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190 Publications

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    05/01/12 | Genetic architecture and adaptive significance of the selfing syndrome in Capsella.
    Slotte T, Hazzouri KM, Stern D, Andolfatto P, Wright SI
    Evolution: International Journal of Organic Evolution. 2012 May;66(5):1360-74. doi: 10.1111/j.1558-5646.2011.01540.x

    The transition from outcrossing to predominant self-fertilization is one of the most common evolutionary transitions in flowering plants. This shift is often accompanied by a suite of changes in floral and reproductive characters termed the selfing syndrome. Here, we characterize the genetic architecture and evolutionary forces underlying evolution of the selfing syndrome in Capsella rubella following its recent divergence from the outcrossing ancestor C. grandiflora. We conduct genotyping by multiplexed shotgun sequencing and map floral and reproductive traits in a large (N= 550) F2 population. Our results suggest that in contrast to previous studies of the selfing syndrome, changes at a few loci, some with major effects, have shaped the evolution of the selfing syndrome in Capsella. The directionality of QTL effects, as well as population genetic patterns of polymorphism and divergence at 318 loci, is consistent with a history of directional selection on the selfing syndrome. Our study is an important step toward characterizing the genetic basis and evolutionary forces underlying the evolution of the selfing syndrome in a genetically accessible model system.

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    Looger LabSvoboda Lab
    04/26/12 | Multiple dynamic representations in the motor cortex during sensorimotor learning.
    Huber D, Gutnisky DA, Peron S, O’Connor DH, Wiegert JS, Tian L, Oertner TG, Looger L, Svoboda K
    Nature. 2012 Apr 26;484(7395):473-8. doi: 10.1038/nature11039

    The mechanisms linking sensation and action during learning are poorly understood. Layer 2/3 neurons in the motor cortex might participate in sensorimotor integration and learning; they receive input from sensory cortex and excite deep layer neurons, which control movement. Here we imaged activity in the same set of layer 2/3 neurons in the motor cortex over weeks, while mice learned to detect objects with their whiskers and report detection with licking. Spatially intermingled neurons represented sensory (touch) and motor behaviours (whisker movements and licking). With learning, the population-level representation of task-related licking strengthened. In trained mice, population-level representations were redundant and stable, despite dynamism of single-neuron representations. The activity of a subpopulation of neurons was consistent with touch driving licking behaviour. Our results suggest that ensembles of motor cortex neurons couple sensory input to multiple, related motor programs during learning.

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    04/19/12 | Differential uptake of MRI contrast agents indicates charge-selective blood-brain interface in the crayfish
    Otopalik AG, Shin J, Beltz BS, Sandeman DC, Kolodny NH
    Cell and Tissue Research. 2012 Apr 19;349(2):493 - 503. doi: 10.1007/s00441-012-1413-9

    This study provides a new perspective on the long-standing problem of the nature of the decapod crustacean blood-brain interface. Previous studies of crustacean blood-brain interface permeability have relied on invasive histological, immunohistochemical and electrophysiological techniques, indicating a leaky non-selective blood-brain barrier. The present investigation involves the use of magnetic resonance imaging (MRI), a method for non-invasive longitudinal tracking of tracers in real-time. Differential uptake rates of two molecularly distinct MRI contrast agents, namely manganese (Mn(II)) and Magnevist® (Gd-DTPA), were observed and quantified in the crayfish, Cherax destructor. Contrast agents were injected into the pericardium and uptake was observed with longitudinal MRI for approximately 14.5 h. Mn(II) was taken up quickly into neural tissue (within 6.5 min), whereas Gd-DTPA was not taken up into neural tissue and was instead restricted to the intracerebral vasculature or excreted into nearby sinuses. Our results provide evidence for a charge-selective intracerebral blood-brain interface in the crustacean nervous system, a structural characteristic once considered too complex for a lower-order arthropod.

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    04/19/12 | Intracellular spatial localization regulated by the microtubule network.
    Chen J, Lippincott-Schwartz J, Liu J
    PloS one. 2012;7(4):e34919. doi: 10.1371/journal.pone.0034919

    The commonly recognized mechanisms for spatial regulation inside the cell are membrane-bounded compartmentalization and biochemical association with subcellular organelles. We use computational modeling to investigate another spatial regulation mechanism mediated by the microtubule network in the cell. Our results demonstrate that the mitotic spindle can impose strong sequestration and concentration effects on molecules with binding affinity for microtubules, especially dynein-directed cargoes. The model can recapitulate the essence of three experimental observations on distinct microtubule network morphologies: the sequestration of germ plasm components by the mitotic spindles in the Drosophila syncytial embryo, the asymmetric cell division initiated by the time delay in centrosome maturation in the Drosophila neuroblast, and the diffusional block between neighboring energids in the Drosophila syncytial embryo. Our model thus suggests that the cell cycle-dependent changes in the microtubule network are critical for achieving different spatial regulation effects. The microtubule network provides a spatially extensive docking platform for molecules and gives rise to a "structured cytoplasm", in contrast to a free and fluid environment.

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    04/17/12 | Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.
    Kopek BG, Shtengel G, Xu CS, Clayton DA, Hess HF
    Proceedings of the National Academy of Science of the United States of America. 2012 Apr 17;109(16):6136-41. doi: 10.1073/pnas.1121558109

    Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.

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    04/15/12 | Bioimage informatics: a new category in Bioinformatics.
    Peng H, Bateman A, Valencia A, Wren JD
    Bioinformatics. 2012 Apr 15;28(8):1057. doi: 10.1093/bioinformatics/bts111
    04/09/12 | Using translational enhancers to increase transgene expression in Drosophila.
    Pfeiffer BD, Truman JW, Rubin GM
    Proceedings of the National Academy of Sciences of the United States of America. 2012 Apr 9;109(17):6626-31. doi: 10.1073/pnas.1204520109

    The ability to specify the expression levels of exogenous genes inserted in the genomes of transgenic animals is critical for the success of a wide variety of experimental manipulations. Protein production can be regulated at the level of transcription, mRNA transport, mRNA half-life, or translation efficiency. In this report, we show that several well-characterized sequence elements derived from plant and insect viruses are able to function in Drosophila to increase the apparent translational efficiency of mRNAs by as much as 20-fold. These increases render expression levels sufficient for genetic constructs previously requiring multiple copies to be effective in single copy, including constructs expressing the temperature-sensitive inactivator of neuronal function Shibire(ts1), and for the use of cytoplasmic GFP to image the fine processes of neurons.

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    Gonen Lab
    04/05/12 | The influence of lipids on voltage-gated ion channels.
    Jiang Q, Gonen T
    Current Opinion in Structural Biology. 2012 Apr 5;22(4):529-36. doi: 10.1016/j.sbi.2012.03.009

    Voltage-gated ion channels are responsible for transmitting electrochemical signals in both excitable and non-excitable cells. Structural studies of voltage-gated potassium and sodium channels by X-ray crystallography have revealed atomic details on their voltage-sensor domains (VSDs) and pore domains, and were put in context of disparate mechanistic views on the voltage-driven conformational changes in these proteins. Functional investigation of voltage-gated channels in membranes, however, showcased a mechanism of lipid-dependent gating for voltage-gated channels, suggesting that the lipids play an indispensible and critical role in the proper gating of many of these channels. Structure determination of membrane-embedded voltage-gated ion channels appears to be the next frontier in fully addressing the mechanism by which the VSDs control channel opening. Currently electron crystallography is the only structural biology method in which a membrane protein of interest is crystallized within a complete lipid-bilayer mimicking the native environment of a biological membrane. At a sufficiently high resolution, an electron crystallographic structure could reveal lipids, the channel and their mutual interactions at the atomic level. Electron crystallography is therefore a promising avenue toward understanding how lipids modulate channel activation through close association with the VSDs.

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    04/03/12 | Lmo4 in the basolateral complex of the amygdala modulates fear learning.
    Maiya R, Kharazia V, Lasek AW, Heberlein U
    PLoS One. 2012 Apr 3;7(4):e34559. doi: 10.1371/journal.pone.0034559

    Pavlovian fear conditioning is an associative learning paradigm in which mice learn to associate a neutral conditioned stimulus with an aversive unconditioned stimulus. In this study, we demonstrate a novel role for the transcriptional regulator Lmo4 in fear learning. LMO4 is predominantly expressed in pyramidal projection neurons of the basolateral complex of the amygdala (BLC). Mice heterozygous for a genetrap insertion in the Lmo4 locus (Lmo4gt/+), which express 50% less Lmo4 than their wild type (WT) counterparts display enhanced freezing to both the context and the cue in which they received the aversive stimulus. Small-hairpin RNA-mediated knockdown of Lmo4 in the BLC, but not the dentate gyrus region of the hippocampus recapitulated this enhanced conditioning phenotype, suggesting an adult- and brain region-specific role for Lmo4 in fear learning. Immunohistochemical analyses revealed an increase in the number of c-Fos positive puncta in the BLC of Lmo4gt/+ mice in comparison to their WT counterparts after fear conditioning. Lastly, we measured anxiety-like behavior in Lmo4gt/+ mice and in mice with BLC-specific downregulation of Lmo4 using the elevated plus maze, open field, and light/dark box tests. Global or BLC-specific knockdown of Lmo4 did not significantly affect anxiety-like behavior. These results suggest a selective role for LMO4 in the BLC in modulating learned but not unlearned fear.

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    Cui Lab
    04/03/12 | Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination.
    Fiolka R, Shao L, Rego EH, Davidson MW, Gustafsson MG
    Proceedings of the National Academy of Sciences of the United States of America. 2012 Apr 3;109(14):5311-5. doi: 10.1073/pnas.1119262109

    Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.

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