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6 Janelia Publications

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    01/01/24 | Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers using chemistry.
    Pratik Kumar , Alina Gutu , Amelia Waring , Timothy A. Brown , Luke D. Lavis , Alison G. Tebo
    bioRxiv. 2024 Jan 01:. doi: 10.1101/2023.12.30.573644

    Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

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    11/29/23 | A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction
    Xiao Li , Rico Gamuyao , Ming-Lun Wu , Woo Jung Cho , Nathan B. Kurtz , Sharon V. King , R.A. Petersen , Daniel R. Stabley , Caleb Lindow , Leslie Climer , Abbas Shirinifard , Francesca Ferrara , Robert E. Throm , Camenzind G. Robinson , Alex Carisey , Alison G. Tebo , Chi-Lun Chang
    bioRxiv. 2023 Nov 29:. doi: 10.1101/2023.11.29.569289

    Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen- Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

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    10/18/23 | A blue-shifted genetically encoded Ca2+ indicator with enhanced two-photon absorption
    Abhi Aggarwal , Smrithi Sunil , Imane Bendifallah , Michael Moon , Mikhail Drobizhev , Landon Zarowny , Jihong Zheng , Sheng-Yi Wu , Alexander W. Lohman , Alison G. Tebo , Valentina Emiliani , Kaspar Podgorski , Yi Shen , Robert E. Campbell
    bioRxiv. 2023 Oct 18:. doi: 10.1101/2023.10.12.562058

    Significance Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.

    Aim We describe the development and applications of T-GECO1 – a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1.

    Approach We used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices.

    Results The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.

    Conclusion T-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

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    06/27/23 | Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging.
    Usama SM, Marker SC, Li D, Caldwell DR, Stroet M, Patel NL, Tebo AG, Hernot S, Kalen JD, Schnermann M
    Journal of the American Chemical Society. 2023 Jun 27;145(27):14647–14659. doi: 10.1021/jacs.3c01765

    Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

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    01/15/22 | Fluorescent chemigenetic actuators and indicators for use in living animals.
    Farrants H, Tebo AG
    Current Opinion in Pharmacology. 2022 Jan 15;62:159-167. doi: 10.1016/j.coph.2021.12.007

    Fluorescent indicators and actuators provide a means to optically observe and perturb dynamic events in living animals. Although chemistry and protein engineering have contributed many useful tools to observe and perturb cells, an emerging strategy is to use chemigenetics: systems in which a small molecule dye interacts with a genetically encoded protein domain. Here we review chemigenetic strategies that have been successfully employed in living animals as photosensitizers for photoablation experiments, fluorescent cell cycle indicators, and fluorescent indicators for studying dynamic biological signals. Although these strategies at times suffer from challenges, e.g. delivery of the small molecule and assembly of the chemigenetic unit in living animals, the advantages of using small molecules with high brightness, low photobleaching, no chromophore maturation time and expanded color palette, combined with the ability to genetically target them to specific cell types, make chemigenetic fluorescent actuators and indicators an attractive strategy for use in living animals.

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    02/04/21 | Structure-Function Dataset Reveals Environment Effects within a Fluorescent Protein Model System.
    De Zitter E, Hugelier S, Duwé S, Vandenberg W, Tebo AG, Van Meervelt L, Dedecker P
    Angew Chemie (International Edition English). 2021 Feb 04:. doi: 10.1002/anie.202015201

    Anisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure-function relationships. We examined this using fluorescent proteins as easy-to-modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

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