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1287 Janelia Publications

Showing 81-90 of 1287 results
07/18/17 | A connectome of a learning and memory center in the adult Drosophila brain.
Takemura S, Aso Y, Hige T, Wong AM, Lu Z, Xu CS, Rivlin PK, Hess HF, Zhao T, Parag T, Berg S, Huang G, Katz WT, Olbris DJ, Plaza SM, Umayam LA, Aniceto R, Chang L, Lauchie S, et al
eLife. 2017 Jul 18;6:e26975. doi: 10.7554/eLife.26975

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB’s α lobe, using a dataset of isotropic 8-nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only six percent of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall.

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07/17/17 | A consensus view of ESCRT-mediated Human Immunodeficiency Virus Type 1 abscission.
Lippincott-Schwartz J, Freed EO, van Engelenburg SB
Annual Review of Virology. 2017 Jul 17;4(1):309-25. doi: 10.1146/annurev-virology-101416-041840

The strong dependence of retroviruses, such as human immunodeficiency virus type 1 (HIV-1), on host cell factors is no more apparent than when the endosomal sorting complex required for transport (ESCRT) machinery is purposely disengaged. The resulting potent inhibition of retrovirus release underscores the importance of understanding fundamental structure-function relationships at the ESCRT-HIV-1 interface. Recent studies utilizing advanced imaging technologies have helped clarify these relationships, overcoming hurdles to provide a range of potential models for ESCRT-mediated virus abscission. Here, we discuss these models in the context of prior work detailing ESCRT machinery and the HIV-1 release process. To provide a template for further refinement, we propose a new working model for ESCRT-mediated HIV-1 release that reconciles disparate and seemingly conflicting studies. Expected final online publication date for the Annual Review of Virology Volume 4 is September 29, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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07/13/17 | Chemistry is dead. Long live chemistry!
Lavis LD
Biochemistry. 2017 Jul 13;56(39):5165-70. doi: 10.1021/acs.biochem.7b00529

Chemistry, once king of fluorescence microscopy, was usurped by the field of fluorescent proteins. The increased demands of modern microscopy techniques on the “photon budget” requires better and brighter fluorophores. Here, we review the recent advances in biochemistry, protein engineering, and organic synthesis that have allowed a triumphant return of chemical dyes to modern biological imaging.

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07/13/17 | Mapping the neural substrates of behavior.
Robie AA, Hirokawa J, Edwards AW, Umayam LA, Lee A, Phillips ML, Card GM, Korff W, Rubin GM, Simpson JH, Reiser MB, Branson KM
Cell. 2017-07-13;170(2):393-406. doi: 10.1016/j.cell.2017.06.032

Assigning behavioral functions to neural structures has long been a central goal in neuroscience and is a necessary first step toward a circuit-level understanding of how the brain generates behavior. Here, we map the neural substrates of locomotion and social behaviors for Drosophila melanogaster using automated machine-vision and machine-learning techniques. From videos of 400,000 flies, we quantified the behavioral effects of activating 2,204 genetically targeted populations of neurons. We combined a novel quantification of anatomy with our behavioral analysis to create brain-behavior correlation maps, which are shared as browsable web pages and interactive software. Based on these maps, we generated hypotheses of regions of the brain causally related to sensory processing, locomotor control, courtship, aggression, and sleep. Our maps directly specify genetic tools to target these regions, which we used to identify a small population of neurons with a role in the control of walking.

•We developed machine-vision methods to broadly and precisely quantify fly behavior•We measured effects of activating 2,204 genetically targeted neuronal populations•We created whole-brain maps of neural substrates of locomotor and social behaviors•We created resources for exploring our results and enabling further investigation

Machine-vision analyses of large behavior and neuroanatomy data reveal whole-brain maps of regions associated with numerous complex behaviors.

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07/10/17 | Expression, purification, and contaminant detection for structural studies of Ralstonia metallidurance ClC protein rm1.
Abeyrathne PD, Grigorieff N
PloS One. 2017;12(7):e0180163. doi: 10.1371/journal.pone.0180163

Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E. coli) BL21(DE3) strain. Mass spectrometry and electron microscopy analyses of purified samples revealed multiple contaminants that can obfuscate results of subsequent high-resolution structural analysis. Here we describe the systematic optimization of sample preparation procedures, including expression systems, solubilization techniques, purification protocols, and contamination detection. We found that expressing ClC-rm1 in E. coli BL21(DE3) and using n-dodecyl-β-D-maltopyranoside as a detergent for solubilization and purification steps resulted in the highest quality samples of those we tested. However, although protein yield, sample stability, and the resolution of structural detail were improved following these changes, we still detected contaminants including Acriflavine resistant protein AcrB. AcrB was particularly difficult to remove as it co-purified with ClC-rm1 due to four intrinsic histidine residues at its C-terminus that bind to affinity resins. We were able to obtain properly folded pure ClC-rm1 by adding eGFP to the C-terminus and overexpressing the protein in the ΔacrB variant of the JW0451-2 E. coli strain.

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07/06/17 | Building bridges between cellular and molecular structural biology.
Patwardhan A, Brandt R, Butcher SJ, Collinson L, Gault D, Grünewald K, Hecksel C, Huiskonen JT, Iudin A, Jones ML, Korir PK, Koster AJ, Lagerstedt I, Lawson CL, Mastronarde D, McCormick M, Parkinson H, Rosenthal PB, Saalfeld S, Saibil HR, Sarntivijai S, Solanes Valero I, Subramaniam S, Swedlow JR, Tudose I, Winn M, Kleywegt GJ
eLife. 2017 Jul 06;6:. doi: 10.7554/eLife.25835

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.

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07/05/17 | Ion Channels: History, Diversity, and Impact.
Brenowitz S, Duguid I, Kammermeier PJ
Cold Spring Harbor Protocols. 2017 Jul 05;2017(7):pdb.top092288. doi: 10.1101/pdb.top092288

From patch-clamp techniques to recombinant DNA technologies, three-dimensional protein modeling, and optogenetics, diverse and sophisticated methods have been used to study ion channels and how they determine the electrical properties of cells.

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07/04/17 | The candidate multi-cut for cell segmentation.
Funke J, Zhang C, Pietzsch T, Saalfeld S
arXiv. 2017 Jul 04:1707.00907

Two successful approaches for the segmentation of biomedical images are (1) the selection of segment candidates from a merge-tree, and (2) the clustering of small superpixels by solving a Multi-Cut problem. In this paper, we introduce a model that unifies both approaches. Our model, the Candidate Multi-Cut (CMC), allows joint selection and clustering of segment candidates from a merge-tree. This way, we overcome the respective limitations of the individual methods: (1) the space of possible segmentations is not constrained to candidates of a merge-tree, and (2) the decision for clustering can be made on candidates larger than superpixels, using features over larger contexts. We solve the optimization problem of selecting and clustering of candidates using an integer linear program. On datasets of 2D light microscopy of cell populations and 3D electron microscopy of neurons, we show that our method generalizes well and generates more accurate segmentations than merge-tree or Multi-Cut methods alone.

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07/01/17 | mRNA quantification using single-molecule FISH in Drosophila embryos.
Trcek T, Lionnet T, Shroff H, Lehmann R
Nature Protocols. 2017 Jul;12(7):1326-1348. doi: 10.1038/nprot.2017.030

Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4-5 d.

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07/01/17 | Opening a path to commercialization.
Optics and Photonics News. 2017 Jul 01;28(7):42-9. doi: 10.1364/OPN.28.7.000042

To smooth the academic-to-industry transition, one institution is experimenting with offering biomedical researchers pre-commercial open access to new optical imaging systems still under development. The approach, the authors of this case study suggest, can be a win on both sides.

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