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2721 Janelia Publications

Showing 2521-2530 of 2721 results
05/18/22 | Three Mutations Convert the Selectivity of a Protein Sensor From Nicotinic Agonists to S-Methadone For Use in Cells, Organelles, and Biofluids.
Anand K. Muthusamy , Charlene H. Kim , Scott C. Virgil , Hailey J. Knox , Jonathan S. Marvin , Aaron L. Nichols , Bruce N. Cohen , Dennis A. Dougherty , Loren L. Looger , Henry A. Lester
Journal of the American Chemical Society. 2022 May 18;144(19):. doi: 10.1101/2022.02.24.481226

We report a reagentless, intensity-based S-methadone fluorescent sensor, iS-methadoneSnFR, consisting of a circularly permuted GFP inserted within the sequence of a mutated bacterial periplasmic binding protein (PBP). We used directed evolution to convert a previously reported nicotine-binding PBP to a selective S-methadone-binding sensor, via three mutations in the PBP’s second shell and hinge regions. iS-methadoneSnFR displays sensitivity across the pharmacologically relevant range and selectivity against endogenous analytes and other opioids. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Genetic encoding and imaging in mammalian demonstrated the acid trapping of S-methadone in the Golgi apparatus where opioid receptors can signal. This work shows a straightforward strategy in adapting existing PBPs to serve real-time applications ranging from subcellular to personal pharmacokinetics.

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Sternson Lab
11/28/16 | Three pillars for the neural control of appetite.
Sternson SM, Eiselt A
Annual Review of Physiology. 2016 Nov 28;79:401-23. doi: 10.1146/annurev-physiol-021115-104948

The neural control of appetite is important for understanding motivated behavior along with the present rising prevalence of obesity. Over the past several years, new tools for cell type-specific neuron activity monitoring and perturbation have enabled increasingly detailed analyses of the mechanisms underlying appetite-control systems. Three major neural circuits strongly and acutely influence appetite but with notably different characteristics. Although these circuits interact, they have distinct properties and thus appear to contribute to separate but interlinked processes influencing appetite, thereby forming three pillars of appetite control. Here, we summarize some of the key characteristics of appetite circuits that are emerging from recent work and synthesize the findings into a provisional framework that can guide future studies. Expected final online publication date for the Annual Review of Physiology Volume 79 is February 10, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Gonen Lab
11/19/13 | Three-dimensional electron crystallography of protein microcrystals.
Shi D, Nannenga BL, Iadanza MG, Gonen T
eLife. 2013 Nov 19;2:01345. doi: 10.7554/eLife.01345

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology.

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Cui Lab
09/01/13 | Three-dimensional live microscopy beyond the diffraction limit.
Fiolka R
Journal of Optics. 2013 Sep;15:094002. doi: 10.1088/2040-8978/15/9/094002

In fluorescence microscopy it has become possible to fundamentally overcome the diffraction limited resolution in all three spatial dimensions. However, to have the most impact in biological sciences, new optical microscopy techniques need to be compatible with live cell imaging: image acquisition has to be fast enough to capture cellular dynamics at the new resolution limit while light exposure needs to be minimized to prevent photo-toxic effects. With increasing spatial resolution, these requirements become more difficult to meet, even more so when volumetric imaging is performed. In this review, techniques that have been successfully applied to three-dimensional, super-resolution live microscopy are presented and their relative strengths and weaknesses are discussed.

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06/05/09 | Three-dimensional nanoscopy of biological samples.
Vaziri A, Tang J, Shroff H, Shank CV
2009 Conference on Lasers and Electro-Optics and Quantum Electronics and Laser Science Conference (CLEO/QELS 2009), Vols. 1-5. 2009 Jun 5;1-5:147-8

We have demonstrated super-resolution imaging of protein distributions in cells at depth at multiple layers with a lateral localization precision better than 50 nm. The approach is based on combining photoactivated localization microscopy with temporal focusing.

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10/18/23 | Three-dimensional reconstructions of mechanosensory end organs suggest a unifying mechanism underlying dynamic, light touch
Annie Handler , Qiyu Zhang , Song Pang , Tri M. Nguyen , Michael Iskols , Michael Nolan-Tamariz , Stuart Cattel , Rebecca Plumb , Brianna Sanchez , Karyl Ashjian , Aria Shotland , Bartianna Brown , Madiha Kabeer , Josef Turecek , Genelle Rankin , Wangchu Xiang , Elisa C. Pavarino , Nusrat Africawala , Celine Santiago , Wei-Chung Allen Lee , C. Shan Xu , David D. Ginty
Neuron. 2023 Oct 18:. doi: 10.1016/j.neuron.2023.08.023

Specialized mechanosensory end organs within mammalian skin—hair follicle-associated lanceolate complexes, Meissner corpuscles, and Pacinian corpuscles—enable our perception of light, dynamic touch1. In each of these end organs, fast-conducting mechanically sensitive neurons, called Aβ low-threshold mechanoreceptors (Aβ LTMRs), associate with resident glial cells, known as terminal Schwann cells (TSCs) or lamellar cells, to form complex axon ending structures. Lanceolate-forming and corpuscle-innervating Aβ LTMRs share a low threshold for mechanical activation, a rapidly adapting (RA) response to force indentation, and high sensitivity to dynamic stimuli16. How mechanical stimuli lead to activation of the requisite mechanotransduction channel Piezo2715 and Aβ RA-LTMR excitation across the morphologically dissimilar mechanosensory end organ structures is not understood. Here, we report the precise subcellular distribution of Piezo2 and high-resolution, isotropic 3D reconstructions of all three end organs formed by Aβ RA-LTMRs determined by large volume enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging. We found that within each end organ, Piezo2 is enriched along the sensory axon membrane and is minimally or not expressed in TSCs and lamellar cells. We also observed a large number of small cytoplasmic protrusions enriched along the Aβ RA-LTMR axon terminals associated with hair follicles, Meissner corpuscles, and Pacinian corpuscles. These axon protrusions reside within close proximity to axonal Piezo2, occasionally contain the channel, and often form adherens junctions with nearby non-neuronal cells. Our findings support a unified model for Aβ RA-LTMR activation in which axon protrusions anchor Aβ RA-LTMR axon terminals to specialized end organ cells, enabling mechanical stimuli to stretch the axon in hundreds to thousands of sites across an individual end organ and leading to activation of proximal Piezo2 channels and excitation of the neuron.

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03/12/24 | Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)
Talon Chandler , Min Guo , Yijun Su , Jiji Chen , Yicong Wu , Junyu Liu , Atharva Agashe , Robert S. Fischer , Shalin B. Mehta , Abhishek Kumar , Tobias I. Baskin , Valentin Jamouille , Huafeng Liu , Vinay Swaminathan , Amrinder Nain , Rudolf Oldenbourg , Patrick La Riviere , Hari Shroff
bioRxiv. 2024 Mar 12:. doi: 10.1101/2024.03.09.584243

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.Competing Interest StatementH.S., A.K., S.M., P.L.R., R.O., Y.W., and T.C. hold US Patent #11428632.

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07/26/09 | Three-dimensional super-resolution imaging of thick biological samples.
Vaziri A, Tang J, Shroff H, Shank C
Microscopy and Microanalysis. 2009 Jul 26;15:36-7. doi: 10.1017/S1431927609092368
10/01/15 | Three-dimensional tracking of plus-tips by lattice light-sheet microscopy permits the quantification of microtubule growth trajectories within the mitotic apparatus.
Yamashita N, Morita M, Legant WR, Chen B, Betzig E, Yokota H, Mimori-Kiyosue Y
Journal of Biomedical Optics. 2015 Oct 1;20(10):101206. doi: 10.1117/1.JBO.20.10.101206
Ji Lab
04/15/18 | Three-photon fluorescence microscopy with an axially elongated Bessel focus.
Rodriguez C, Liang Y, Lu R, Ji N
Optics Letters. 2018 Apr 15;43(8):1914-1917. doi: 10.1364/OL.43.001914

Volumetric imaging tools that are simple to adopt, flexible, and robust are in high demand in the field of neuroscience, where the ability to image neurons and their networks with high spatiotemporal resolution is essential. Using an axially elongated focus approximating a Bessel beam, in combination with two-photon fluorescence microscopy, has proven successful at such an endeavor. Here, we demonstrate three-photon fluorescence imaging with an axially extended Bessel focus. We use an axicon-based module that allowed for the generation of Bessel foci of varying numerical apertures and axial lengths, and apply this volumetric imaging tool to image mouse brain slices and for in vivo imaging of the mouse brain.

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