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205 Janelia Publications

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    Gonen Lab
    12/26/18 | MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat.
    Purdy MD, Shi D, Chrustowicz J, Hattne J, Gonen T, Yeager M
    Proceedings of the National Academy of Sciences of the United States of America. 2018 Dec 26;115(52):13258-63. doi: 10.1073/pnas.1806806115

    HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

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    12/18/18 | Interleukin 2 modulates thymic-derived regulatory T cell epigenetic landscape.
    Chorro L, Suzuki M, Chin SS, Williams TM, Snapp EL, Odagiu L, Labrecque N, Lauvau G
    Nature Communications. 2018 Dec 18;9(1):5368. doi: 10.1038/s41467-018-07806-6

    Foxp3CD4 regulatory T (T) cells are essential for preventing fatal autoimmunity and safeguard immune homeostasis in vivo. While expression of the transcription factor Foxp3 and IL-2 signals are both required for the development and function of T cells, the commitment to the T cell lineage occurs during thymic selection upon T cell receptor (TCR) triggering, and precedes the expression of Foxp3. Whether signals beside TCR contribute to establish T cell epigenetic and functional identity is still unknown. Here, using a mouse model with reduced IL-2 signaling, we show that IL-2 regulates the positioning of the pioneer factor SATB1 in CD4 thymocytes and controls genome wide chromatin accessibility of thymic-derived T cells. We also show that T cells receiving only low IL-2 signals can suppress endogenous but not WT autoreactive T cell responses in vitro and in vivo. Our findings have broad implications for potential therapeutic strategies to reprogram T cells in vivo.

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    12/18/18 | Mapping Neurotransmitter Identity in the Whole-Mount Brain Using Multiplex High-Throughput Fluorescence Hybridization.
    Meissner GW, Nern A, Singer RH, Wong AM, Malkesman O, Long X
    Genetics. 2018 Dec 18;211(2):473-82. doi: 10.1534/genetics.118.301749

    Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter co-expression, reveal neurotransmitter phenotypes of specific cell types and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large scale FISH detection of any mRNA in whole-mount fly brains.

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    12/14/18 | Motor cortex is an input-driven dynamical system controlling dexterous movement.
    Sauerbrei B, Guo J, Mischiati M, Guo W, Kabra M, Verma N, Branson KM, Hantman AW
    bioRxiv. 2018-12-14:266320. doi: 10.1101/266320

    Skillful control of movement is central to our ability to sense and manipulate the world. A large body of work in nonhuman primates has demonstrated that motor cortex provides flexible, time-varying activity patterns that control the arm during reaching and grasping. Previous studies have suggested that these patterns are generated by strong local recurrent dynamics operating autonomously from inputs during movement execution. An alternative possibility is that motor cortex requires coordination with upstream brain regions throughout the entire movement in order to yield these patterns. Here, we developed an experimental preparation in the mouse to directly test these possibilities using optogenetics and electrophysiology during a skilled reach-to-grab-to-eat task. To validate this preparation, we first established that a specific, time-varying pattern of motor cortical activity was required to produce coordinated movement. Next, in order to disentangle the contribution of local recurrent motor cortical dynamics from external input, we optogenetically held the recurrent contribution constant, then observed how motor cortical activity recovered following the end of this perturbation. Both the neural responses and hand trajectory varied from trial to trial, and this variability reflected variability in external inputs. To directly probe the role of these inputs, we used optogenetics to perturb activity in the thalamus. Thalamic perturbation at the start of the trial prevented movement initiation, and perturbation at any stage of the movement prevented progression of the hand to the target; this demonstrates that input is required throughout the movement. By comparing motor cortical activity with and without thalamic perturbation, we were able to estimate the effects of external inputs on motor cortical population activity. Thus, unlike pattern-generating circuits that are local and autonomous, such as those in the spinal cord that generate left-right alternation during locomotion, the pattern generator for reaching and grasping is distributed across multiple, strongly-interacting brain regions.

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    12/13/18 | Gene flow mediates the role of sex chromosome meiotic drive during complex speciation.
    Meiklejohn CD, Landeen EL, Gordon KE, Rzatkiewicz T, Kingan SB, Geneva AJ, Vedanayagam JP, Muirhead CA, Garrigan D, Stern DL, Presgraves DC
    eLife. 2018 Dec 13;7:. doi: 10.7554/eLife.35468

    During speciation, sex chromosomes often accumulate interspecific genetic incompatibilities faster than the rest of the genome. The drive theory posits that sex chromosomes are susceptible to recurrent bouts of meiotic drive and suppression, causing the evolutionary build-up of divergent cryptic sex-linked drive systems and, incidentally, genetic incompatibilities. To assess the role of drive during speciation, we combine high-resolution genetic mapping of X-linked hybrid male sterility with population genomics analyses of divergence and recent gene flow between the fruitfly species, and . Our findings reveal a high density of genetic incompatibilities and a corresponding dearth of gene flow on the X chromosome. Surprisingly, we find that a known drive element recently migrated between species and, rather than contributing to interspecific divergence, caused a strong reduction in local sequence divergence, undermining the evolution of hybrid sterility. Gene flow can therefore mediate the effects of selfish genetic elements during speciation.

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    12/12/18 | Reactive oxygen species regulate activity-dependent neuronal structural plasticity in Drosophila.
    Oswald MC, Brooks PS, Zwart MF, Mukherjee A, West RJ, Morarach K, Sweeney ST, Landgraf M
    eLife. 2018 Dec 12:. doi: 10.7554/eLife.39393

    Neurons are inherently plastic, adjusting their structure, connectivity and excitability in response to changes in activity. How neurons sense changes in their activity level and then transduce these to structural changes remains to be fully elucidated. Working with the Drosophila larval locomotor network, we show that neurons use reactive oxygen species (ROS), metabolic byproducts, to monitor their activity. ROS signals are both necessary and sufficient for activity-dependent structural adjustments of both pre- and postsynaptic terminals and for network output, as measured by larval crawling behavior. We find the highly conserved Parkinsons disease-linked protein DJ-1b acts as a redox sensor in neurons where it regulates pre- and postsynaptic structural plasticity, in part via modulation of the PTEN-PI3Kinase pathway. Neuronal ROS thus play an important physiological role as second messengers required for neuronal and network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction.

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    12/12/18 | Structure of Plasmodium falciparum Rh5-CyRPA-Ripr invasion complex.
    Wong W, Huang R, Menant S, Hong C, Sandow JJ, Birkinshaw RW, Healer J, Hodder AN, Kanjee U, Tonkin CJ, Heckmann D, Soroka V, Søgaard TM, Jørgensen T, Duraisingh MT, Czabotar PE, de Jongh WA, Tham W, Webb AI, Yu Z, Cowman AF
    Nature. 2018 Dec 12;565(7737):118-21. doi: 10.1038/s41586-018-0779-6

    Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the β-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.

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    12/11/18 | Convergence of monosynaptic and polysynaptic sensory paths onto common motor outputs in a feeding connectome.
    Miroschnikow A, Schlegel P, Schoofs A, Hueckesfeld S, Li F, Schneider-Mizell CM, Fetter RD, Truman JW, Cardona A, Pankratz MJ
    eLife. 2018 Dec 11;7:. doi: 10.7554/eLife.40247

    We reconstructed, from a whole CNS EM volume, the synaptic map of input and output neurons that underlie food intake behavior of larvae. Input neurons originate from enteric, pharyngeal and external sensory organs and converge onto seven distinct sensory synaptic compartments within the CNS. Output neurons consist of feeding motor, serotonergic modulatory and neuroendocrine neurons. Monosynaptic connections from a set of sensory synaptic compartments cover the motor, modulatory and neuroendocrine targets in overlapping domains. Polysynaptic routes are superimposed on top of monosynaptic connections, resulting in divergent sensory paths that converge on common outputs. A completely different set of sensory compartments is connected to the mushroom body calyx. The mushroom body output neurons are connected to interneurons that directly target the feeding output neurons. Our results illustrate a circuit architecture in which monosynaptic and multisynaptic connections from sensory inputs traverse onto output neurons via a series of converging paths.

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    12/11/18 | MYC induces a hybrid energetics program early in cell reprogramming.
    Prieto J, Seo AY, León M, Santacatterina F, Torresano L, Palomino-Schätzlein M, Giménez K, Vallet-Sánchez A, Ponsoda X, Pineda-Lucena A, Cuezva JM, Lippincott-Schwartz J, Torres J
    Stem Cell Reports. 2018 Dec 11;11(6):1479-92. doi: 10.1016/j.stemcr.2018.10.018

    Cell reprogramming is thought to be associated with a full metabolic switch from an oxidative- to a glycolytic-based metabolism. However, neither the dynamics nor the factors controlling this metabolic switch are fully understood. By using cellular, biochemical, protein array, metabolomic, and respirometry analyses, we found that c-MYC establishes a robust bivalent energetics program early in cell reprogramming. Cells prone to undergo reprogramming exhibit high mitochondrial membrane potential and display a hybrid metabolism. We conclude that MYC proteins orchestrate a rewiring of somatic cell metabolism early in cell reprogramming, whereby somatic cells acquire the phenotypic plasticity necessary for their transition to pluripotency in response to either intrinsic or external cues.

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    12/10/18 | Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores.
    Spahn C, Grimm JB, Lavis LD, Lampe M, Heilemann M
    Nano Letters. 2018 Dec 10;19(1):500-5. doi: 10.1021/acs.nanolett.8b04385

    We demonstrate STED microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy.

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