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7 Janelia Publications

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    Singer Lab
    02/28/17 | RNP transport in cell biology: the long and winding road.
    Eliscovich C, Singer RH
    Current Opinion in Cell Biology. 2017 Feb 28;45:38-46. doi: 10.1016/

    Regulation of gene expression is key determinant to cell structure and function. RNA localization, where specific mRNAs are transported to subcellular regions and then translated, is highly conserved in eukaryotes ranging from yeast to extremely specialized and polarized cells such as neurons. Messenger RNA and associated proteins (mRNP) move from the site of transcription in the nucleus to their final destination in the cytoplasm both passively through diffusion and actively via directed transport. Dysfunction of RNA localization, transport and translation machinery can lead to pathology. Single-molecule live-cell imaging techniques have revealed unique features of this journey with unprecedented resolution. In this review, we highlight key recent findings that have been made using these approaches and possible implications for spatial control of gene function.

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    02/27/17 | A variant Sp1 (R218Q) transcription factor might enhance HbF expression in β(0) -thalassaemia homozygotes.
    Jiang Z, Luo H, Farrell JJ, Zhang Z, Schulz VP, Albarawi D, Steinberg MH, Al-Allawi NA, Gallagher PG, Forget BG, Chui DH
    British Journal of Haematology. 2017 Feb 27;180(5):755-7. doi: 10.1111/bjh.14445
    02/27/17 | Video-rate volumetric functional imaging of the brain at synaptic resolution.
    Lu R, Sun W, Liang Y, Kerlin A, Bierfeld J, Seelig JD, Wilson DE, Scholl B, Mohar B, Tanimoto M, Koyama M, Fitzpatrick D, Orger MB, Ji N
    Nature Neuroscience. 2017 Feb 27;20(4):620-8. doi: 10.1038/nn.4516

    Neurons and neural networks often extend hundreds of micrometers in three dimensions. Capturing the calcium transients associated with their activity requires volume imaging methods with subsecond temporal resolution. Such speed is a challenge for conventional two-photon laser-scanning microscopy, because it depends on serial focal scanning in 3D and indicators with limited brightness. Here we present an optical module that is easily integrated into standard two-photon laser-scanning microscopes to generate an axially elongated Bessel focus, which when scanned in 2D turns frame rate into volume rate. We demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice and ferrets in vivo. Calcium signals in objects as small as dendritic spines could be resolved at video rates, provided that the samples were sparsely labeled to limit overlap in their axially projected images.

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    02/16/17 | An unsupervised method for quantifying the behavior of interacting individuals.
    Klibaite U, Berman GJ, Cande J, Stern DL
    Physical Biology. 2017 Feb16;14(1):1609.09345. doi: 10.1088/1478-3975/aa5c50

    Behaviors involving the interaction of multiple individuals are complex and frequently crucial for an animal's survival. These interactions, ranging across sensory modalities, length scales, and time scales, are often subtle and difficult to characterize. Contextual effects on the frequency of behaviors become even more difficult to quantify when physical interaction between animals interferes with conventional data analysis, e.g. due to visual occlusion. We introduce a method for quantifying behavior in fruit fly interaction that combines high-throughput video acquisition and tracking of individuals with recent unsupervised methods for capturing an animal's entire behavioral repertoire. We find behavioral differences between solitary flies and those paired with an individual of the opposite sex, identifying specific behaviors that are affected by social and spatial context. Our pipeline allows for a comprehensive description of the interaction between two individuals using unsupervised machine learning methods, and will be used to answer questions about the depth of complexity and variance in fruit fly courtship.

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    Gonen Lab
    02/13/17 | Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.
    de la Cruz MJ, Hattne J, Shi D, Seidler P, Rodriguez J, Reyes FE, Sawaya MR, Cascio D, Weiss SC, Kim SK, Hinck CS, Hinck AP, Calero G, Eisenberg D, Gonen T
    Nature Methods. 2017 Feb 13;14(4):399-402. doi: 10.1038/nmeth.4178

    Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

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    02/13/17 | Self-organizing actin patterns shape membrane architecture but not cell mechanics.
    Fritzsche M, Li D, Colin-York H, Chang VT, Moeendarbary E, Felce JH, Sezgin E, Charras G, Betzig E, Eggeling C
    Nature Communications. 2017 Feb 13;8:14347. doi: 10.1038/ncomms14347

    Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

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    02/08/17 | Cilia-mediated Hedgehog signaling controls form and function in the mammalian larynx.
    Tabler JM, Rigney MM, Berman GJ, Gopalakrishnan S, Heude E, Al-Lami HA, Yannakoudakis BZ, Fitch RD, Carter CM, Vokes SA, Liu KJ, Tajbakhsh S, Egnor SR, Wallingford JB
    eLife. 2017 Feb 08;6:. doi: 10.7554/eLife.19153

    Acoustic communication is fundamental to social interactions among animals, including humans. In fact, deficits in voice impair the quality of life for a large and diverse population of patients. Understanding the molecular genetic mechanisms of development and function in the vocal apparatus is thus an important challenge with relevance both to the basic biology of animal communication and to biomedicine. However, surprisingly little is known about the developmental biology of the mammalian larynx. Here, we used genetic fate mapping to chart the embryological origins of the tissues in the mouse larynx, and we describe the developmental etiology of laryngeal defects in mice with disruptions in cilia-mediated Hedgehog signaling. In addition, we show that mild laryngeal defects correlate with changes in the acoustic structure of vocalizations. Together, these data provide key new insights in the molecular genetics of form and function in the mammalian vocal apparatus.

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