@article {69833, title = {Glutamate indicators with increased sensitivity and tailored deactivation rates}, journal = {Nat Methods}, year = {2025}, month = {2025 Dec 23}, abstract = {

Understanding how neurons integrate signals from thousands of input synapses requires methods to monitor neurotransmission across many sites simultaneously. The fluorescent protein glutamate indicator iGluSnFR enables visualization of synaptic signaling, but the sensitivity, scale and speed of such measurements are limited by existing variants. Here we developed two highly sensitive fourth-generation iGluSnFR variants with fast activation and tailored deactivation rates: iGluSnFR4f for tracking rapid dynamics, and iGluSnFR4s for recording from large populations of synapses. These indicators detect glutamate with high spatial specificity and single-vesicle sensitivity in vivo. We used them to record natural patterns of synaptic transmission across multiple experimental contexts in mice, including two-photon imaging in cortical layers 1{\textendash}4 and hippocampal CA1, and photometry in the midbrain. The iGluSnFR4 variants extend the speed, sensitivity and scalability of glutamate imaging, enabling direct observation of information flow through neural networks in the intact brain.

}, doi = {10.1038/s41592-025-02965-z}, url = {https://doi.org/10.1038/s41592-025-02965-z}, author = {Aggarwal, Abhi and Negrean, Adrian and Chen, Yang and Iyer, Rishyashring and Reep, Daniel and Liu, Anyi and Palutla, Anirudh and Xie, Michael E. and MacLennan, Bryan J. and Hagihara, Kenta M. and Kinsey, Lucas W. and Sun, Julianna L. and Yao, Pantong and Zheng, Jihong and Tsang, Arthur and Tsegaye, Getahun and Zhang, Yonghai and Patel, Ronak H. and Arthur, Benjamin J. and Hiblot, Julien and Leippe, Philipp and Tarnawski, Miroslaw and Marvin, Jonathan S. and Vevea, Jason D. and Turaga, Srinivas C. and Tebo, Alison G. and Carandini, Matteo and Rossi, L. F. and Kleinfeld, David and Konnerth, Arthur and Svoboda, Karel and Turner, Glenn C. and Hasseman, Jeremy and Podgorski, Kaspar} } @article {70170, title = {NanoBlocks: creating fluorescent biosensors from affinity binders using competitive binding}, journal = {bioRxiv}, year = {2025}, month = {2025 October 28}, abstract = {

Genetically-encoded fluorescent biosensors have revolutionized our understanding of complex systems by permitting the in situ observation of chemical activities. However, only a comparatively small set of chemical activities can be monitored, largely due to the need to identify protein domains that undergo conformational and/or association changes in response to a stimulus. Here, we present a strategy that can convert {\textquoteright}simple{\textquoteright} affinity binders such as nanobodies into biosensors for their innate targets by introducing a peptide sequence that competes for the binding site. We demonstrate proof-of-concept implementations of this {\textquoteright}NanoBlock{\textquoteright} design, developing sensors based on the ALFA nanobody and on the PDZ domain of Erbin. We show that these sensors can reliably detect their targets in vitro, in mammalian cells, and as part of fluorescence-activated cell sorting (FACS) experiments. In doing so, our strategy offers a way to strongly expand the range of cellular processes that can be probed using fluorescent biosensors.

}, doi = {10.1101/2025.10.28.685015}, url = {https://www.biorxiv.org/content/early/2025/10/28/2025.10.28.685015}, author = {Denis, Silke and Van Deuren, Vincent and Frecot, Desiree I. and Rothbauer, Ulrich and Ismail, Shehab and Tebo, Alison G. and Dedecker, Peter} } @article {67826, title = {Nitrite reductase activity within an antiparallel de novo scaffold.}, journal = {JBIC Journal of Biological Inorganic Chemistry}, volume = {26}, year = {2021}, month = {09/2021}, pages = {855 - 862}, abstract = {

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis3\ binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis3\ binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis3\ binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

}, issn = {0949-8257}, doi = {10.1007/s00775-021-01889-1}, url = {https://link.springer.com/10.1007/s00775-021-01889-1}, author = {Koebke, Karl J. and Tebo, Alison G. and Manickas, Elizabeth C. and Deb, Aniruddha and Penner-Hahn, James E. and Pecoraro, Vincent L.} } @article {67825, title = {The power of peer networking for improving STEM faculty job applications: a successful pilot program}, journal = {bioRxiv}, year = {2021}, month = {10/2021}, abstract = {

In order to successfully obtain a faculty position, postdoctoral fellows or {\textquoteleft}postdocs{\textquoteright}, must submit an application which requires considerable time and effort to produce. These job applications are often reviewed by mentors and colleagues, but rarely are postdocs offered the opportunity to solicit feedback multiple times from reviewers with the same breadth of expertise often found on an academic search committee. To address this gap, this manuscript describes an international peer reviewing program for small groups of postdocs with a broad range of expertise to reciprocally and iteratively provide feedback to each other on their application materials. Over 145 postdocs have participated, often multiple times, over three years. A survey of participants in this program revealed that nearly all participants would recommend participation in such a program to other faculty applicants. Furthermore, this program was more likely to attract participants who struggled to find mentoring and support elsewhere, either because they changed fields or because of their identity as a woman or member of an underrepresented population in STEM. Participation in programs like this one could provide early career academics like postdocs with a diverse and supportive community of peer mentors during the difficult search for a faculty position. Such psychosocial support and encouragement has been shown to prevent attrition of individuals from these populations and programs like this one target the largest {\textquoteleft}leak{\textquoteright} in the pipeline, that of postdoc to faculty. Implementation of similar peer reviewing programs by universities or professional scientific societies could provide a valuable mechanism of support and increased chances of success for early-career academics in their search for independence.Competing Interest StatementThe authors have declared no competing interest.

}, doi = {10.1101/2021.10.16.464662}, url = {https://www.biorxiv.org/content/early/2021/10/18/2021.10.16.464662}, author = {Guardia, Carlos M. and Kane, Erin and Tebo, Alison G. and Sanders, Anna A.W.M. and Kaya, Devrim and Grogan, Kathleen E.} } @article {65762, title = {Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins}, journal = {bioRxiv}, year = {2020}, month = {09/2020}, pages = {2020.09.25.313528}, abstract = {

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

}, doi = {10.1101/2020.09.25.313528}, author = {Zitter, Elke De and Hugelier, Siewert and Duw{\'e}, Sam and Vandenberg, Wim and Tebo, Alison G. and Meervelt, Luc Van and Dedecker, Peter} } @article {65761, title = {Orthogonal fluorescent chemogenetic reporters for multicolor imaging}, journal = {Nature Chemical Biology}, year = {2020}, month = {04/2020}, pages = {1{\textendash}9}, abstract = {

Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein{\textendash}protein interactions by live-cell fluorescence microscopy. The fluorescent chemogenetic reporters greenFAST and redFAST were engineered by protein engineering. They display orthogonal fluorogen recognition and spectral properties allowing efficient multicolor imaging of proteins in live cells and organisms.

}, issn = {1552-4450}, doi = {10.1038/s41589-020-0611-0}, author = {Tebo, Alison G. and Moeyaert, Benjamien and Thauvin, Marion and Carlon-Andres, Irene and B{\"o}ken, Dorothea and Volovitch, Michel and Padilla-Parra, Sergi and Dedecker, Peter and Vriz, Sophie and Gautier, Arnaud} } @article {65760, title = {Sensing cellular biochemistry with fluorescent chemical{\textendash}genetic hybrids}, journal = {Current Opinion in Chemical Biology}, volume = {57}, year = {2020}, month = {08/2020}, pages = {58{\textendash}64}, abstract = {

Fluorescent biosensors are powerful tools\ for the detection of biochemical events inside cells with high spatiotemporal resolution. Biosensors based on fluorescent proteins often suffer from issues with photostability and brightness. On the other hand, hybrid, chemical{\textendash}genetic systems present unique opportunities to combine the strengths of synthetic, organic chemistry with biological macromolecules to generate exquisitely tailored semisynthetic sensors.

}, issn = {1367-5931}, doi = {10.1016/j.cbpa.2020.04.005}, author = {Gautier, Arnaud and Tebo, Alison G.} } @article {65770, title = {Fluorogenic Protein-Based Strategies for Detection, Actuation, and Sensing}, journal = {BioEssays}, volume = {40}, year = {2018}, month = {08/2018}, pages = {1800118}, abstract = {

Fluorescence imaging has become an indispensable tool in cell and molecular biology. GFP-like fluorescent proteins have revolutionized fluorescence microscopy, giving experimenters exquisite control over the localization and specificity of tagged constructs. However, these systems present certain drawbacks and as such, alternative systems based on a fluorogenic interaction between a chromophore and a protein have been developed. While these systems are initially designed as fluorescent labels, they also present new opportunities for the development of novel labeling and detection strategies. This review focuses on new labeling protocols, actuation methods, and biosensors based on fluorogenic protein systems. This review presents recently developed fluorogenic protein-based systems made of a protein tag incorporating an external chromophore. Beyond addressing some limitations of classical fluorescent proteins, these unique systems present characteristics than can be used to creatively push the limits of biological imaging, in particular for the development of new labeling protocols, actuation methods and biosensors.

}, issn = {0265-9247}, doi = {10.1002/bies.201800118}, url = {https://onlinelibrary-wiley-com.accesdistant.sorbonne-universite.fr/doi/pdf/10.1002/bies.201800118}, author = {Gautier, Arnaud and Tebo, Alison G.} } @article {65780, title = {Discovery of chemoautotrophic symbiosis in the giant shipworm Kuphus polythalamia (Bivalvia: Teredinidae) extends wooden-steps theory}, journal = {Proceedings of the National Academy of Sciences}, volume = {114}, year = {2017}, month = {05/2017}, pages = {E3652{\textendash}E3658}, abstract = {

Certain marine invertebrates harbor chemosynthetic bacterial symbionts, giving them the remarkable ability to consume inorganic chemicals such as hydrogen sulfide (H2S) rather than organic matter as food. These chemosynthetic animals are found near geochemical (e.g., hydrothermal vents) or biological (e.g., decaying wood or large animal carcasses) sources of H2S on the seafloor. Although many such symbioses have been discovered, little is known about how or where they originated. Here, we demonstrate a new chemosynthetic symbiosis in the giant teredinid bivalve (shipworm) Kuphus polythalamia and show that this symbiosis arose in a wood-eating ancestor via the displacement of ancestral cellulolytic symbionts by sulfur-oxidizing invaders. Here, wood served as an evolutionary stepping stone for a dramatic transition from heterotrophy to chemoautotrophy.The {\textquotedblleft}wooden-steps{\textquotedblright} hypothesis [Distel DL, et al. (2000) Nature 403:725{\textendash}726] proposed that large chemosynthetic mussels found at deep-sea hydrothermal vents descend from much smaller species associated with sunken wood and other organic deposits, and that the endosymbionts of these progenitors made use of hydrogen sulfide from biogenic sources (e.g., decaying wood) rather than from vent fluids. Here, we show that wood has served not only as a stepping stone between habitats but also as a bridge between heterotrophic and chemoautotrophic symbiosis for the giant mud-boring bivalve Kuphus polythalamia. This rare and enigmatic species, which achieves the greatest length of any extant bivalve, is the only described member of the wood-boring bivalve family Teredinidae (shipworms) that burrows in marine sediments rather than wood. We show that K. polythalamia harbors sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria instead of the cellulolytic symbionts that allow other shipworm species to consume wood as food. The characteristics of its symbionts, its phylogenetic position within Teredinidae, the reduction of its digestive system by comparison with other family members, and the loss of morphological features associated with wood digestion indicate that K. polythalamia is a chemoautotrophic bivalve descended from wood-feeding (xylotrophic) ancestors. This is an example in which a chemoautotrophic endosymbiosis arose by displacement of an ancestral heterotrophic symbiosis and a report of pure culture of a thioautotrophic endosymbiont.

}, issn = {0027-8424}, doi = {10.1073/pnas.1620470114}, url = {https://www.pnas.org/content/114/18/E3652}, author = {Distel, Daniel L. and Altamia, Marvin A. and Lin, Zhenjian and Shipway, J. Reuben and Han, Andrew and Forteza, Imelda and Antemano, Rowena and Limbaco, Ma. Gwen J. Pe{\~n}aflor and Tebo, Alison G. and Dechavez, Rande and Albano, Julie and Rosenberg, Gary and Concepcion, Gisela P. and Schmidt, Eric W. and Haygood, Margo G.} } @article {65777, title = {Fluorogenic Labeling Strategies for Biological Imaging}, journal = {International Journal of Molecular Sciences}, volume = {18}, year = {2017}, month = {07/2017}, pages = {1473 {\textendash} 11}, abstract = {

The spatiotemporal fluorescence imaging of biological processes requires effective tools to label intracellular biomolecules in living systems. This review presents a brief overview of recent labeling strategies that permits one to make protein and RNA strongly fluorescent using synthetic fluorogenic probes. Genetically encoded tags selectively binding the exogenously applied molecules ensure high labeling selectivity, while high imaging contrast is achieved using fluorogenic chromophores that are fluorescent only when bound to their cognate tag, and are otherwise dark. Beyond avoiding the need for removal of unbound synthetic dyes, these approaches allow the development of sophisticated imaging assays, and open exciting prospects for advanced imaging, particularly for multiplexed imaging and super-resolution microscopy.

}, doi = {10.3390/ijms18071473}, url = {http://www.mdpi.com/1422-0067/18/7/1473}, author = {Li, Chenge and Tebo, Alison G. and Gautier, Arnaud} } @inbook {65784, title = {Porphyrins and Metalloporphyrins at Components in Artificial Photosynthesis Research}, booktitle = {Handbook of Porphyrin Science}, volume = {34}, year = {2014}, pages = {196 {\textendash} 233}, publisher = {World Scientific Pulishing}, organization = {World Scientific Pulishing}, isbn = {978-981-4425-10-0}, doi = {10.1142/9789814417297_0016}, author = {Tebo, Alison G. and Herrero, C and Aukauloo, A}, editor = {Kadish, Karl M and Smith, Kevin M and undefined, and Guilard, Roger} }