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1765 Janelia Publications

Showing 61-70 of 1765 results
02/13/20 | The Neuropixels probe: A CMOS based integrated microsystems platform for neuroscience and brain-computer interfaces.
Dutta B, Trautmann EM, Welkenhuysen M, Shenoy KV, Andrei A, Harris TD, Lopez CM, O'Callahan J, Putzeys J, Raducanu BC, Severi S, Stavisky SD
2019 IEEE International Electron Devices Meeting (IEDM). 2020 Feb 13:. doi: 10.1109/IEDM19573.201910.1109/IEDM19573.2019.8993611

We review recent progress in neural probes for brain recording, with a focus on the Neuropixels platform. Historically the number of neurons’ recorded simultaneously, follows a Moore’s law like behavior, with numbers doubling every 6.7 years. Using traditional techniques of probe fabrication, continuing to scale up electrode densities is very challenging. We describe a custom CMOS process technology that enables electrode counts well beyond 1000 electrodes; with the aim to characterize large neural populations with single neuron spatial precision and millisecond timing resolution. This required integrating analog and digital circuitry with the electrode array, making it a standalone integrated electrophysiology recording system. Input referred noise and power per channel is 7.5µV and <50µW respectively to ensure tissue heating <1°C. This approach enables doubling the number of measured neurons every 12 months.

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02/08/20 | A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies.
Borden P, Zhang P, Shivange AV, Marvin JS, Cichon J, Dan C, Podgorski K, Figueiredo A, Novak O, Tanimoto M, Shigetomi E, Lobas MA, Kim H, Zhu P, Zhang Y, Zheng WS, Fan C, Wang G, Xiang B, Gan L, Zhang G, Guo K, Lin L, Cai Y, Yee AG, Aggarwal A, Ford CP, Rees DC, Dietrich D, Khakh BS, Dittman JS, Gan W, Koyama M, Jayaraman V, Cheer JF, Lester HA, Zhu JJ, Looger LL
bioRxiv. 2020 Feb 8:. doi: https://doi.org/10.1101/2020.02.07.939504

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

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05/01/20 | Estimating the power of sequence covariation for detecting conserved RNA structure.
Rivas E, Clements J, Eddy SR, Ponty Y
Bioinformatics. 2020 May 01;36(10):3072-76. doi: 10.1093/bioinformatics/btaa080

Pairwise sequence covariations are a signal of conserved RNA secondary structure. We describe a method for distinguishing when lack of covariation signal can be taken as evidence against a conserved RNA structure, as opposed to when a sequence alignment merely has insufficient variation to detect covariations. We find that alignments for several long noncoding RNAs previously shown to lack covariation support do have adequate covariation detection power, providing additional evidence against their proposed conserved structures.

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02/06/20 | A versatile vector system for the fast generation of knock-in cell lines with CRISPR.
Perez-Leal O, Nixon-Abell J, Barrero CA, Gordon J, Rico MC
bioRxiv. 2020 Feb 06:. doi: 10.1101/2020.02.06.927384

Until recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from ~8 weeks to <15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.

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02/03/20 | Cellpose: a generalist algorithm for cellular segmentation
Stringer C, Michaelos M, Pachitariu M
bioRxiv. 2020 Feb 03:. doi: 10.1101/2020.02.02.931238

Many biological applications require the segmentation of cell bodies, membranes and nuclei from microscopy images. Deep learning has enabled great progress on this problem, but current methods are specialized for images that have large training datasets. Here we introduce a generalist, deep learning-based segmentation algorithm called Cellpose, which can very precisely segment a wide range of image types out-of-the-box and does not require model retraining or parameter adjustments. We trained Cellpose on a new dataset of highly-varied images of cells, containing over 70,000 segmented objects. To support community contributions to the training data, we developed software for manual labelling and for curation of the automated results, with optional direct upload to our data repository. Periodically retraining the model on the community-contributed data will ensure that Cellpose improves constantly.

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02/03/20 | Wiring patterns from auditory sensory neurons to the escape and song-relay pathways in fruit flies.
Kim H, Horigome M, Ishikawa Y, Li F, Lauritzen JS, Card G, Bock DD, Kamikouchi A
Journal of Comparative Neurology. 2020 Feb 03:. doi: 10.1002/cne.24877

Many animals rely on acoustic cues to decide what action to take next. Unraveling the wiring patterns of the auditory neural pathways is prerequisite for understanding such information processing. Here we reconstructed the first step of the auditory neural pathway in the fruit fly brain, from primary to secondary auditory neurons, at the resolution of transmission electron microscopy. By tracing axons of two major subgroups of auditory sensory neurons in fruit flies, low-frequency tuned Johnston's organ (JO)-B neurons and high-frequency tuned JO-A neurons, we observed extensive connections from JO-B neurons to the main second-order neurons in both the song-relay and escape pathways. In contrast, JO-A neurons connected strongly to a neuron in the escape pathway. Our findings suggest that heterogeneous JO neuronal populations could be recruited to modify escape behavior whereas only specific JO neurons contribute to courtship behavior. We also found that all JO neurons have postsynaptic sites at their axons. Presynaptic modulation at the output sites of JO neurons could affect information processing of the auditory neural pathway in flies. This article is protected by copyright. All rights reserved.

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02/01/20 | In situ structure determination at nanometer resolution using TYGRESS.
Song K, Shang Z, Fu X, Lou X, Grigorieff N, Nicastro D
Nature Methods. 2020 Feb 01;17(2):201-08. doi: 10.1038/s41592-019-0651-0

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called 'tomography-guided 3D reconstruction of subcellular structures' (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 Å. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.

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01/01/20 | Aurora B functions at the apical surface after specialized cytokinesis during morphogenesis in C. elegans.
Bai X, Melesse M, Sorensen Turpin CG, Sloan D, Chen C, Wang W, Lee P, Simmons JR, Nebenfuehr B, Mitchell D, Klebanow LR, Mattson N, Betzig E, Chen B, Cheerambathur D, Bembenek JN
Development. 2020 Jan;147(1):1-16. doi: 10.1242/dev.181099

While cytokinesis has been intensely studied, the way it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant embryonic divisions and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we found several unexpected alterations to cytokinesis including apical midbody migration in polarizing epithelial cells of the gut, pharynx and sensory neurons. Aurora B kinase, which is essential for several aspects of cytokinesis, remains apically localized in each of these tissues after internalization of midbody ring components. Aurora B inactivation disrupts cytokinesis and causes defects in apical structures, even if inactivated post-mitotically. Therefore, cytokinesis is implemented in a specialized way during epithelial polarization and Aurora B has a new role in the formation of the apical surface.

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01/27/20 | Structure and assembly of calcium homeostasis modulator proteins.
Syrjanen JL, Michalski K, Chou T, Grant T, Rao S, Simorowski N, Tucker SJ, Grigorieff N, Furukawa H
Nature Structural and Molecular Biology. 2020 Jan 27;27(2):150-9. doi: 10.1038/s41594-019-0369-9

The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.

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01/27/20 | Imaging Cellular Proteins and Structures
Arias IM, Alter HJ, Boyer JL, Cohen DE, Shafritz DA, Thorgeirsson SS, Wolkoff AW, Weigel AV, Snapp EL
The Liver : Biology and Pathobiology:965 - 978. doi: 10.1002/978111943681210.1002/9781119436812.ch72

This chapter describes many of the technologies, which have the potential to provide new insights into fundamental aspects of liver biology. Imaging live liver tissue in an animal with multiphoton microscopy coupled with photoactivatable fluorescent proteins and/or additional fluorescent proteins could be used to follow the lineage and fates of individual transplanted stem cells or developing transgenic cells in liver. Proteins or other molecules are labeled with a dye that can be excited with light source. Cells and proteins are generally too small to detect with the naked eye, relatively transparent when imaged by light microscopy, and are highly dynamic. With the increased signal to noise, isotropic and volumetric imaging and high speeds lattice light sheet allows for 3D super‐resolution microscopy, as well. Photomultiplier tubes, while capable of detecting and counting single photons, are less useful for high‐speed imaging because they normally only detect a single pixel at a time.

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