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2041 Janelia Publications

Showing 71-80 of 2041 results
09/02/21 | Electrode pooling can boost the yield of extracellular recordings with switchable silicon probes.
Lee KH, Ni Y, Colonell J, Karsh B, Putzeys J, Pachitariu M, Harris TD, Meister M
Nature Communications. 2021 Sep 02;12(1):5245. doi: 10.1038/s41467-021-25443-4

State-of-the-art silicon probes for electrical recording from neurons have thousands of recording sites. However, due to volume limitations there are typically many fewer wires carrying signals off the probe, which restricts the number of channels that can be recorded simultaneously. To overcome this fundamental constraint, we propose a method called electrode pooling that uses a single wire to serve many recording sites through a set of controllable switches. Here we present the framework behind this method and an experimental strategy to support it. We then demonstrate its feasibility by implementing electrode pooling on the Neuropixels 1.0 electrode array and characterizing its effect on signal and noise. Finally we use simulations to explore the conditions under which electrode pooling saves wires without compromising the content of the recordings. We make recommendations on the design of future devices to take advantage of this strategy.

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09/02/21 | Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.
Nguyen VQ, Ranjan A, Liu S, Tang X, Ling YH, Wisniewski J, Mizuguchi G, Li KY, Jou V, Zheng Q, Lavis LD, Lionnet T, Wu C
Molecular Cell. 2021 Sep 02;81(17):3560-3575. doi: 10.1016/j.molcel.2021.07.022

Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

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09/01/21 | Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes.
Timothy A. Brown , Katherine S. Schaefer , Arthur Tsang , Hyun Ah Yi , Jonathan B. Grimm , Andrew L. Lemire , Fadi M. Jradi , Charles Kim , Kevin McGowan , Kimberly Ritola , Derek T. Armstrong , Heba H. Mostafa , Wyatt Korff , Ronald D. Vale , Luke D. Lavis
Journal of Biomolecular Techniques. 2021 Sep 01;32(3):121-133. doi: https://doi.org/10.1101/2020.12.26.20248878

The worldwide COVID-19 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce two pH sensitive ‘LAMPshade’ dyes as novel readouts in an isothermal RT- LAMP amplification assay for SARS-CoV-2 RNA. The resulting JaneliaLAMP (jLAMP) assay is robust, simple, inexpensive, has low technical requirements and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.

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08/30/21 | A genetic screen for Drosophila social isolation mutants and analysis of sex pistol.
Eddison M
Scientific Reports. 2021 Aug 30;11(1):17395. doi: 10.1038/s41598-021-96871-x

Prolonged periods of forced social isolation is detrimental to well-being, yet we know little about which genes regulate susceptibility to its effects. In the fruit fly, Drosophila melanogaster, social isolation induces stark changes in behavior including increased aggression, locomotor activity, and resistance to ethanol sedation. To identify genes regulating sensitivity to isolation, I screened a collection of sixteen hundred P-element insertion lines for mutants with abnormal levels of all three isolation-induced behaviors. The screen identified three mutants whose affected genes are likely central to regulating the effects of isolation in flies. One mutant, sex pistol (sxp), became extremely aggressive and resistant to ethanol sedation when socially isolated. sxp also had a high level of male-male courtship. The mutation in sxp reduced the expression of two minor isoforms of the actin regulator hts (adducin), as well as mildly reducing expression of CalpA, a calcium-dependent protease. As a consequence, sxp also had increased expression of the insulin-like peptide, dILP5. Analysis of the social behavior of sxp suggests that these minor hts isoforms function to limit isolation-induced aggression, while chronically high levels of dILP5 increase male-male courtship.

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Cardona Lab
08/27/21 | Synchronous and opponent thermosensors use flexible cross-inhibition to orchestrate thermal homeostasis.
Hernandez-Nunez L, Chen A, Budelli G, Berck ME, Richter V, Rist A, Thum AS, Cardona A, Klein M, Garrity P, Samuel AD
Science Advances. 2021 Aug 27;7(35):. doi: 10.1126/sciadv.abg6707

Body temperature homeostasis is essential and reliant upon the integration of outputs from multiple classes of cooling- and warming-responsive cells. The computations that integrate these outputs are not understood. Here, we discover a set of warming cells (WCs) and show that the outputs of these WCs combine with previously described cooling cells (CCs) in a cross-inhibition computation to drive thermal homeostasis in larval WCs and CCs detect temperature changes using overlapping combinations of ionotropic receptors: Ir68a, Ir93a, and Ir25a for WCs and Ir21a, Ir93a, and Ir25a for CCs. WCs mediate avoidance to warming while cross-inhibiting avoidance to cooling, and CCs mediate avoidance to cooling while cross-inhibiting avoidance to warming. Ambient temperature-dependent regulation of the strength of WC- and CC-mediated cross-inhibition keeps larvae near their homeostatic set point. Using neurophysiology, quantitative behavioral analysis, and connectomics, we demonstrate how flexible integration between warming and cooling pathways can orchestrate homeostatic thermoregulation.

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08/25/21 | Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.
Arts JJ, Mahlandt EK, Grönloh M, Schimmel L, Noordstra I, Gordon E, van Steen AC, Tol S, Walzog B, van Rijssel J, Nolte MA, Postma M, Khuon S, Heddleston JM, Wait E, Chew TL, Winter M, Montanez E, Goedhart J, van Buul JD
eLife. 2021 Aug 25;10:. doi: 10.7554/eLife.66074

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

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08/24/21 | Campylobacter jejuni Triggers Signaling through Host Cell Focal Adhesions To Inhibit Cell Motility.
Klappenbach CM, Negretti NM, Aaron J, Chew T, Konkel ME
mBio. 2021 Aug 24:e0149421. doi: 10.1128/mBio.01494-21

Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblotting, and superresolution interferometric photoactivated localization microscopy (iPALM). We found that C. jejuni infection of epithelial cells results in increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction in collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of a protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially reestablishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions. Campylobacter jejuni is a major foodborne pathogen that causes severe gastritis. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected epithelial cells. Focal adhesions act as signaling complexes that connect the extracellular matrix to the intracellular cytoskeleton. The key findings of this study show that C. jejuni changes the structure (size and position), composition, and function of cellular focal adhesions using a combination of virulence factors. Mechanistically, we found that the changes in focal adhesion dynamics are dependent upon the activation of host cell signaling pathways, which affect the assembly and disassembly of cellular proteins from the focal adhesion. To summarize, we have identified a new cellular phenotype in C. jejuni-infected cells that may be responsible for the restricted intestinal repair observed in C. jejuni-infected animals.

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08/24/21 | Campylobacter jejuni Triggers Signaling through Host Cell Focal Adhesions To Inhibit Cell Motility.
Klappenbach CM, Negretti NM, Aaron J, Chew T, Konkel ME
mBio. 2021 Aug 24;12(4):e0149421. doi: 10.1128/mBio.01494-21

Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblotting, and superresolution interferometric photoactivated localization microscopy (iPALM). We found that C. jejuni infection of epithelial cells results in increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction in collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of a protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially reestablishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions. Campylobacter jejuni is a major foodborne pathogen that causes severe gastritis. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected epithelial cells. Focal adhesions act as signaling complexes that connect the extracellular matrix to the intracellular cytoskeleton. The key findings of this study show that C. jejuni changes the structure (size and position), composition, and function of cellular focal adhesions using a combination of virulence factors. Mechanistically, we found that the changes in focal adhesion dynamics are dependent upon the activation of host cell signaling pathways, which affect the assembly and disassembly of cellular proteins from the focal adhesion. To summarize, we have identified a new cellular phenotype in C. jejuni-infected cells that may be responsible for the restricted intestinal repair observed in C. jejuni-infected animals.

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08/20/21 | Imaging Africa: a strategic approach to optical microscopy training in Africa.
Reiche MA, Warner DF, Aaron J, Khuon S, Fletcher DA, Hahn K, Rogers KL, Mhlanga M, Koch A, Quaye W, Chew T
Nature Methods. 2021 Aug 20;18(8):847-855. doi: 10.1038/s41592-021-01227-y
01/08/18 | Development and Applications of Fluorescent Proteins for Correlative Light and Electron Microscopy
Paez-Segala MG, Wang Y, Iyer N, Li W, Rivlin PK, Looger LL
Microscopy and Microanalysis. 01/2018;24(S1):2318 - 2319. doi: 10.1017/S1431927618012072

Recent advances in super-resolution microscopy have pushed the resolution limit of light microscopy closer to that of electron microscopy. However, as they invariably rely on fluorescence, light microscopy techniques only visualize whatever gets labeled. On the other hand, while electron microscopy reveals cellular structures at the highest resolution, it offers no specificity. The information gap between the two imaging modalities can only be bridged by correlative light and electron microscopy (CLEM). Previously we have developed a probe (mEos4) whose fluorescence and photoconversion survive 0.5-1% OsO4 fixation, allowing super-resolution visualization of organelles and fused proteins in the context of resinembedded ultrastructure in both transmission EM (TEM) and scanning EM (SEM) [1,2].

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