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18 Publications

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    11/30/21 | Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging.
    Benaissa H, Ounoughi K, Aujard I, Fischer E, Goïame R, Nguyen J, Tebo AG, Li C, Le Saux T, Bertolin G, Tramier M, Danglot L, Pietrancosta N, Morin X, Jullien L, Gautier A
    Nature Communications. 2021 Nov 30;12(1):6989. doi: 10.1038/s41467-021-27334-0

    Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Förster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques.

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    10/31/21 | Versatile On-Demand Fluorescent Labeling of Fusion Proteins Using Fluorescence-Activating and Absorption-Shifting Tag (FAST).
    Gautier A, Jullien L, Li C, Plamont M, Tebo AG, Thauvin M, Volovitch M, Vriz S
    Methods Mol Biol. 2021;2350:253-265. doi: 10.1007/978-1-0716-1593-5_16

    Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible.

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    10/19/21 | Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis.
    Aguilera-Castrejon A, Hanna JH
    J Vis Exp. 10/2021(176):. doi: 10.3791/63160

    Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

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    10/18/21 | The power of peer networking for improving STEM faculty job applications: a successful pilot program
    Guardia CM, Kane E, Tebo AG, Sanders AA, Kaya D, Grogan KE
    bioRxiv. 10/2021:. doi: 10.1101/2021.10.16.464662

    In order to successfully obtain a faculty position, postdoctoral fellows or ‘postdocs’, 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 ‘leak’ 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.

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    09/11/21 | Erratum: Label-free imaging of fibroblast membrane interfaces and protein signatures with vibrational infrared photothermal and phase signals: publisher's note.
    Samolis PD, Langley D, O'Reilly BM, Oo Z, Hilzenrat G, Erramilli S, Sgro AE, McArthur S, Sander MY
    Biomed Opt Express. 09/2021;12(9):5400. doi: 10.1364/BOE.438946

    [This corrects the article on p. 303 in vol. 12, PMID: 33520386.].

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    09/06/21 | Nitrite reductase activity within an antiparallel de novo scaffold.
    Koebke KJ, Tebo AG, Manickas EC, Deb A, Penner-Hahn JE, Pecoraro VL
    JBIC Journal of Biological Inorganic Chemistry. 09/2021;26(7):855 - 862. doi: 10.1007/s00775-021-01889-1

    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.

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    09/02/21 | Principles of signaling pathway modulation for enhancing human naive pluripotency induction.
    Bayerl J, Ayyash M, Shani T, Manor YS, Gafni O, Massarwa R, Kalma Y, Aguilera-Castrejon A, Zerbib M, Amir H, Sheban D, Geula S, Mor N, Weinberger L, Naveh Tassa S, Krupalnik V, Oldak B, Livnat N, Tarazi S, Tawil S, Wildschutz E, Ashouokhi S, Lasman L, Rotter V, Hanna S, Ben-Yosef D, Novershtern N, Viukov S, Hanna JH
    Cell Stem Cell. 09/2021;28(9):1549-1565.e12. doi: 10.1016/j.stem.2021.04.001

    Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

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    08/19/21 | Ventral stress fibers induce plasma membrane deformation in human fibroblasts.
    Ghilardi SJ, Aronson MS, Sgro AE
    Mol Biol Cell. 08/2021;32(18):1707-1723. doi: 10.1091/mbc.E21-03-0096

    Interactions between the actin cytoskeleton and the plasma membrane are important in many eukaryotic cellular processes. During these processes, actin structures deform the cell membrane outward by applying forces parallel to the fiber's major axis (as in migration) or they deform the membrane inward by applying forces perpendicular to the fiber's major axis (as in the contractile ring during cytokinesis). Here we describe a novel actin-membrane interaction in human dermal myofibroblasts. When labeled with a cytosolic fluorophore, the myofibroblasts displayed prominent fluorescent structures on the ventral side of the cell. These structures are present in the cell membrane and colocalize with ventral actin stress fibers, suggesting that the stress fibers bend the membrane to form a "cytosolic pocket" that the fluorophores diffuse into, creating the observed structures. The existence of this pocket was confirmed by transmission electron microscopy. While dissolving the stress fibers, inhibiting fiber protein binding, or inhibiting myosin II binding of actin removed the observed pockets, modulating cellular contractility did not remove them. Taken together, our results illustrate a novel actin-membrane bending topology where the membrane is deformed outward rather than being pinched inward, resembling the topological inverse of the contractile ring found in cytokinesis.

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    05/26/21 | An Accumulation-of-Evidence Task Using Visual Pulses for Mice Navigating in Virtual Reality
    Pinto L, Koay SA, Engelhard B, Yoon AM, Deverett B, Thiberge SY, Witten IB, Tank DW, Brody CD
    Frontiers in Behavioral Neuroscience. Jun-03-2018;12:. doi: 10.3389/fnbeh.2018.00036

    The gradual accumulation of sensory evidence is a crucial component of perceptual decision making, but its neural mechanisms are still poorly understood. Given the wide availability of genetic and optical tools for mice, they can be useful model organisms for the study of these phenomena; however, behavioral tools are largely lacking. Here, we describe a new evidence-accumulation task for head-fixed mice navigating in a virtual reality (VR) environment. As they navigate down the stem of a virtual T-maze, they see brief pulses of visual evidence on either side, and retrieve a reward on the arm with the highest number of pulses. The pulses occur randomly with Poisson statistics, yielding a diverse yet well-controlled stimulus set, making the data conducive to a variety of computational approaches. A large number of mice of different genotypes were able to learn and consistently perform the task, at levels similar to rats in analogous tasks. They are sensitive to side differences of a single pulse, and their memory of the cues is stable over time. Moreover, using non-parametric as well as modeling approaches, we show that the mice indeed accumulate evidence: they use multiple pulses of evidence from throughout the cue region of the maze to make their decision, albeit with a small overweighting of earlier cues, and their performance is affected by the magnitude but not the duration of evidence. Additionally, analysis of the mice's running patterns revealed that trajectories are fairly stereotyped yet modulated by the amount of sensory evidence, suggesting that the navigational component of this task may provide a continuous readout correlated to the underlying cognitive variables. Our task, which can be readily integrated with state-of-the-art techniques, is thus a valuable tool to study the circuit mechanisms and dynamics underlying perceptual decision making, particularly under more complex behavioral contexts.

     
     
     

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    05/19/21 | Multimodal patterns of inhibitory activity in cerebellar cortex
    Chie Satou , Rainer W. Friedrich
    Neuron. 05/2021;109:1590-1592. doi: https://doi.org/10.1016/j.neuron.2021.04.029

    In this issue of Neuron, Gurnani and Silver (2021) report that activity across Golgi cells, a major type of inhibitory interneuron in the cerebellar cortex, is multidimensional and modulated by behavior. These results suggest multiple functions for inhibition in cerebellar computations.

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