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1832 Janelia Publications

Showing 91-100 of 1832 results
05/19/20 | mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding.
Bao C, Loerch S, Ling C, Korostelev AA, Grigorieff N, Ermolenko DN
eLife. 2020 May 19;9:. doi: 10.7554/eLife.55799

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from mRNA and the transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

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05/18/20 | Freeze-frame imaging of synaptic activity using SynTagMA.
Perez-Alvarez A, Fearey BC, Schulze C, O'Toole RJ, Moeyaert B, Mohr MA, Arganda-Carreras I, Yang W, Wiegert JS, Schreiter ER, Gee CE, Hoppa MB, Oertner TG
Nature Communications. 2020 May 18;11(1):2464. doi: 10.1038/s41467-020-16315-4

Information within the brain travels from neuron to neuron across synapses. At any given moment, only a few synapses within billions will be active and are thought to transmit key information about the environment, a behavior being executed or memory being recalled. Here we present SynTagMA, which marks active synapses within a ~2 s time window. Upon violet illumination, the genetically expressed tag converts from green to red fluorescence if bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we developed an analysis program that automatically identifies and tracks the fluorescence of thousands of individual synapses in tissue. Together, these tools provide a high throughput method for repeatedly mapping active synapses in vitro and in vivo.

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05/14/20 | Detecting the Starting Frame of Actions in Video
Kwak IS, Guo J, Hantman A, Branson K, Kriegman D
2020 IEEE Winter Conference on Applications of Computer Vision (WACV). 2020 May 14:. doi: 10.1109/WACV45572.202010.1109/WACV45572.2020.9093405

In this work, we address the problem of precisely localizing key frames of an action, for example, the precise time that a pitcher releases a baseball, or the precise time that a crowd begins to applaud. Key frame localization is a largely overlooked and important action-recognition problem, for example in the field of neuroscience, in which we would like to understand the neural activity that produces the start of a bout of an action. To address this problem, we introduce a novel structured loss function that properly weights the types of errors that matter in such applications: it more heavily penalizes extra and missed action start detections over small misalignments. Our structured loss is based on the best matching between predicted and labeled action starts. We train recurrent neural networks (RNNs) to minimize differentiable approximations of this loss. To evaluate these methods, we introduce the Mouse Reach Dataset, a large, annotated video dataset of mice performing a sequence of actions. The dataset was collected and labeled by experts for the purpose of neuroscience research. On this dataset, we demonstrate that our method outperforms related approaches and baseline methods using an unstructured loss.

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05/15/20 | Unanticipated stressful and rewarding experiences engage the same prefrontal cortex and ventral tegmental area neuronal populations.
Del Arco A, Park J, Moghaddam B
eNeuro. 2020 May 08:. doi: 10.1523/ENEURO.0029-20.2020

Brain networks that mediate motivated behavior in the context of aversive and rewarding experiences involve the prefrontal cortex (PFC) and ventral tegmental area (VTA). Neurons in both regions are activated by stress and reward, and by learned cues that predict aversive or appetitive outcomes. Recent studies have proposed that separate neuronal populations and circuits in these regions encode learned aversive versus appetitive contexts. But how about the actual experience? Do the same or different PFC and VTA neurons encode unanticipated aversive and appetitive experiences? To address this, we recorded unit activity and local field potentials (LFP) in the dorsomedial PFC (dmPFC) and VTA of male rats as they were exposed, in the same recording session, to reward (sucrose) or stress (tail pinch) spaced one hour apart. As expected, experience-specific neuronal responses were observed. About 15-25% of single units in each region responded by excitation or inhibition to either stress or reward, and only stress increased LFP theta oscillation power in both regions and coherence between regions. But the largest number of responses (29% dmPFC and 30% VTA units) involved dual-valence neurons that responded to both stress and reward exposure. Moreover, the temporal profile of neuronal population activity in dmPFC and VTA as assessed by principal component analysis were similar during both types of experiences. These results reveal that aversive and rewarding experiences engage overlapping neuronal populations in the dmPFC and the VTA. These populations may provide a locus of vulnerability for stress related disorders, which are often associated with anhedonia. Animals must recognize unexpected harmful and rewarding events in order to survive. How the brain represents these competing experiences is not fully understood. Two interconnected brain regions implicated in encoding both rewarding and stressful events are the dmPFC and the VTA. In either region, separate neurons and associated circuitry are assumed to respond to events with positive or negative valence. We find, however, that a significant subpopulation of neurons in dmPFC and VTA encode both rewarding and aversive experiences. These dual-valence neurons may provide a computational advantage for flexible planning of behavior when organisms face unexpected rewarding and harmful experiences.

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05/06/20 | Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy.
Ueda HR, Dodt H, Osten P, Economo MN, Chandrashekar J, Keller PJ
Neuron. 2020 May 06;106(3):369-387. doi: 10.1016/j.neuron.2020.03.004

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.

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05/04/20 | FMNL2 regulates dynamics of fascin in filopodia.
Pfisterer K, Levitt J, Lawson CD, Marsh RJ, Heddleston JM, Wait E, Ameer-Beg SM, Cox S, Parsons M
Journal of Cell Biology. 2020 May 04;219(5):. doi: 10.1083/jcb.201906111

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

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05/03/20 | Co-evolving wing spots and mating displays are genetically separable traits in Drosophila.
Massey JH, Rice GR, Firdaus A, Chen C, Yeh S, Stern DL, Wittkopp PJ
Evolution. 2020 May 03:. doi: 10.1111/evo.13990

The evolution of sexual traits often involves correlated changes in morphology and behavior. For example, in Drosophila, divergent mating displays are often accompanied by divergent pigment patterns. To better understand how such traits co-evolve, we investigated the genetic basis of correlated divergence in wing pigmentation and mating display between the sibling species Drosophila elegans and D. gunungcola. Drosophila elegans males have an area of black pigment on their wings known as a wing spot and appear to display this spot to females by extending their wings laterally during courtship. By contrast, D. gunungcola lost both of these traits. Using Multiplexed Shotgun Genotyping (MSG), we identified a ∼440 kb region on the X chromosome that behaves like a genetic switch controlling the presence or absence of male-specific wing spots. This region includes the candidate gene optomotor-blind (omb), which plays a critical role in patterning the Drosophila wing. The genetic basis of divergent wing display is more complex, with at least two loci on the X chromosome and two loci on autosomes contributing to its evolution. Introgressing the X-linked region affecting wing spot development from D. gunungcola into D. elegans reduced pigmentation in the wing spots but did not affect the wing display, indicating that these are genetically separable traits. Consistent with this observation, broader sampling of wild D. gunungcola populations confirmed the wing spot and wing display are evolving independently: some D. gunungcola males performed wing displays similar to D. elegans despite lacking wing spots. These data suggest that correlated selection pressures rather than physical linkage or pleiotropy are responsible for the coevolution of these morphological and behavioral traits. They also suggest that the change in morphology evolved prior to the change in behavior. This article is protected by copyright. All rights reserved.

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05/01/20 | Effect of circuit structure on odor representation in the insect olfactory system.
Rajagopalan A, Assisi C
eNeuro. 2020 May;7(3):1-12. doi: 10.1523/ENEURO.0130-19.2020

In Neuroscience, the structure of a circuit has often been used to intuit function - an inversion of Louis Kahn's famous dictum, `Form follows function' (Kristan and Katz 2006). However, different brain networks may utilize different network architectures to solve the same problem. The olfactory circuits of two insects, the Locust, and the fruit fly, , serve the same function - to identify and discriminate odors. The neural circuitry that achieves this shows marked structural differences. Projection neurons (PN) in the antennal lobe (AL) innervate Kenyon cells (KC) of the mushroom body (MB). In locust, each KC receives inputs from ∼50% PNs, a scheme that maximizes the difference between inputs to any two of ∼50,000 KCs. In contrast, in drosophila, this number is only 5% and appears sub-optimal. Using a computational model of the olfactory system, we show the activity of KCs is sufficiently high-dimensional that it can separate similar odors regardless of the divergence of PN-KC connections. However, when temporal patterning encodes odor attributes, dense connectivity outperforms sparse connections.Increased separability comes at the cost of reliability. The disadvantage of sparse connectivity can be mitigated by incorporating other aspects of circuit architecture seen in drosophila. Our simulations predict that drosophila and locust circuits lie at different ends of a continuum where the drosophila gives up on the ability to resolve similar odors to generalize across varying environments, while the locust separates odor representations but risks misclassifying noisy variants of the same odor. How does the structure of a network affect its function? We address this question in the context of two olfactory systems that serve the same function, to distinguish the attributes of different odorants, but do so using markedly distinct architectures. In the locust, the probability of connections between projection neurons and Kenyon cells - a layer downstream - is nearly 50%. In contrast, this number is merely 5% in drosophila. We developed computational models of these networks to understand the relative advantages of each connectivity. Our analysis reveals that the two systems exist along a continuum of possibilities that balance two conflicting goals - separating the representations of similar odors while grouping together noisy variants of the same odor.

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05/01/20 | Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation.
Samuels TJ, Arava Y, Järvelin AI, Robertson F, Lee JY, Yang L, Yang C, Lee T, Ish-Horowicz D, Davis I
Biology Open. 2020 May;9(5):. doi: 10.1242/bio.049684

During and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent hybridisation to show that larval neurons selectively transcribe a long mRNA isoform containing a 15 kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the mRNA stability of the long isoform, which allows an upregulation of Prospero protein production. Adult flies selectively lacking the long isoform show abnormal behaviour that could result from impaired locomotor or neurological activity. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.

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05/01/20 | Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.
Trzaskoma P, Ruszczycki B, Lee B, Pels KK, Krawczyk K, Bokota G, Szczepankiewicz AA, Aaron J, Walczak A, Śliwińska MA, Magalska A, Kadlof M, Wolny A, Parteka Z, Arabasz S, Kiss-Arabasz M, Plewczyński D, Ruan Y, Wilczyński GM
Nature Communications. 2020 May 01;11(1):2120. doi: 10.1038/s41467-020-15987-2

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states.

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