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1416 Publications

Showing 101-110 of 1416 results
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    09/11/18 | Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy
    Tebo AG, Pimenta FM, Zhang Y, Gautier A
    Biochemistry. 11/2018;57:5648 – 5653. doi: 10.1021/acs.biochem.8b00649

    Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The fluorescence-activating and absorption shifting tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (so-called fluorogens). Here, we use rational design to modify the binding pocket of the protein and screen for improved fluorescence performances with four different fluorogens. The introduction of a single mutation results in improvements in both quantum yield and dissociation constant with nearly all fluorogens tested. Our improved FAST (iFAST) allowed the generation of a tandem iFAST (td-iFAST) that forms green and red fluorescent reporters 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, while having a comparable size.

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    09/06/18 | Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency.
    Mor N, Rais Y, Sheban D, Peles S, Aguilera-Castrejon A, Zviran A, Elinger D, Viukov S, Geula S, Krupalnik V, Zerbib M, Chomsky E, Lasman L, Shani T, Bayerl J, Gafni O, Hanna S, Buenrostro JD, Hagai T, Masika H, Vainorius G, Bergman Y, Greenleaf WJ, Esteban MA, Elling U, Levin Y, Massarwa R, Merbl Y, Novershtern N, Hanna JH
    Cell Stem Cell. 09/2018;23(3):412-425.e10. doi: 10.1016/j.stem.2018.07.004

    Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.

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    08/28/18 | Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing
    Gautier A, Tebo AG
    BioEssays. 08/2018;40:1800118. doi: 10.1002/bies.201800118

    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.

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    Grigorieff Lab
    08/24/18 | Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation.
    Vemu A, Szczesna E, Zehr EA, Spector JO, Grigorieff N, Deaconescu AM, Roll-Mecak A
    Science (New York, N.Y.). 2018 Aug 24;361(6404):. doi: 10.1126/science.aau1504

    Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)-tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

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    08/23/18 | Stepwise wiring of the Drosophila olfactory map requires specific Plexin B levels
    Li J, Guajardo R, Xu C, Wu B, Li H, Li T, Luginbuhl DJ, Xie X, Luo L
    Elife. 08/2018;7:. doi: 10.7554/eLife.39088

    The precise assembly of a neural circuit involves many consecutive steps. The conflict between a limited number of wiring molecules and the complexity of the neural network impels each molecule to execute multiple functions at different steps. Here, we examined the cell-type specific distribution of endogenous levels of axon guidance receptor Plexin B (PlexB) in the developing antennal lobe, the first olfactory processing center in . We found that different classes of olfactory receptor neurons (ORNs) express PlexB at different levels in two wiring steps - axonal trajectory choice and subsequent target selection. In line with its temporally distinct patterns, the proper levels of PlexB control both steps in succession. Genetic interactions further revealed that the effect of high-level PlexB is antagonized by its canonical partner Sema2b. Thus, PlexB plays a multifaceted role in instructing the assembly of the olfactory circuit through temporally-regulated expression patterns and expression level-dependent effects.

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    08/13/18 | Cerebellar involvement in an evidence-accumulation decision-making task
    Deverett B, Koay SA, Oostland M, Wang SS
    eLife. 08/2018;7:. doi: 10.7554/eLife.36781

    To make successful evidence-based decisions, the brain must rapidly and accurately transform sensory inputs into specific goal-directed behaviors. Most experimental work on this subject has focused on forebrain mechanisms. Using a novel evidence-accumulation task for mice, we performed recording and perturbation studies of crus I of the lateral posterior cerebellum, which communicates bidirectionally with numerous forebrain regions. Cerebellar inactivation led to a reduction in the fraction of correct trials. Using two-photon fluorescence imaging of calcium, we found that Purkinje cell somatic activity contained choice/evidence-related information. Decision errors were represented by dendritic calcium spikes, which in other contexts are known to drive cerebellar plasticity. We propose that cerebellar circuitry may contribute to computations that support accurate performance in this perceptual decision-making task.

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    08/08/18 | Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors.
    Tebo AG, Pimenta FM, Zoumpoulaki M, Kikuti C, Sirkia H, Plamont M, Houdusse A, Gautier A
    ACS Chemical Biology. 09/2018;13:2392 – 2397. doi: 10.1021/acschembio.8b00417

    Fluorescent reporters are essential components for the design of optical biosensors that are able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca2+ in living mammalian cells in real time.

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    07/10/18 | CYK-4 functions independently of its centralspindlin partner ZEN-4 to cellularize oocytes in germline syncytia
    Lee K, Green RA, Gutierrez E, Gomez-Cavazos JS, Kolotuev I, Wang S, Desai A, Groisman A, Oegema K, Balasubramanian MK, Akhmanova A
    eLife. 07/2018;7:e36919. doi: 10.7554/eLife.36919

    Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Gamete formation ultimately requires bridge closure, yet how bridges are reactivated to close is not known. The most conserved bridge component is centralspindlin, a complex of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6. Here, we show that oocyte production by the syncytial \textitCaenorhabditis elegans germline requires CYK-4 but not ZEN-4, which contrasts with cytokinesis, where both are essential. Longitudinal imaging after conditional inactivation revealed that CYK-4 activity is important for oocyte cellularization, but not for the cytokinesis-like events that generate syncytial compartments. CYK-4’s lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were both required to target CYK-4 to intercellular bridges and to cellularize oocytes. These results suggest that the conserved C1-GAP region of CYK-4 constitutes a targeting module required for closure of intercellular bridges in germline syncytia.

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    06/11/18 | A cerebellar role in evidence-guided decision-making
    Deverett B, Koay SA, Oostland M, Wang SS
    bioRxiv. 06/2018:. doi: 10.1101/343095

    To make successful evidence-based decisions, the brain must rapidly and accurately transform sensory inputs into specific goal-directed behaviors. Most experimental work on this subject has focused on forebrain mechanisms. Here we show that during perceptual decision-making over a period of seconds, decision-, sensory-, and error-related information converge on the lateral posterior cerebellum in crus I, a structure that communicates bidirectionally with numerous forebrain regions. We trained mice on a novel evidence-accumulation task and demonstrated that cerebellar inactivation reduces behavioral accuracy without impairing motor parameters of action. Using two-photon calcium imaging, we found that Purkinje cell somatic activity encoded choice- and evidence-related variables. Decision errors were represented by dendritic calcium spikes, which are known to drive plasticity. We propose that cerebellar circuitry may contribute to the set of distributed computations in the brain that support accurate perceptual decision-making.

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    06/05/18 | CalmAn: An open source tool for scalable Calcium Imaging data Analysis
    Giovannucci A, Friedrich J, Gunn P, Kalfon J, Koay SA, Taxidis J, Najafi F, Gauthier JL, Zhou P, Tank DW, Chklovskii D, Pnevmatikakis EA
    bioRxiv. 06/2018:. doi: 10.1101/339564

    Advances in fluorescence microscopy enable monitoring larger brain areas in-vivo with finer time resolution. The resulting data rates require reproducible analysis pipelines that are reliable, fully automated, and scalable to datasets generated over the course of months. Here we present CaImAn, an open-source library for calcium imaging data analysis. CaImAn provides automatic and scalable methods to address problems common to pre-processing, including motion correction, neural activity identification, and registration across different sessions of data collection. It does this while requiring minimal user intervention, with good performance on computers ranging from laptops to high-performance computing clusters. CaImAn is suitable for two-photon and one-photon imaging, and also enables real-time analysis on streaming data. To benchmark the performance of CaImAn we collected a corpus of ground truth annotations from multiple labelers on nine mouse two-photon datasets. We demonstrate that CaImAn achieves near-human performance in detecting locations of active neurons.

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