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139 Publications

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    07/15/08 | Molecular characterization of pea aphid facultative parthenogenesis
    Dayalan G. Srinivasan , Greg K. Davis , David L. Stern
    Developmental Biology. 07/2008;319(2):494-495. doi: 10.1016/j.ydbio.2008.05.098

    Meiosis is a highly conserved process in which a diploid genome is recombined and assorted into haploid gametes. Remarkably, the pea aphid Acyrthosiphon pisum exhibits a reproductive polyphenism whereby environmental signals trigger a switch between apomixis (parthenogenetic reproduction) and meiosis (sexual reproduction). Aphid apomixis results in daughter embryo clones with 2n genome content without male contribution or recombination. This important adaptation allows aphid populations to not only rapidly expand upon abundant resources during summer but also survive winter. How aphids have evolved this ability to switch between parthenogenesis and sexual meiosis is unknown. To arrive at a mechanistic explanation for this developmental plasticity, I determined meiosis gene activity in sexuals and asexuals. I first identified homologs of a core set of meiosis genes from the pea aphid genome. Next, I tested the expression of these core meiosis genes by PCR spanning across at least one intron from cDNA isolated from asexual and sexual ovaries. Surprisingly, meiosis specific genes (e.g., Spo11, Msh4, Msh5, Hop2 and Mnd1) are expressed in asexual ovaries. Additionally, the Spo11 PCR product contained intronic sequence, thus representing unspliced mRNA. Future experiments looking at the quantities and localizations of mRNA and protein will help to distinguish among several possible explanations for these results. Further molecular characterization of this phenotypic plasticity will be helpful in understanding how multiple interacting pathways can evolve to create alternate developmental phenotypes.

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    07/15/08 | Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator.
    Fiolka R, Beck M, Stemmer A
    Optics Letters. 2008 Jul 15;33(14):1629-31

    In wide-field fluorescence microscopy, illuminating the specimen with evanescent standing waves increases lateral resolution more than twofold. We report a versatile setup for standing-wave illumination in total internal reflection fluorescence microscopy. An adjustable diffraction grating written on a phase-only spatial light modulator controls the illumination field. Selecting appropriate diffraction orders and displaying a sheared (tilted) diffraction grating allows one to tune the penetration depth in very fine steps. The setup achieves 91 nm lateral resolution for green emission.

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    07/15/08 | Tools for neuroanatomy and neurogenetics in Drosophila.
    Pfeiffer BD, Jenett A, Hammonds AS, Ngo TB, Misra S, Murphy C, Scully A, Carlson JW, Wan KH, Laverty TR, Mungall C, Svirskas R, Kadonaga JT, Doe CQ, Eisen MB, Celniker SE, Rubin GM
    Proceedings of the National Academy of Sciences of the United States of America. 2008 Jul 15;105:9715-20. doi:

    We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.

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    Sternson Lab
    07/09/08 | A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping.
    Atasoy D, Aponte Y, Su HH, Sternson SM
    The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2008 Jul 9;28(28):7025-30. doi: 10.1523/JNEUROSCI.1954-08.2008
    Svoboda Lab
    07/04/08 | The spread of Ras activity triggered by activation of a single dendritic spine.
    Harvey CD, Yasuda R, Zhong H, Svoboda K
    Science. 2008 Jul 4;321(5885):136-40. doi: 10.1126/science.1159675

    In neurons, individual dendritic spines isolate N-methyl-d-aspartate (NMDA) receptor-mediated calcium ion (Ca2+) accumulations from the dendrite and other spines. However, the extent to which spines compartmentalize signaling events downstream of Ca2+ influx is not known. We combined two-photon fluorescence lifetime imaging with two-photon glutamate uncaging to image the activity of the small guanosine triphosphatase Ras after NMDA receptor activation at individual spines. Induction of long-term potentiation (LTP) triggered robust Ca2+-dependent Ras activation in single spines that decayed in approximately 5 minutes. Ras activity spread over approximately 10 micrometers of dendrite and invaded neighboring spines by diffusion. The spread of Ras-dependent signaling was necessary for the local regulation of the threshold for LTP induction. Thus, Ca2+-dependent synaptic signals can spread to couple multiple synapses on short stretches of dendrite.

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    Looger LabSchreiter Lab
    07/01/08 | Crystallization and preliminary x-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2.
    Rodríguez Guilbe MM, Alfaro Malavé EC, Akerboom J, Marvin JS, Looger LL, Schreiter ER
    Acta Crystallographica. Section F, Structural Biology and Crystallization Communications. 2008 Jul 1;64:629-31. doi: 10.1107/S1744309108016059

    Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1.

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    Gonen Lab
    07/01/08 | Electron crystallography of aquaporins.
    Andrews S, Reichow SL, Gonen T
    IUBMB Life. 2008 Jul;60(7):430-6. doi: 10.1002/iub.53

    Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.

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    07/01/08 | Electrostatic readout of DNA microarrays with charged microspheres.
    Clack NG, Salaita K, Groves JT
    Nature Biotechnology. 2008 Jul;26(7):825-30. doi: 10.1038/nbt1416

    DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care. Here, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-mum lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays.

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    Gonen Lab
    07/01/08 | Interactions of lipids with aquaporin-0 and other membrane proteins.
    Hite RK, Gonen T, Harrison SC, Walz T
    Pflügers Archiv - European Journal of Physiology. 2008 Jul;456(4):651-61. doi: 10.1007/s00424-007-0353-9

    The structure of aquaporin-0 (AQP0) has recently been determined by electron crystallography of two-dimensional (2D) crystals and by X-ray crystallography of three-dimensional (3D) crystals. The electron crystallographic structure revealed nine lipids per AQP0 monomer, which form an almost complete bilayer. The lipids adopt a wide variety of conformations and tightly fill the space between adjacent AQP0 tetramers. The conformations of the lipid acyl chains appear to be determined not only by the protein surface but also by the acyl chains of adjacent lipid molecules. In the X-ray structure, the hydrophobic region of the protein is surrounded by a detergent micelle, with two ordered detergent molecules per AQP0 monomer. Despite the different environments, the electron crystallographic and X-ray structures of AQP0 are virtually identical, but they differ in the temperature factors of the atoms that either contact the lipids in the 2D crystals or are exposed to detergents in the 3D crystals. The temperature factors are higher in the X-ray structure, suggesting that the detergent-exposed AQP0 residues are less ordered than the corresponding ones contacting lipids in the 2D crystals. An examination of ordered detergent molecules in crystal structures of other aquaporins and of lipid molecules in 2D and 3D crystals of bacteriorhodopsin suggests that the increased conformational variability of detergent-exposed residues compared to lipid-contacting residues is a general feature.

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    07/01/08 | Locomotor control by the central complex in Drosophila-an analysis of the tay bridge mutant.
    Poeck B, Triphan T, Neuser K, Strauss R
    Developmental Neurobiology. 2008 Jul;68(8):1046-58. doi: 10.1002/dneu.20643

    Several aspects of locomotor control have been ascribed to the central complex of the insect brain; however, the role of distinct substructures of this complex is not well known. The tay bridge1 (tay1) mutant of Drosophila melanogaster was originally isolated on the basis of reduced walking speed and activity. In addition, tay1 is defective in the compensation of rotatory stimuli during walking and histologically, tay1 causes a mid-sagittal constriction of the protocerebral bridge, a constituent of the central complex. Cloning of the tay gene revealed that it encodes a novel protein with no significant homology to any known protein. To associate the behavioral phenotypes with the anatomical defect in the protocerebral bridge, we used different driver lines to express the tay cDNA in various neuronal subpopulations of the central brain in tay1-mutant flies. These experiments showed an association of the aberrant walking speed and activity with the structural defect in the protocerebral bridge. In contrast, the compensation of rotatory stimuli during walking was rescued without a restoration of the protocerebral bridge. The results of our differential rescue approach are supported by neuronal silencing experiments using conditional tetanus toxin expression in the same subset of neurons. These findings show for the first time that the walking speed and activity is controlled by different substructures of the central brain than the compensatory locomotion for rotatory stimuli.

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