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5 Publications

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    05/17/23 | Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator
    Abdelfattah AS, Zheng J, Singh A, Huang Y, Reep D, Tsegaye G, Tsang A, Arthur BJ, Rehorova M, Olson CV, Shuai Y, Zhang L, Fu T, Milkie DE, Moya MV, Weber TD, Lemire AL, Baker CA, Falco N, Zheng Q, Grimm JB, Yip MC, Walpita D, Chase M, Campagnola L, Murphy GJ, Wong AM, Forest CR, Mertz J, Economo MN, Turner GC, Koyama M, Lin B, Betzig E, Novak O, Lavis LD, Svoboda K, Korff W, Chen T, Schreiter ER, Hasseman JP, Kolb I
    Neuron. 2023 May 17;111(10):1547-1563. doi: 10.1016/j.neuron.2023.03.009

    The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

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    04/01/21 | The HaloTag as a general scaffold for far-red tunable chemigenetic indicators.
    Deo C, Abdelfattah AS, Bhargava HK, Berro AJ, Falco N, Farrants H, Moeyaert B, Chupanova M, Lavis LD, Schreiter ER
    Nature Chemical Biology. 2021 Apr 01:. doi: 10.1038/s41589-021-00775-w

    Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

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    01/09/20 | Bright and tunable far-red chemigenetic indicators.
    Deo C, Abdelfattah AS, Bhargava HK, Berro AJ, Falco N, Moeyaert B, Chupanova M, Lavis LD, Schreiter ER
    bioRxiv. 2020 Jan 9:
    08/13/19 | Bright and photostable chemigenetic indicators for extended in vivo voltage imaging.
    Abdelfattah AS, Kawashima T, Singh A, Novak O, Liu H, Shuai Y, Huang Y, Campagnola L, Seeman SC, Yu J, Zheng J, Grimm JB, Patel R, Friedrich J, Mensh BD, Paninski L, Macklin JJ, Murphy GJ, Podgorski K, Lin B, Chen T, Turner GC, Liu Z, Koyama M, Svoboda K, Ahrens MB, Lavis LD, Schreiter ER
    Science. 2019 Aug 13;365(6454):699-704. doi: 10.1126/science.aav6416

    Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

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    05/24/16 | Design and synthesis of a calcium-sensitive photocage.
    Heckman LM, Grimm JB, Schreiter ER, Kim C, Verdecia MA, Shields BC, Lavis LD
    Angewandte Chemie (International ed. in English). 2016 May 24:. doi: 10.1002/anie.201602941

    Photolabile protecting groups (or "photocages") enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input-output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca(2+) -sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca(2+) ]. Caging a fluorophore with this ion-sensitive moiety yields an "ion integrator" that permanently marks cells undergoing high Ca(2+) flux during an illumination-defined time period. Our general design concept demonstrates a new class of light-sensitive material for cellular imaging, sensing, and targeted molecular delivery.

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