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Permanent link (DOI): https://doi.org/10.7939/R3FX74822

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

Descriptions

Other title
Subject/Keyword
fluorescent protein
GECO
biosensor
glutamate indicator
fluorescence Ca2+ imaging
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Wu, Jiahui
Supervisor and department
Campbell, Robert (Department of Chemistry)
Examining committee member and department
Gibbs-Davis, Julianne (Department of Chemistry)
Li, Liang (Department of Chemistry)
Vederas, John (Department of Chemistry)
Palmer, Amy (Department of Chemistry & Biochemistry, University of Colorado, Boulder)
Campbell, Robert (Department of Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2014-09-04T11:45:15Z
Graduation date
2014-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R3FX74822
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. 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.
Citation for previous publication
S. C. Alford, J. Wu, Y. Zhao, R. E. Campbell, and T. Knöpfel, “Optogenetic reporters”. Biol. Cell., 105, 14–29 (2013).J. Wu, L. Liu, T. Matsuda, Y. Zhao, A. Rebane, M. Drobizhev, Y-F. Chang, S. Araki, Y. Arai, K. March, T. E. Hughes, K. Sagou, T. Miyata, T. Nagai, W-h. Li, R. E. Campbell, “Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications”, ACS Chem. Neurosci., 4, 963–972 (2013).J. Wu, D. L. Prole, Y. Shen, Z. Lin, A. Gnanasekaran, Y. Liu, L. Chen, H. Zhou, S. R. W. Chen, Y. M. Usachev, C. W. Taylor and R. E. Campbell, “Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum”, Biochem. J. Immediate Publication, doi:10.1042/BJ20140931, (2014).

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