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2041 Janelia Publications

Showing 91-100 of 2041 results
07/23/21 | YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.
Feliciano D, Ott CM, Espinosa-Medina I, Weigel AV, Benedetti L, Milano KM, Tang Z, Lee T, Kliman HJ, Guller SM, Lippincott-Schwartz J
Nature Communications. 2021 Jul 23;12(1):4502. doi: 10.1038/s41467-021-24708-2

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

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07/22/21 | Molecular characterization of projection neuron subtypes in the mouse olfactory bulb.
Zeppilli S, Ackels T, Attey R, Klimpert N, Kimberly Ritola D, Boeing S, Crombach A, Schaefer AT, Fleischmann A
eLife. 2021 Jul 22;10:. doi: 10.7554/eLife.65445

Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data, and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

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07/21/21 | Enabling In Vivo Photocatalytic Activation of Rapid Bioorthogonal Chemistry by Repurposing Silicon-Rhodamine Fluorophores as Cytocompatible Far-Red Photocatalysts.
Wang C, Zhang H, Zhang T, Zou X, Wang H, Rosenberger JE, Vannam R, Trout WS, Grimm JB, Lavis LD, Thorpe C, Jia X, Li Z, Fox JM
Journal of the American Chemical Society. 2021 Jul 21;143(28):10793-10803. doi: 10.1021/jacs.1c05547

Chromophores that absorb in the tissue-penetrant far-red/near-infrared window have long served as photocatalysts to generate singlet oxygen for photodynamic therapy. However, the cytotoxicity and side reactions associated with singlet oxygen sensitization have posed a problem for using long-wavelength photocatalysis to initiate other types of chemical reactions in biological environments. Herein, silicon-Rhodamine compounds (SiRs) are described as photocatalysts for inducing rapid bioorthogonal chemistry using 660 nm light through the oxidation of a dihydrotetrazine to a tetrazine in the presence of cyclooctene dienophiles. SiRs have been commonly used as fluorophores for bioimaging but have not been applied to catalyze chemical reactions. A series of SiR derivatives were evaluated, and the Janelia Fluor-SiR dyes were found to be especially effective in catalyzing photooxidation (typically 3%). A dihydrotetrazine/tetrazine pair is described that displays high stability in both oxidation states. A protein that was site-selectively modified by cyclooctene was quantitatively conjugated upon exposure to 660 nm light and a dihydrotetrazine. By contrast, a previously described methylene blue catalyst was found to rapidly degrade the protein. SiR-red light photocatalysis was used to cross-link hyaluronic acid derivatives functionalized by dihydrotetrazine and cyclooctenes, enabling 3D culture of human prostate cancer cells. Photoinducible hydrogel formation could also be carried out in live mice through subcutaneous injection of a Cy7-labeled hydrogel precursor solution, followed by brief irradiation to produce a stable hydrogel. This cytocompatible method for using red light photocatalysis to activate bioorthogonal chemistry is anticipated to find broad applications where spatiotemporal control is needed in biological environments.

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07/20/21 | Method for Rapid Enzymatic Cleaning for Reuse of Patch Clamp Pipettes: Increasing Throughput by Eliminating Manual Pipette Replacement between Patch Clamp Attempts.
Landry CR, Yip MC, Kolb I, Stoy WA, Gonzalez MM, Forest CR
Bio Protocol. 2021 Jul 20;11(14):e4085. doi: 10.21769/BioProtoc.4085

The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This article presents an optimized protocol for pipette cleaning, which enables it to be performed quickly (< 30 s), resulting in a high yield of whole-cell recording success rate (> 90%) for over 100 reuses of a single pipette. For most patch-clamp experiments (< 30 whole-cell recordings per day), this method enables a single pipette to be used for an entire day of experiments. In addition, we describe easily implementable hardware and software as well as troubleshooting tips to help other labs implement this method in their own experiments. Pipette cleaning enables patch-clamp experiments to be performed with higher throughput, whether manually or in an automated fashion, by eliminating the tedious and skillful task of replacing pipettes. From our experience with numerous electrophysiology laboratories, pipette cleaning can be integrated into existing patch-clamp setups in approximately one day using the hardware and software described in this article. Graphic abstract: Rapid enzymatic cleaning for reuse of patch-clamp pipettes.

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07/16/21 | An inexpensive, high-precision, modular spherical treadmill setup optimized for experiments.
Loesche F, Reiser MB
Frontiers in Behavioral Neuroscience. 2021 Jul 16;15:689573. doi: 10.3389/fnbeh.2021.689573

To pursue a more mechanistic understanding of the neural control of behavior, many neuroethologists study animal behavior in controlled laboratory environments. One popular approach is to measure the movements of restrained animals while presenting controlled sensory stimulation. This approach is especially powerful when applied to genetic model organisms, such as , where modern genetic tools enable unprecedented access to the nervous system for activity monitoring or targeted manipulation. While there is a long history of measuring the behavior of body- and head-fixed insects walking on an air-supported ball, the methods typically require complex setups with many custom components. Here we present a compact, simplified setup for these experiments that achieves high-performance at low cost. The simplified setup integrates existing hardware and software solutions with new component designs. We replaced expensive optomechanical and custom machined components with off-the-shelf and 3D-printed parts, and built the system around a low-cost camera that achieves 180 Hz imaging and an inexpensive tablet computer to present view-angle-corrected stimuli updated through a local network. We quantify the performance of the integrated system and characterize the visually guided behavior of flies in response to a range of visual stimuli. In this paper, we thoroughly document the improved system; the accompanying repository incorporates CAD files, parts lists, source code, and detailed instructions. We detail a complete ~$300 system, including a cold-anesthesia tethering stage, that is ideal for hands-on teaching laboratories. This represents a nearly 50-fold cost reduction as compared to a typical system used in research laboratories, yet is fully featured and yields excellent performance. We report the current state of this system, which started with a 1-day teaching lab for which we built seven parallel setups and continues toward a setup in our lab for larger-scale analysis of visual-motor behavior in flies. Because of the simplicity, compactness, and low cost of this system, we believe that high-performance measurements of tethered insect behavior should now be widely accessible and suitable for integration into many systems. This access enables broad opportunities for comparative work across labs, species, and behavioral paradigms.

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07/06/21 | Adhesion-mediated mechanosignaling forces mitohormesis.
Tharp KM, Higuchi-Sanabria R, Timblin GA, Ford B, Garzon-Coral C, Schneider C, Muncie JM, Stashko C, Daniele JR, Moore AS, Frankino PA, Homentcovschi S, Manoli SS, Shao H, Richards AL, Chen K, Hoeve JT, Ku GM, Hellerstein M, Nomura DK, Saijo K, Gestwicki J, Dunn AR, Krogan NJ, Swaney DL, Dillin A, Weaver VM
Cell Metabolism. 2021 July 6;33(7):1322. doi: 10.1016/j.cmet.2021.04.017

Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programed cell death. The biochemical milieu is known to affect mitochondrial function and contribute to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of aging. However, the physical characteristics of the extracellular matrix are also altered in cancerous and aging tissues. Here, we demonstrate that cells sense the physical properties of the extracellular matrix and activate a mitochondrial stress response that adaptively tunes mitochondrial function via solute carrier family 9 member A1-dependent ion exchange and heat shock factor 1-dependent transcription. Overall, our data indicate that adhesion-mediated mechanosignaling may play an unappreciated role in the altered mitochondrial functions observed in aging and cancer.

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07/05/21 | Lipid droplets in the nervous system.
Ralhan I, Chang C, Lippincott-Schwartz J, Ioannou MS
Journal of Cell Biology. 2021 Jul 05;220(7):. doi: 10.1083/jcb.202102136

Lipid droplets are dynamic intracellular lipid storage organelles that respond to the physiological state of cells. In addition to controlling cell metabolism, they play a protective role for many cellular stressors, including oxidative stress. Despite prior descriptions of lipid droplets appearing in the brain as early as a century ago, only recently has the role of lipid droplets in cells found in the brain begun to be understood. Lipid droplet functions have now been described for cells of the nervous system in the context of development, aging, and an increasing number of neuropathologies. Here, we review the basic mechanisms of lipid droplet formation, turnover, and function and discuss how these mechanisms enable lipid droplets to function in different cell types of the nervous system under healthy and pathological conditions.

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07/01/21 | Automatic Detection of Synaptic Partners in a Whole-Brain Drosophila EM Dataset
Buhmann J, Sheridan A, Gerhard S, Krause R, Nguyen T, Heinrich L, Schlegel P, Lee WA, Wilson R, Saalfeld S, Jefferis G, Bock D, Turaga S, Cook M, Funke J
Nature Methods. 2021 Jul 1;18(7):771-4. doi: 10.1038/s41592-021-01183-7

The study of neural circuits requires the reconstruction of neurons and the identification of synaptic connections between them. To scale the reconstruction to the size of whole-brain datasets, semi-automatic methods are needed to solve those tasks. Here, we present an automatic method for synaptic partner identification in insect brains, which uses convolutional neural networks to identify post-synaptic sites and their pre-synaptic partners. The networks can be trained from human generated point annotations alone and requires only simple post-processing to obtain final predictions. We used our method to extract 244 million putative synaptic partners in the fifty-teravoxel full adult fly brain (FAFB) electron microscopy (EM) dataset and evaluated its accuracy on 146,643 synapses from 702 neurons with a total cable length of 312 mm in four different brain regions. The predicted synaptic connections can be used together with a neuron segmentation to infer a connectivity graph with high accuracy: 96% of edges between connected neurons are correctly classified as weakly connected (less than five synapses) and strongly connected (at least five synapses). Our synaptic partner predictions for the FAFB dataset are publicly available, together with a query library allowing automatic retrieval of up- and downstream neurons.

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07/01/21 | Biomolecular Condensates and Their Links to Cancer Progression.
Cai D, Liu Z, Lippincott-Schwartz J
Trends in Biochemical Sciences. 2021 Jul 01;46(7):535-549. doi: 10.1016/j.tibs.2021.01.002

Liquid-liquid phase separation (LLPS) has emerged in recent years as an important physicochemical process for organizing diverse processes within cells via the formation of membraneless organelles termed biomolecular condensates. Emerging evidence now suggests that the formation and regulation of biomolecular condensates are also intricately linked to cancer formation and progression. We review the most recent literature linking the existence and/or dissolution of biomolecular condensates to different hallmarks of cancer formation and progression. We then discuss the opportunities that this condensate perspective provides for cancer research and the development of novel therapeutic approaches, including the perturbation of condensates by small-molecule inhibitors.

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07/01/21 | Bursting potentiates the neuro-AI connection.
Sun W, Zhao X, Spruston N
Nature Neuroscience. 2021 Jul 01;24(7):905-6. doi: 10.1038/s41593-021-00844-2