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194 Publications

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    Gonen Lab
    02/01/13 | HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation.
    Filbin ME, Vollmar BS, Shi D, Gonen T, Kieft JS
    Nature Structural & Molecular Biology. 2013 Feb;20(2):150-8. doi: 10.1038/nsmb.2465

    The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit’s decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome’s structure and function can be altered by bound RNAs, including those derived from cellular invaders.

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    02/01/13 | Intensity discrimination deficits cause habituation changes in middle-aged Caenorhabditis elegans.
    Timbers TA, Giles AC, Ardiel EL, Kerr RA, Rankin CH
    Neurobiology of Aging. 2013 Feb;34(2):621-31. doi: 10.1016/j.neurobiolaging.2012.03.016

    The ability to learn and remember is critical for all animals to survive in the ever-changing environment. As we age, many of our biological faculties decay and of these, decline in learning and memory can be the most distressing. To carefully define age-dependent changes in learning during reproductive age in the nematode Caenorhabditis elegans, we performed a parametric behavioral study of habituation to nonlocalized mechanical stimuli (petri plate taps) over a range of intensities in middle-aged worms. We found that as worms age (from the onset of reproduction to the end of egg laying), response probability habituation increases (at both 10- and 60-second interstimulus intervals) and that these age-related changes were associated with a decrease in the discrimination between stimuli of different intensities. We also used optogenetics to investigate where these age-dependent changes occur. Our data suggest that the changes occur upstream of mechanosensory neuron depolarization. These data support the idea that declines in stimulus intensity discrimination abilities during aging may be one variable underlying age-related cognitive deficits.

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    02/01/13 | Optogenetics in a transparent animal: circuit function in the larval zebrafish.
    Portugues R, Severi KE, Wyart C, Ahrens MB
    Current Opinion in Neurobiology. 2013 Feb;23(1):119-26. doi: 10.1016/j.conb.2012.11.001

    Optogenetic tools can be used to manipulate neuronal activity in a reversible and specific manner. In recent years, such methods have been applied to uncover causal relationships between activity in specified neuronal circuits and behavior in the larval zebrafish. In this small, transparent, genetic model organism, noninvasive manipulation and monitoring of neuronal activity with light is possible throughout the nervous system. Here we review recent work in which these new tools have been applied to zebrafish, and discuss some of the existing challenges of these approaches.

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    Singer Lab
    02/01/13 | Single-molecule analysis of gene expression using two-color RNA labeling in live yeast.
    Hocine S, Raymond P, Zenklusen D, Chao JA, Singer RH
    Nature Methods. 2013 Feb;10(2):119-21. doi: 10.1038/nmeth.2305

    Live-cell imaging of mRNA yields important insights into gene expression, but it has generally been limited to the labeling of one RNA species and has never been used to count single mRNAs over time in yeast. We demonstrate a two-color imaging system with single-molecule resolution using MS2 and PP7 RNA labeling. We use this methodology to measure intrinsic noise in mRNA levels and RNA polymerase II kinetics at a single gene.

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    02/01/13 | Taok2 controls behavioral response to ethanol in mice.
    Kapfhamer D, Taylor S, Zou ME, Lim JP, Kharazia V, Heberlein U
    Genes, Brain, and Behavior. 2013 Feb;12(1):87-97. doi: 10.1111/j.1601-183X.2012.00834.x

    Despite recent advances in the understanding of ethanol's biological action, many of the molecular targets of ethanol and mechanisms behind ethanol's effect on behavior remain poorly understood. In an effort to identify novel genes, the products of which regulate behavioral responses to ethanol, we recently identified a mutation in the dtao gene that confers resistance to the locomotor stimulating effect of ethanol in Drosophila. dtao encodes a member of the Ste20 family of serine/threonine kinases implicated in MAP kinase signaling pathways. In this study, we report that conditional ablation of the mouse dtao homolog, Taok2, constitutively and specifically in the nervous system, results in strain-specific and overlapping alterations in ethanol-dependent behaviors. These data suggest a functional conservation of dtao and Taok2 in mediating ethanol's biological action and identify Taok2 as a putative candidate gene for ethanol use disorders in humans.

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    01/31/13 | Multi-channel acoustic recording and automated analysis of Drosophila courtship songs.
    Arthur BJ, Sunayama-Morita T, Coen P, Murthy M, Stern DL
    BMC Biology. 2013 Jan 31;11:11. doi: 10.1186/1741-7007-11-11

    Drosophila melanogaster has served as a powerful model system for genetic studies of courtship songs. To accelerate research on the genetic and neural mechanisms underlying courtship song, we have developed a sensitive recording system to simultaneously capture the acoustic signals from 32 separate pairs of courting flies as well as software for automated segmentation of songs.

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    01/23/13 | Multiple interactions control synaptic layer specificity in the Drosophila visual system.
    Pecot MY, Tadros W, Nern A, Bader M, Chen Y, Zipursky SL
    Neuron. 2013 Jan 23;77(2):299-310. doi: 10.1016/j.neuron.2012.11.007

    How neurons form synapses within specific layers remains poorly understood. In the Drosophila medulla, neurons target to discrete layers in a precise fashion. Here we demonstrate that the targeting of L3 neurons to a specific layer occurs in two steps. Initially, L3 growth cones project to a common domain in the outer medulla, overlapping with the growth cones of other neurons destined for a different layer through the redundant functions of N-Cadherin (CadN) and Semaphorin-1a (Sema-1a). CadN mediates adhesion within the domain and Sema-1a mediates repulsion through Plexin A (PlexA) expressed in an adjacent region. Subsequently, L3 growth cones segregate from the domain into their target layer in part through Sema-1a/PlexA-dependent remodeling. Together, our results and recent studies argue that the early medulla is organized into common domains, comprising processes bound for different layers, and that discrete layers later emerge through successive interactions between processes within domains and developing layers.

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    01/23/13 | Regularization and nonlinearities for neural language models: when are they needed?
    Pachitariu M, Sahani M
    arXiv. 2013 Jan 23:arXiv:1301.5650

    Neural language models (LMs) based on recurrent neural networks (RNN) are some of the most successful word and character-level LMs. Why do they work so well, in particular better than linear neural LMs? Possible explanations are that RNNs have an implicitly better regularization or that RNNs have a higher capacity for storing patterns due to their nonlinearities or both. Here we argue for the first explanation in the limit of little training data and the second explanation for large amounts of text data. We show state-of-the-art performance on the popular and small Penn dataset when RNN LMs are regularized with random dropout. Nonetheless, we show even better performance from a simplified, much less expressive linear RNN model without off-diagonal entries in the recurrent matrix. We call this model an impulse-response LM (IRLM). Using random dropout, column normalization and annealed learning rates, IRLMs develop neurons that keep a memory of up to 50 words in the past and achieve a perplexity of 102.5 on the Penn dataset. On two large datasets however, the same regularization methods are unsuccessful for both models and the RNN's expressivity allows it to overtake the IRLM by 10 and 20 percent perplexity, respectively. Despite the perplexity gap, IRLMs still outperform RNNs on the Microsoft Research Sentence Completion (MRSC) task. We develop a slightly modified IRLM that separates long-context units (LCUs) from short-context units and show that the LCUs alone achieve a state-of-the-art performance on the MRSC task of 60.8%. Our analysis indicates that a fruitful direction of research for neural LMs lies in developing more accessible internal representations, and suggests an optimization regime of very high momentum terms for effectively training such models.

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    01/15/13 | Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions.
    Legant WR, Choi CK, Miller JS, Shao L, Gao L, Betzig E, Chen CS
    Proceedings of the National Academy of Sciences of the United States of America. 2013 Jan 15;110(3):881-6. doi: 10.1073/pnas.1207997110

    Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.

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    Grigorieff Lab
    01/09/13 | Location of the dsRNA-dependent polymerase, VP1, in rotavirus particles.
    Estrozi LF, Settembre EC, Goret G, McClain B, Zhang X, Chen JZ, Grigorieff N, Harrison SC
    Journal of Molecular Biology. 2013 Jan 9;425(1):124-32. doi: 10.1016/j.jmb.2012.10.011

    Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner double-layered particle (DLP) into the cytosol. DLP structures determined by X-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a "fivefold hub" of more structured density projecting inward from the VP2 shell of the DLP along each of the twelve 5-fold axes. Analysis of the X-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the X-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the 5-fold axis. The template strand enters from within the particle, and the dsRNA product of the initial replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.

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