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802 Janelia Publications

Showing 1-10 of 802 results
11/01/15 | Structural basis for the antipolymer activity of Hb ζ22βsζ2βs2 trapped in a tense conformation.
Safo MK, Ko T, Schreiter ER, Russell JEric
Journal of Molecular Structure. 2015 Nov;1099:99-107. doi: 10.1016/j.molstruc.2015.06.047

The phenotypical severity of sickle cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb S, Hb α2β2s) to contain embryonic ζ globin in place of adult α-globin subunits (Hb ζ2β2s). Crystallographical analyses of liganded Hb ζζ2β2s, though, demonstrate a tense (T-state) quaternary structure that paradoxically predicts its participation in--rather than its exclusion from--pathological deoxyHb S polymers. We resolved this structure-function conundrum by examining the effects of α → ζ exchange on the characteristics of specific amino acids that mediate sickle polymer assembly. Superposition analyses of the βs subunits of T-state deoxyHb α2β2s and T-state CO-liganded Hb ζ2β2s reveal significant displacements of both mutant βsVal6 and conserved β-chain contact residues, predicting weakening of corresponding polymer-stabilizing interactions. Similar comparisons of the α- and ζ-globin subunits implicate four amino acids that are either repositioned or undergo non-conservative substitution, abrogating critical polymer contacts. CO-Hb ζ2βs2 additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological βsVal6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ2β2s suggests that it does not co-assemble with deoxyHb S in vivo  . In sum, the antipolymer activities of Hb ζ2β2s appear to arise from both repositioning and replacement of specific α- and βs-chain residues, favoring an alternate T-state solution structure that is excluded from pathological deoxyHb S polymers. These data account for the antipolymer activity of Hb ζ2β2s, and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.

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08/28/15 | Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.
Li D, Shao L, Chen B, Zhang X, Zhang M, Moses B, Milkie DE, Beach JR, Hammer JA, Pasham M, Kirchhausen T, Baird MA, Davidson MW, Xu P, Betzig E
Science (New York, N.Y.). 2015 Aug 28;349(6251):. doi: 10.1126/science.aab3500

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

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08/26/15 | Optogenetics: 10 years after ChR2 in neurons-views from the community.
Adamantidis A, Arber S, Bains JS, Bamberg E, Bonci A, Buzsáki G, Cardin JA, Costa RM, Dan Y, Goda Y, Graybiel AM, Häusser M, Hegemann P, Huguenard JR, Insel TR, Janak PH, Johnston D, Josselyn SA, Koch C, Kreitzer AC, Lüscher C, Malenka RC, Miesenböck G, Nagel G, Roska B, Schnitzer MJ, Shenoy KV, Soltesz I, Sternson SM, Tsien RW, Tsien RY, Turrigiano GG, Tye KM, Wilson RI
Nature Neuroscience. 2015 Aug 26;18(9):1202-12. doi: 10.1038/nn.4106
08/25/15 | Genome-wide errant targeting by Hairy.
Kok K, Ay A, Li LM, Arnosti DN
eLife. 2015 Aug 25;4:. doi: 10.7554/eLife.06394

Metazoan transcriptional repressors regulate chromatin through diverse histone modifications. Contributions of individual factors to the chromatin landscape in development is difficult to establish, as global surveys reflect multiple changes in regulators. Therefore, we studied the conserved Hairy/Enhancer of Split family repressor Hairy, analyzing histone marks and gene expression in Drosophila embryos. This long-range repressor mediates histone acetylation and methylation in large blocks, with highly context-specific effects on target genes. Most strikingly, Hairy exhibits biochemical activity on many loci that are uncoupled to changes in gene expression. Rather than representing inert binding sites, as suggested for many eukaryotic factors, many regions are targeted errantly by Hairy to modify the chromatin landscape. Our findings emphasize that identification of active cis-regulatory elements must extend beyond the survey of prototypical chromatin marks. We speculate that this errant activity may provide a path for creation of new regulatory elements, facilitating the evolution of novel transcriptional circuits.

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08/22/15 | C/D box sRNA-guided 2'-O-methylation patterns of archaeal rRNA molecules.
Dennis PP, Tripp V, Lui L, Lowe T, Randau L
BMC Genomics. 2015 Aug 22;16(1):632. doi: 10.1186/s12864-015-1839-z

BACKGROUND: In archaea and eukaryotes, ribonucleoprotein complexes containing small C/D box s(no)RNAs use base pair complementarity to target specific sites within ribosomal RNA for 2'-O-ribose methylation. These modifications aid in the folding and stabilization of nascent rRNA molecules and their assembly into ribosomal particles. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures.

RESULTS: We have identified the complete sets of C/D box sRNAs from seven archaea using RNA-Seq methodology. In total, 489 C/D box sRNAs were identified, each containing two guide regions. A combination of computational and manual analyses predicts 719 guide interactions with 16S and 23S rRNA molecules. This first pan-archaeal description of guide sequences identifies (i) modified rRNA nucleotides that are frequently conserved between species and (ii) regions within rRNA that are hotspots for 2'-O-methylation. Gene duplication, rearrangement, mutational drift and convergent evolution of sRNA genes and guide sequences were observed. In addition, several C/D box sRNAs were identified that use their two guides to target locations distant in the rRNA sequence but close in the secondary and tertiary structure. We propose that they act as RNA chaperones and facilitate complex folding events between distant sequences.

CONCLUSIONS: This pan-archaeal analysis of C/D box sRNA guide regions identified conserved patterns of rRNA 2'-O-methylation in archaea. The interaction between the sRNP complexes and the nascent rRNA facilitates proper folding and the methyl modifications stabilize higher order rRNA structure within the assembled ribosome.

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08/19/15 | Novel Behavioral Paradigm Reveals Lower Temporal Limits on Mouse Olfactory Decisions.
Resulaj A, Rinberg D
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 2015 Aug 19;35(33):11667-73. doi: 10.1523/JNEUROSCI.4693-14.2015

UNLABELLED: Temporal limits on perceptual decisions set strict boundaries on the possible underlying neural computations. How odor information is encoded in the olfactory system is still poorly understood. Here, we sought to define the limit on the speed of olfactory processing. To achieve this, we trained mice to discriminate different odor concentrations in a novel behavioral setup with precise odor delivery synchronized to the sniffing cycle. Mice reported their choice by moving a horizontal treadmill with their front limbs. We found that mice reported discriminations of 75% accuracy in 70-90 ms after odor inhalation. For a low concentration and nontrigeminal odorant, this time was 90-140 ms, showing that mice process odor information rapidly even in the absence of trigeminal stimulation. These response times establish, after accounting for odor transduction and motor delays, that olfactory processing can take tens of milliseconds. This study puts a strong limit on the underlying neural computations and suggests that the action potentials forming the neural basis for these decisions are fired in a few tens of milliseconds.

SIGNIFICANCE STATEMENT: Understanding how sensory information is processed requires different approaches that span multiple levels of investigation from genes to neurons to behavior. Limits on behavioral performance constrain the possible neural mechanisms responsible for specific computations. Using a novel behavioral paradigm, we established that mice can make decisions about odor intensity surprisingly fast. After accounting for sensory and motor delays, the limit on some olfactory neural computations can be as low as a few tens of milliseconds, which suggests that only the first action potentials across a population of neurons contribute to these computations.

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08/13/15 | CTFFIND4: Fast and accurate defocus estimation from electron micrographs.
Rohou A, Grigorieff N
Journal of Structural Biology. 2015 Aug 13:. doi: 10.1016/j.jsb.2015.08.008

CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope's contrast transfer function (CTF) to an image's amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.

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08/13/15 | Lighting up genes in single cells at scale.
Liu Z
Cell. 2015 Aug 13;162(4):705-7. doi: 10.1016/j.cell.2015.07.052
08/11/15 | Whole-central nervous system functional imaging in larval Drosophila.
Lemon WC, Pulver SR, Höckendorf B, McDole K, Branson KM, Freeman J, Keller PJ
Nature Communications. 2015 Aug 11;6:7924. doi: 10.1038/ncomms8924

Understanding how the brain works in tight concert with the rest of the central nervous system (CNS) hinges upon knowledge of coordinated activity patterns across the whole CNS. We present a method for measuring activity in an entire, non-transparent CNS with high spatiotemporal resolution. We combine a light-sheet microscope capable of simultaneous multi-view imaging at volumetric speeds 25-fold faster than the state-of-the-art, a whole-CNS imaging assay for the isolated Drosophila larval CNS and a computational framework for analysing multi-view, whole-CNS calcium imaging data. We image both brain and ventral nerve cord, covering the entire CNS at 2 or 5 Hz with two- or one-photon excitation, respectively. By mapping network activity during fictive behaviours and quantitatively comparing high-resolution whole-CNS activity maps across individuals, we predict functional connections between CNS regions and reveal neurons in the brain that identify type and temporal state of motor programs executed in the ventral nerve cord.

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08/06/15 | Automatic Adaptation to Fast Input Changes in a Time-Invariant Neural Circuit.
Bharioke A, Chklovskii DB
PLoS Computational Biology. 2015 Aug 6;11(8):e1004315. doi: 10.1371/journal.pcbi.1004315