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18 Janelia Publications

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    06/27/17 | Cryo-electron tomography reveals novel features of a viral RNA replication compartment.
    Ertel KJ, Benefield D, Castaño-Diez D, Pennington J, Horswill M, den Boon JA, Otegui M, Ahlquist P
    eLife. 2017 Jun 27;6:. doi: 10.7554/eLife.25940

    Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments is largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule's necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control.

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    06/29/17 | Desensitized D2 autoreceptors are resistant to trafficking.
    Robinson BG, Bunzow JR, Grimm JB, Lavis LD, Dudman JT, Brown J, Neve KA, Williams JT
    Scientific Reports. 2017 Jun 29;7(1):4379. doi: 10.1038/s41598-017-04728-z

    Dendritic release of dopamine activates dopamine D2 autoreceptors, which are inhibitory G protein-coupled receptors (GPCRs), to decrease the excitability of dopamine neurons. This study used tagged D2 receptors to identify the localization and distribution of these receptors in living midbrain dopamine neurons. GFP-tagged D2 receptors were found to be unevenly clustered on the soma and dendrites of dopamine neurons within the substantia nigra pars compacta (SNc). Physiological signaling and desensitization of the tagged receptors were not different from wild type receptors. Unexpectedly, upon desensitization the tagged D2 receptors were not internalized. When tagged D2 receptors were expressed in locus coeruleus neurons, a desensitizing protocol induced significant internalization. Likewise, when tagged µ-opioid receptors were expressed in dopamine neurons they too were internalized. The distribution and lack of agonist-induced internalization of D2 receptors on dopamine neurons indicate a purposefully regulated localization of these receptors.

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    06/29/17 | Rational engineering of photoconvertible fluorescent proteins for dual-color fluorescence nanoscopy enabled by a triplet-state mechanism of primed conversion.
    Mohr MA, Kobitski AY, Sabater LR, Nienhaus K, Obara CJ, Lippincott-Schwartz J, Nienhaus GU, Pantazis P
    Angewandte Chemie (International ed. in English). 2017 Jun 29;56(38):11628-33. doi: 10.1002/anie.201706121

    Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs.

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    Gonen Lab
    06/24/17 | MicroED structure of Au146(p-MBA)57 at subatomic resolution reveals a twinned FCC cluster.
    Vergara S, Lukes DA, Martynowycz MW, Santiago U, Plascencia-Villa G, Weiss SC, de la Cruz MJ, Black DM, Alvarez MM, Lopez-Lozano X, Barnes CO, Lin G, Weissker H, Whetten RL, Gonen T, Calero G
    Journal of Physical Chemistry Letters. 2017 Oct 31;8(5523-30):arXiv:1706.07902 [physics.atm-clus]. doi: 10.1021/acs.jpclett.7b02621

    Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. We report the structure of Au146(p-MBA)57 at subatomic resolution (0.85 {\AA}) using electron diffraction (MicroED) and atomic resolution by X-ray diffraction. The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.

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    06/22/17 | 3D Bayesian cluster analysis of super-resolution data reveals LAT recruitment to the T cell synapse.
    Griffié J, Shlomovich L, Williamson DJ, Shannon M, Aaron J, Khuon S, L Burn G, Boelen L, Peters R, Cope AP, Cohen EA, Rubin-Delanchy P, Owen DM
    Scientific Reports. 2017 Jun 22;7(1):4077. doi: 10.1038/s41598-017-04450-w

    Single-molecule localisation microscopy (SMLM) allows the localisation of fluorophores with a precision of 10-30 nm, revealing the cell's nanoscale architecture at the molecular level. Recently, SMLM has been extended to 3D, providing a unique insight into cellular machinery. Although cluster analysis techniques have been developed for 2D SMLM data sets, few have been applied to 3D. This lack of quantification tools can be explained by the relative novelty of imaging techniques such as interferometric photo-activated localisation microscopy (iPALM). Also, existing methods that could be extended to 3D SMLM are usually subject to user defined analysis parameters, which remains a major drawback. Here, we present a new open source cluster analysis method for 3D SMLM data, free of user definable parameters, relying on a model-based Bayesian approach which takes full account of the individual localisation precisions in all three dimensions. The accuracy and reliability of the method is validated using simulated data sets. This tool is then deployed on novel experimental data as a proof of concept, illustrating the recruitment of LAT to the T-cell immunological synapse in data acquired by iPALM providing ~10 nm isotropic resolution.

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    Gonen Lab
    06/22/17 | Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets.
    Hughes MP, Sawaya MR, Goldschmidt L, Rodriguez JA, Cascio D, Gonen T, Eisenberg DS
    bioRxiv. 2017 Jun 22:. doi: 10.1101/153817

    Control of metabolism by compartmentation is a widespread feature of higher cells. Recent studies have focused on dynamic intracellular bodies such as stress granules, P-bodies, nucleoli, and metabolic puncta. These bodies appear as separate phases, some containing reversible, amyloid-like fibrils formed by interactions of low-complexity protein domains. Here we report five atomic structures of segments of low-complexity domains from granule-forming proteins, one determined to 1.1 Å resolution by micro-electron diffraction. Four of these interacting protein segments show common characteristics, all in contrast to pathogenic amyloid: kinked peptide backbones, small surface areas of interaction, and predominate attractions between aromatic side-chains. By computationally threading the human proteome on three of our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such reversible interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, keratins, and cornified envelope proteins, consistent with the capacity of cells to form a wide variety of dynamic intracellular bodies.

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    Gonen Lab
    06/22/17 | Taking the measure of MicroED.
    Rodriguez JA, Eisenberg DS, Gonen T
    Current Opinion in Structural Biology. 2017 Jun 22;46:79-86. doi: 10.1016/

    It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.

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    Gonen Lab
    06/21/17 | A method to minimize condenser lens-induced hysteresis effects in a JEOL JEM-3200FSC microscope to enable stable cryoEM low-dose operations.
    de la Cruz MJ, Martynowycz M, Hattne J, Shi D, Gonen T
    bioRxiv. 2017 Jun 21:. doi: 10.1101/153395

    Low dose imaging procedures are key for a successful cryoEM experiment (whether by electron cryotomography, single particle analysis, electron crystallography, or MicroED). We present a method to minimize magnetic hysteresis of the condenser lens system in the JEOL JEM-3200FSC transmission electron microscope (TEM) in order to maintain a stable optical axis for the beam path of low-dose imaging. The simple procedure involves independent voltage ramping of the CL1 and CL2 lenses immediately before switching to the focusing and exposure beam settings for data collection.

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    06/21/17 | Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation.
    Fritzsche M, Fernandes RA, Chang VT, Colin-York H, Clausen MP, Felce JH, Galiani S, Erlenkämper C, Santos AM, Heddleston JM, Pedroza-Pacheco I, Waithe D, de la Serna JB, Lagerholm BC, Liu T, Chew T, Betzig E, Davis SJ, Eggeling C
    Science Advances. 2017 Jun 21;3(6):e1603032. doi: 10.1126/sciadv.1603032

    T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions.

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    06/21/17 | Feature integration drives probabilistic behavior in the Drosophila escape response.
    von Reyn CR, Nern A, Williamson WR, Breads P, Wu M, Namiki S, Card GM
    Neuron. 2017 Jun 21;94(6):1190-204. doi: 10.1016/j.neuron.2017.05.036

    Animals rely on dedicated sensory circuits to extract and encode environmental features. How individual neurons integrate and translate these features into behavioral responses remains a major question. Here, we identify a visual projection neuron type that conveys predator approach information to the Drosophila giant fiber (GF) escape circuit. Genetic removal of this input during looming stimuli reveals that it encodes angular expansion velocity, whereas other input cell type(s) encode angular size. Motor program selection and timing emerge from linear integration of these two features within the GF. Linear integration improves size detection invariance over prior models and appropriately biases motor selection to rapid, GF-mediated escapes during fast looms. Our findings suggest feature integration, and motor control may occur as simultaneous operations within the same neuron and establish the Drosophila escape circuit as a model system in which these computations may be further dissected at the circuit level.

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