from 12 µM to more than 540 µM. We demonstrate that these indicators can be used to image cell compartments with high Ca2+ concentration or with a broad range of Ca2+ change, such as the endoplasmic reticulum (ER) and mitochondria. We also demonstrate these new red Ca2+ indicators with low affinities can be used to monitor ER and mitochondrial Ca2+ in combination with a green fluorescent protein (GFP)-based reporter. We also report in this thesis the development, optimization and characterization of the first red fluorescent protein (RFP)-based glutamate indicator, GltR1. We demonstrate GltR1 can detect glutamate changes on the surface of cultured human cells, as well as the glutamate dynamics during spontaneous activities of dissociated rat hippocampal neurons.

• Subjects / Keywords

  • Biosensors
  • Fluorescence Ca2+ imaging
  • Glutamate indicator
  • Fluorescent proteins
  • GECO

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3FX74822

• 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.