Expanding the palette of genetically-encoded calcium ion indicators for monitoring neural activity

  • Author / Creator
    Qian, Yong
  • Light-emitting proteins, including both fluorescent proteins, luciferases, and their derivative indicators, have equipped scientists with a variety of genetically-encoded tools for non-invasively visualizing cellular signaling networks. Calcium ion (Ca2+) imaging is one of the most widely used imaging technologies due to the pivotal roles that Ca2+ plays in cell biology. In neuroscience, Ca2+ imaging with genetically encoded Ca2+ indicators (GECIs) is a robust approach to monitor neural activity. Furthermore, the combined use of GECIs with optogenetic actuators (i.e., channelrhodopsins) for simultaneously measuring and controlling neural activity in the nervous system could, in principle, provide critical insights into molecular mechanism behind brain networks. However, currently available fluorescent GECIs exhibit substantial spectral overlap with optogenetic actuators, which makes it challenging to image the GECI without also activating optogenetic actuators. In this thesis, I describe two approaches for overcoming this challenge: the development of a bioluminescent Ca2+ indicator and near-infrared (NIR) fluorescent Ca2+ indicator.
    In this thesis, I first describe my efforts to develop a ratiometric bioluminescent Ca2+ indicator, LUCI-GECO1, based on one of the brightest luciferase, Nanoluc, and a topological variant of GCaMP6s, ncpGCaMP6s. LUCI-GECO1 retains the high Ca2+ affinity of ncpGCaMP6s and outperformed another ratiometric bioluminescent Ca2+ indicator CalfluxVTN in histamine-treated HeLa cells. Due to the lack of external excitation, LUCI-GECO1 is compatible with channelrhodopsins.
    I also describe a genetically-encoded NIR fluorescent Ca2+ indicator, NIR-GECO1, with excitation and emission maxima at 678 nm and 704 nm, respectively. NIR-GECO1 was engineered based on an monomeric near-infrared FP, mIFP, through extensive direction evolution. Working with collaborators, we demonstrated that NIR-GECO1 was able to reliably report Ca2+ transients in cultured neurons, in acute brain slice and in mouse brain in vivo at mesoscale. Due to the highly red-shifted spectra, imaging of NIR-GECO1 has essentially no crosstalk with the stimulation of the high photocurrent channelrhodopsin CoChR. NIR-GECO1 also enabled multiparameter imaging in conjunction with other fluorescent-protein-based intensiometric and ratiometric indicators.
    Finally, I describe efforts to further improve the properties of NIR-GECO1. I performed three additional rounds of directed evolution and selected a new variant, NIR-GECO2. Compared to NIR-GECO1, NIR-GECO2 enables more sensitive Ca2+ imaging in cultured neurons and acute brain slices with 50% improvement in cellular brightness and a Kd of 102 nM (the Kd of NIR-GECO1 is 215 nM ). Working with collaborators, I expressed NIR-GECO2 in C. elegans and successfully detected spontaneous neural activity in worms in vivo. I anticipate that NIR-GECO2 will be an excellent tool for studying central nervous system (CNS) circuits and complex behaviors of C. elegans and other model organisms.

  • Subjects / Keywords
  • Graduation date
    Fall 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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