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10 Janelia Publications

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    10/24/16 | Bright photoactivatable fluorophores for single-molecule imaging.
    Lavis LD, Grimm JB, English BP, Choi H, Muthusamy AK, Mehl BP, Dong P, Brown TA, Lippincott-Schwartz J, Liu Z, Lionnet T
    Nature Methods. 2016 Oct 24;13(12):985-8. doi: 10.1038/nmeth.4034

    Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing "Janelia Fluor" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

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    09/15/16 | Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.
    Zhang Z, English BP, Grimm JB, Kazane SA, Hu W, Tsai A, Inouye C, You C, Piehler J, Schultz PG, Lavis LD, Revyakin A, Tjian R
    Genes and Development. 2016 Sep 15;30:2106-18. doi: 10.1101/gad.285395.116

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

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    07/12/16 | Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers.
    Breda L, Motta I, Lourenco S, Gemmo C, Deng W, Rupon JW, Abdulmalik OY, Manwani D, Blobel GA, Rivella S
    Blood. 2016 Jul 12:. doi: 10.1182/blood-2016-01-691089

    Overcoming the silencing of the fetal γ-globin gene has been a long standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called LCR, dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells the LCR can be re-directed from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom designed zinc finger proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacological reactivation of fetal hemoglobin (HbF), hematopoietic cells from SCD patients were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double of that observed with decitabine and pomalidomide; butyrate had an intermediate effect while tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle Hb (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared to the drug regimens support its therapeutic potential for the treatment of SCD.

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    05/05/16 | Real-time quantification of single RNA translation dynamics in living cells.
    Morisaki T, Lyon K, DeLuca KF, DeLuca JG, English BP, Zhang Z, Lavis LD, Grimm JB, Viswanathan S, Looger LL
    Science. 2016 May 05;352(6292):1425-9. doi: 10.1126/science.aaf0899

    Although mRNA translation is a fundamental biological process, it has never been imaged in real-time with single molecule precision in vivo. To achieve this, we developed Nascent Chain Tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify single mRNA protein synthesis dynamics. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 s. Polysomes contain ~1 ribosome every 200-900 nucleotides and are globular rather than elongated in shape. By developing multi-color probes, we show most polysomes act independently; however, a small fraction (~5%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

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    05/03/16 | RNA Polymerase II cluster dynamics predict mRNA output in living cells.
    Cho W, Jayanth N, English BP, Inoue T, Andrews JO, Conway W, Grimm JB, Spille J, Lavis LD, Lionnet T, Cisse II
    eLife. 2016 May 03;5:. doi: 10.7554/eLife.13617

    Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.

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    04/29/16 | TRICK: A single-molecule method for imaging the first round of translation in living cells and animals.
    Halstead JM, Wilbertz JH, Wippich F, Lionnet T, Ephrussi A, Chao JA
    Methods in Enzymology. 2016;572:123-57. doi: 10.1016/bs.mie.2016.02.027

    The life of an mRNA is dynamic within a cell. The development of quantitative fluorescent microscopy techniques to image single molecules of RNA has allowed many aspects of the mRNA lifecycle to be directly observed in living cells. Recent advances in live-cell multicolor RNA imaging, however, have now made it possible to investigate RNA metabolism in greater detail. In this chapter, we present an overview of the design and implementation of the translating RNA imaging by coat protein knockoff RNA biosensor, which allows untranslated mRNAs to be distinguished from ones that have undergone a round of translation. The methods required for establishing this system in mammalian cell lines and Drosophila melanogaster oocytes are described here, but the principles may be applied to any experimental system.

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    03/29/16 | A primer on the Bayesian approach to high-density single-molecule trajectories analysis.
    El Beheiry M, Türkcan S, Richly MU, Triller A, Alexandrou A, Dahan M, Masson J
    Biophysical Journal. 2016 Mar 29;110(6):1209-15. doi: 10.1016/j.bpj.2016.01.018

    Tracking single molecules in living cells provides invaluable information on their environment and on the interactions that underlie their motion. New experimental techniques now permit the recording of large amounts of individual trajectories, enabling the implementation of advanced statistical tools for data analysis. In this primer, we present a Bayesian approach toward treating these data, and we discuss how it can be fruitfully employed to infer physical and biochemical parameters from single-molecule trajectories.

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    02/16/16 | Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.
    Abrahamsson S, Ilic R, Wisniewski J, Mehl B, Yu L, Chen L, Davanco M, Oujedi L, Fiche J, Hajj B
    Biomedical Optics Express. 2016 Feb 16;7(3):855-69. doi: 10.1364/BOE.7.000855

    Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a “precise color” MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.


    Additional authors include:

    Xin Jin, Joan Pulupa, Christine Cho, Mustafa Mir, Mohamed El Beheiry, Xavier Darzacq, Marcelo Nollmann, Maxime Dahan, Carl Wu, Timothée Lionnet, J. Alexander Liddle, and Cornelia I. Bargmann


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    02/16/16 | PSF engineering in multifocus microscopy for increased depth volumetric imaging.
    Hajj B, El Beheiry M, Dahan M
    Biomedical Optics Express. 2016 Feb 16;7(3):726-31. doi: 10.1364/BOE.7.000726

    Imaging and localizing single molecules with high accuracy in a 3D volume is a challenging task. Here we combine multifocal microscopy, a recently developed volumetric imaging technique, with point spread function engineering to achieve an increased depth for single molecule imaging. Applications in 3D single molecule localization-based super-resolution imaging is shown over an axial depth of 4 µm as well as for the tracking of diffusing beads in a fluid environment over 8 µm.

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    Tjian LabSinger LabLiu LabTranscription Imaging
    01/16/16 | Imaging transcription: past, present, and future.
    Coleman RA, Liu Z, Darzacq X, Tjian R, Singer RH, Lionnet T
    Cold Spring Harbor Symposia on Quantitative Biology. 2015;80:1-8. doi: 10.1101/sqb.2015.80.027201

    Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

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