Live‐cell imaging of cell signaling using genetically encoded fluorescent reporters DOI Open Access

Qiang Ni,

Sohum Mehta, Jin Zhang

et al.

FEBS Journal, Journal Year: 2017, Volume and Issue: 285(2), P. 203 - 219

Published: June 14, 2017

Synergistic advances in fluorescent protein engineering and live-cell imaging techniques recent years have fueled the concurrent development application of genetically encoded reporters that are tailored for tracking signaling dynamics living systems over multiple length time scales. These biosensors uniquely suited this challenging task, owing to their specificity, sensitivity, versatility, as well noninvasive nondestructive nature fluorescence power genetic encoding. Over past 10 years, a growing number been developed wide range biological signals cells animals, including second messenger metabolite dynamics, enzyme activation activity, cell cycle progression neuronal activity. Many these gaining use proving be indispensable unraveling complex functions individual molecules native environment, cell, shedding new light on structural molecular underpinnings signaling. In review, we highlight likely help expand improve design valuable tools. We then turn our focus specific examples using an important platform advancing understanding G protein-coupled receptor

Language: Английский

Fluorescence resonance energy transfer at the single-molecule level DOI
Taekjip Ha, Jingyi Fei, Sonja Schmid

et al.

Nature Reviews Methods Primers, Journal Year: 2024, Volume and Issue: 4(1)

Published: March 28, 2024

Language: Английский

Citations

18
Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB DOI Open Access
Ben Johnson, Ashley N. Leek, Laura Solé

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2018, Volume and Issue: 115(31)

Published: June 25, 2018

Significance In addition to functioning as a delayed-rectifier K + channel, Kv2.1 interacts with the cortical endoplasmic reticulum (ER) in hippocampal neurons form somatic ER/plasma membrane (ER/PM) junctions. Neuronal activity and insult induce release from ER subsequent withdrawal PM. ER/PM contacts represent >10% of cell surface play roles trafficking, regulation burst firing, Ca 2+ homeostasis, control PM lipid. We report here that Kv2 channel–VAMP-associated protein (VAP) interaction tethers via noncanonical FFAT motif contained within channel C terminus. Since VAPs have wide-ranging interactome, Kv2-induced remodeling VAP concentration at likely central role neuronal physiology.

Language: Английский

Citations

162
Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo DOI Creative Commons
Helmut Bischof, Markus Rehberg, Sarah Stryeck

et al.

Nature Communications, Journal Year: 2017, Volume and Issue: 8(1)

Published: Nov. 6, 2017

Changes in intra- and extracellular potassium ion (K+) concentrations control many important cellular processes related biological functions. However, our current understanding of the spatiotemporal patterns physiological pathological K+ changes is severely limited by lack practicable detection methods. We developed K+-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging dynamics. GEPIIs as purified biosensors are suitable to directly precisely quantify levels different body fluids cell growth media. expressed cells time-lapse recordings global local intracellular signals. Hitherto unknown Ca2+-triggered, organelle-specific were detected pancreatic beta cells. Recombinant also enabled visualization fluctuations vivo with 2-photon microscopy. Therefore, relevant for diverse assays open new avenues live-cell imaging.

Language: Английский

Citations

156
Molecular determinants of ER–Golgi contacts identified through a new FRET–FLIM system DOI Creative Commons
Rossella Venditti, Laura Rita Rega,

Maria Chiara Masone

et al.

The Journal of Cell Biology, Journal Year: 2019, Volume and Issue: 218(3), P. 1055 - 1065

Published: Jan. 18, 2019

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered analysis via optical microscopy as well mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of ERTGoCS. These included ER membrane proteins VAPA VAPB lipid transfer possessing dual (ER TGN) targeting motifs that hypothesized contribute maintenance ERTGoCS, such ceramide protein CERT members oxysterol binding proteins. We found VAP proteins, OSBP1, ORP9, ORP10 are required, with OSBP1 playing redundant role which does not involve its activity, being required due ability phosphatidylserine TGN. Our results indicate both structural tethers proper composition needed ERTGoCS integrity.

Language: Английский

Citations

114
Advances in the synthesis of nitroxide radicals for use in biomolecule spin labelling DOI
Marius M. Haugland, Janet E. Lovett, Edward A. Anderson

et al.

Chemical Society Reviews, Journal Year: 2017, Volume and Issue: 47(3), P. 668 - 680

Published: Dec. 1, 2017

EPR spectroscopy is an increasingly useful analytical tool to probe biomolecule structure, dynamic behaviour, and interactions. Nitroxide radicals are the most commonly used radical in experiments, many methods have been developed for their synthesis, as well incorporation into biomolecules using site-directed spin labelling. In this Tutorial Review, we discuss practical synthesis of nitroxides, focusing on tunability structures, manipulation sidechains labelling handles, installation biomolecules.

Language: Английский

Citations

107
The Cotton Wall-Associated Kinase GhWAK7A Mediates Responses to Fungal Wilt Pathogens by Complexing with the Chitin Sensory Receptors DOI Creative Commons
Ping Wang, Lin Zhou, Pierce Jamieson

et al.

The Plant Cell, Journal Year: 2020, Volume and Issue: 32(12), P. 3978 - 4001

Published: Oct. 9, 2020

Plant receptor-like kinases (RLKs) are important players in response to pathogen infections. Verticillium and Fusarium wilts, caused by dahliae (Vd) oxysporum f. sp vasinfectum (Fov), respectively, among the most devastating diseases cotton (Gossypium spp). To understand these soil-borne fungal pathogens, we performed a genome-wide silico characterization functional screen of diverse RLKs for their involvement wilt diseases. We identified Gossypium hirsutum GhWAK7A, wall-associated kinase, that positively regulates both Vd Fov Chitin, major constituent cell wall, is perceived lysin-motif-containing (LYKs/CERK1), leading activation plant defense against pathogens. A conserved chitin sensing signaling system present cotton, including chitin-induced GhLYK5-GhCERK1 dimerization phosphorylation, contributes Importantly, GhWAK7A directly interacts with GhLYK5 GhCERK1 promotes dimerization. phosphorylates GhLYK5, which itself does not have kinase activity, but requires phosphorylation its function. Consequently, plays crucial role responses. Thus, our data reveal as an component pathogens complexing receptors.

Language: Английский

Citations

103
Supercharging enables organized assembly of synthetic biomolecules DOI
Anna J. Simon, Yi Zhou, Vyas Ramasubramani

et al.

Nature Chemistry, Journal Year: 2019, Volume and Issue: 11(3), P. 204 - 212

Published: Jan. 3, 2019

Language: Английский

Citations

83
A critical comparison of lanthanide based upconversion nanoparticles to fluorescent proteins, semiconductor quantum dots, and carbon dots for use in optical sensing and imaging DOI
Sandy F. Himmelstoß, Thomas Hirsch

Methods and Applications in Fluorescence, Journal Year: 2019, Volume and Issue: 7(2), P. 022002 - 022002

Published: March 1, 2019

The right choice of a fluorescent probe is essential for successful luminescence imaging and sensing especially concerning in vivo vitro applications, the development new classes have gained more attention last years. One most promising class are upconversion nanoparticles (UCNPs)—inorganic nanocrystals capable to convert near-infrared light high energy radiation. In this review we will compare UCNPs with other probes terms (a) optical properties probes, such as their brightness, photostability excitation wavelength; (b) chemical dispersibility, stability under experimental or physiological conditions, availability modification strategies labelling; (c) potential toxicity biocompatibility probe. Thereby want provide better understanding advantages drawbacks address future challenges design nanocrystals.

Language: Английский

Citations

78
Lanthanide-Based Optical Probes of Biological Systems DOI Creative Commons
Ukrae Cho, James Chen

Cell chemical biology, Journal Year: 2020, Volume and Issue: 27(8), P. 921 - 936

Published: July 30, 2020

Language: Английский

Citations

74
Dye‐Loaded Nanoemulsions: Biomimetic Fluorescent Nanocarriers for Bioimaging and Nanomedicine DOI
Andrey S. Klymchenko, Fei Liu, Mayeul Collot

et al.

Advanced Healthcare Materials, Journal Year: 2020, Volume and Issue: 10(1)

Published: Oct. 13, 2020

Abstract Lipid nanoemulsions (NEs), owing to their controllable size (20 500 nm), stability and biocompatibility, are now frequently used in various fields, such as food, cosmetics, pharmaceuticals, drug delivery, even nanoreactors for chemical synthesis. Moreover, being composed of components generally recognized safe (GRAS), they can be considered “green” nanoparticles that mimic closely lipoproteins intracellular lipid droplets. Therefore, attracted attention carriers drugs fluorescent dyes both bioimaging studying the fate cells small animals. In this review, composition dye‐loaded NEs, methods preparation, emerging biological applications described. The design bright NEs with high dye loading minimal aggregation‐caused quenching (ACQ) is focused on. Common issues including leakage discussed, highlighting advanced techniques characterization, Förster resonance energy transfer (FRET) fluorescence correlation spectroscopy (FCS). Attempts functionalize surface also discussed. Thereafter, single‐particle tracking animals well biomedical photodynamic therapy Finally, challenges future perspectives

Language: Английский

Citations

73