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| Open AccessRapid evolutionary change in trait correlations of single proteins
Trait correlations impact evolvability as selection on one trait can influence others. Here, the authors examine trait correlation in two proteins, a fluorescent protein & an antibiotic resistance enzyme, observing rapid evolution of trait correlations through changes in the biophysical properties of these proteins.
- Pouria Dasmeh
- , Jia Zheng
- & Andreas Wagner
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Article
| Open AccessSingle-photon microscopy to study biomolecular condensates
The wide variety of cellular processes involving biomolecular condensation makes their quantification a challenging task. Here, the authors present an integrated platform based on single-photon microscopy to study complex biomolecular processes.
- Eleonora Perego
- , Sabrina Zappone
- & Giuseppe Vicidomini
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Article
| Open AccessDirect regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization
Voltage sensing of ion channels relies on charged transmembrane helices. Here authors use live-cell FLIM-FRET and nonsense suppression-mediated fluorescence labeling to reveal that voltage sensors undergo direct modulation by compartmentalized membrane domains.
- Lucas J. Handlin
- & Gucan Dai
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Article
| Open AccessMulticolor lifetime imaging and its application to HIV-1 uptake
Multicolor imaging employing genetically-encodable fluorescent proteins permits spatiotemporal live cell imaging of multiple cues. Here, authors use multicolor lifetime imaging to visualize quadruple-labelled human immunodeficiency viruses within cellular contexts.
- Tobias Starling
- , Irene Carlon-Andres
- & Sergi Padilla-Parra
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| Open AccessQuantitative real-time in-cell imaging reveals heterogeneous clusters of proteins prior to condensation
The nucleation of biomolecular condensates is seldom quantified in living cells. Here, the authors show how protein clusters form before microscopically visible condensation and find a flat free-energy profile with active blocking of cluster growth.
- Chenyang Lan
- , Juhyeong Kim
- & Thorsten Hugel
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Article
| Open AccessStructural and photophysical characterization of the small ultra-red fluorescent protein
Researchers determined the three-dimensional structure of the small Ultra-Red Fluorescent Protein (smURFP) to understand its properties and the previous directed evolution process. In addition, they show smURFP fluoresces longer than small molecules.
- Atanu Maiti
- , Cosmo Z. Buffalo
- & Erik A. Rodriguez
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Article
| Open AccessFast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging
Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here the authors design spirocyclic rhodamine probes that enable a fluorescent light-up aptamer system suitable for visualizing RNAs in live or fixed cells with two different super-resolution microscopy modalities SMLM and STED.
- Daniel Englert
- , Eva-Maria Burger
- & Murat Sunbul
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Article
| Open AccessCo-crystal structures of the fluorogenic aptamer Beetroot show that close homology may not predict similar RNA architecture
The recently discovered aptamer Beetroot is a homodimeric RNA that binds and activates DFAME, a conditional, red-shifted fluorophore derived from GFP. Here the authors determine the Beetroot-DFAME co-crystal structure, which is distinctively different from that of similar RNA aptamer Corn.
- Luiz F. M. Passalacqua
- , Mary R. Starich
- & Adrian R. Ferré-D’Amaré
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Article
| Open AccessCharacterisation of IL-23 receptor antagonists and disease relevant mutants using fluorescent probes
The single nucleotide polymorphism C115Y in the IL-23 receptor is associated with autoinflammatory diseases. Here the authors demonstrate that this mutation prevents the binding of a fluorescent cyclic peptide and IL-23 to the IL-23 receptor.
- Charles S. Lay
- , Albert Isidro-Llobet
- & Stephen J. Hill
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Article
| Open AccessIllumination of a progressive allosteric mechanism mediating the glycine receptor activation
Glycine receptors are channel receptors mediating signal transduction between neurons that transit between resting and open states upon neurotransmitter binding. Here, the authors illuminate a progressive transition of opening by combining voltage-clamp fluorometry and molecular dynamics.
- Sophie Shi
- , Solène N. Lefebvre
- & Pierre-Jean Corringer
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Article
| Open AccessHigh-precision estimation of emitter positions using Bayesian grouping of localizations
Single-molecule localization microscopy relies on stochastic blinking events, treated as independent events without assignment to a particular emitter. Here, BaGoL takes low precision localizations generated from multiple emitter blinkings during DNAPAINT and dSTORM and finds the underlying emitter positions with high precision.
- Mohamadreza Fazel
- , Michael J. Wester
- & Keith A. Lidke
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Article
| Open AccessBinary-FRET reveals transient excited-state structure associated with activity-dependent CaMKII - NR2B binding and adaptation
FRET can be used to study conformational changes and protein-protein interactions. Here the authors report Binary-FRET for monitoring two FRET reactions, one encoded in the fluorescence lifetime of the donor, another encoded in its anisotropy, and monitor the dynamics of CaMKII and its interaction with NR2B.
- Tuan A. Nguyen
- , Henry L. Puhl III
- & Steven S. Vogel
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Article
| Open AccessInvestigating lytic polysaccharide monooxygenase-assisted wood cell wall degradation with microsensors
It is important to understand the enzymatic degradation of wood biomass by lytic polysaccharide monooxygenase, however, disagreements about the co-substrate exist. Here, the authors use piezo-controlled hydrogen peroxide micro-sensors to demonstrate that even low levels of hydrogen peroxide support the enzymatic degradation of wood cell walls.
- Hucheng Chang
- , Neus Gacias Amengual
- & Roland Ludwig
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Article
| Open AccessFtsN maintains active septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthases in E. coli
FtsN promotes the inward synthesis of septal peptidoglycan (sPG) through the FtsWI complex during bacterial cell division. Here, Lyu et al. apply single-molecule microscopy on E. coli to show that FtsN proteins (I) move processively at a speed similar to that of FtsWI molecules. (II) can be divided into two populations based on their speeds, and (III) their movement is driven exclusively by peptidoglycan synthesis
- Zhixin Lyu
- , Atsushi Yahashiri
- & Jie Xiao
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| Open AccessQuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins
The authors present an in-depth investigation of excited state dynamics and molecular mechanism of the voltage sensing in microbial rhodopsins. Using a combination of spectroscopic investigations and molecular dynamics simulations, the study proposes the voltage-modulated deprotonation of the chromophore as the key event in the voltage sensing. Thus, molecular constraints that may further improve the fluorescence quantum yield and the voltage sensitivity are presented.
- Arita Silapetere
- , Songhwan Hwang
- & Peter Hegemann
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Article
| Open AccessPancreatic α and β cells are globally phase-locked
The Ca2+ modulated pulsatile glucagon and insulin secretions by pancreatic α and β cells are critical in glucose homeostasis. Here the authors show that the Ca2+ oscillations of α and β cells are phase-locked, and that the oscillation pattern is tuned by paracrine interactions between α and β cells.
- Huixia Ren
- , Yanjun Li
- & Chao Tang
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Article
| Open AccessThe antenna of far-red absorbing cyanobacteria increases both absorption and quantum efficiency of Photosystem II
Some cyanobacteria acclimate to far-red light by integrating chlorophyll f into their photosystems. Additional chlorophylls typically slow down charge separation but here the authors show that charge separation in chlorophyll-f-containing Photosystem II is faster in the presence of red-shifted allophycocyanin antennas.
- Vincenzo Mascoli
- , Ahmad Farhan Bhatti
- & Roberta Croce
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Article
| Open AccessStructural basis for the inhibition of IAPP fibril formation by the co-chaperonin prefoldin
Integrated kinetic and structural investigations reveal that the ubiquitous co-chaperonin prefoldin interacts with its coiled-coil helices on the islet amyloid polypeptide fibril surface and fibril ends to inhibit fibril elongation and secondary nucleation.
- Ricarda Törner
- , Tatsiana Kupreichyk
- & Jerome Boisbouvier
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| Open Access4polar-STORM polarized super-resolution imaging of actin filament organization in cells
Single-molecule localisation microscopy does not give orientation information. Here the authors combine Stochastic Optical Reconstruction Microscopy (STORM) with single molecule orientation and wobbling measurements using four-polarisation image splitting, 4polar-STORM.
- Caio Vaz Rimoli
- , Cesar Augusto Valades-Cruz
- & Sophie Brasselet
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Article
| Open AccessGuest-host doped strategy for constructing ultralong-lifetime near-infrared organic phosphorescence materials for bioimaging
Though room-temperature phosphorescence (RTP) in organics is advantageous for bioimaging, designing materials that meet lifetime and wavelength emission requirements is challenging. Here, the authors us a guest-host doped strategy to construct RTP materials with ultralong-lifetime, NIR emission.
- Fuming Xiao
- , Heqi Gao
- & Dan Ding
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Article
| Open AccessQuantification of FRET-induced angular displacement by monitoring sensitized acceptor anisotropy using a dim fluorescent donor
The FRET efficiency usually predominantly depends on the proximity of donor and acceptor. Here the authors report an anisotropy-based mode of FRET detection, FRET-induced Angular Displacement Evaluation via Dim donor (FADED), to allow quantification of the relative angle between donor and acceptor.
- Danai Laskaratou
- , Guillermo Solís Fernández
- & Hideaki Mizuno
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| Open AccessNanoscale imaging of bacterial infections by sphingolipid expansion microscopy
Imaging of lipid bilayers using light microscopy is challenging. Here the authors label cells using a short chain click-compatible ceramide to visualize mammalian and bacterial membranes with expansion microscopy.
- Ralph Götz
- , Tobias C. Kunz
- & Markus Sauer
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Article
| Open AccessSpatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break
53BP1 is a crucial factor involved in double strand break repair which blocks DNA end resection affecting DNA repair pathway choice. Here the authors reveal by live cell nuclear architecture analysis the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response.
- Jieqiong Lou
- , David G. Priest
- & Elizabeth Hinde
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Article
| Open AccessAutomated and optimally FRET-assisted structural modeling
To overcome the limitation of FRET data being too sparse to cover all structural details, FRET experiments need to be carefully designed and complemented with simulations. Here the authors present a toolkit for automated design of FRET experiments, which determines how many and which FRET pairs should be used to maximize the accuracy, and for FRET-assisted structural modeling and refinement at the atomistic level.
- Mykola Dimura
- , Thomas-Otavio Peulen
- & Holger Gohlke
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Article
| Open AccessCooperative transport mechanism of human monocarboxylate transporter 2
Proton-linked monocarboxylate transporters (MCTs) facilitate monocarboxylate efflux in glycolytically active cells and regulate transport down in glycolytically inactive cells. Here authors show a steep dependence of human MCT2 activity on substrate concentration and show the structural basis of cooperative transport.
- Bo Zhang
- , Qiuheng Jin
- & Sheng Ye
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Article
| Open AccessDeep learning enables structured illumination microscopy with low light levels and enhanced speed
Super-resolution microscopy typically requires high laser powers which can induce photobleaching and degrade image quality. Here the authors augment structured illumination microscopy (SIM) with deep learning to reduce the number of raw images required and boost its performance under low light conditions.
- Luhong Jin
- , Bei Liu
- & Klaus M. Hahn
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Article
| Open AccessLive cell imaging of single RNA molecules with fluorogenic Mango II arrays
Fluorogenic RNA aptamers have been used for RNA imaging, but folding and fluorescence stability often limited their use in high resolution applications. Here the authors present an array of stably folding Mango II aptamers for imaging of coding and non-coding RNAs at single-molecule resolution, in both live and fixed cells.
- Adam D. Cawte
- , Peter J. Unrau
- & David S. Rueda
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Article
| Open AccessResolving dynamics and function of transient states in single enzyme molecules
T4 Lysozyme (T4L) is a model protein whose structure is extensively studied. Here the authors combine single-molecule and ensemble FRET measurements, FRET-positioning and screening and EPR spectroscopy to study the structural dynamics of T4L and describe its conformational landscape during the catalytic cycle by an extended Michaelis–Menten mechanism and identify an excited conformational state of the enzyme.
- Hugo Sanabria
- , Dmitro Rodnin
- & Claus A. M. Seidel
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Article
| Open AccessImaging of fluorescence anisotropy during photoswitching provides a simple readout for protein self-association
Performing homo-FRET measurements in cells using a fluorescence microscope is challenging, especially when using high numerical aperture objective lenses. Here the authors present a method for improved homo-FRET measurements based on anisotropy changes in photoswitchable fluorescent proteins.
- Namrata Ojha
- , Kristin H. Rainey
- & George H. Patterson
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Article
| Open AccessProton-dynamic therapy following photosensitiser activation by accelerated protons demonstrated through fluorescence and singlet oxygen production
The authors use accelerated protons on photosensitizers (PS, conventionally excited by light), to generate fluorescence and singlet oxygen which can enhance the efficacy of proton therapy. A pilot study on glioblastoma cells confirms differential cell death upon irradiation in the presence of PS.
- M. Grigalavicius
- , M. Mastrangelopoulou
- & T. A. Theodossiou
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Article
| Open AccessAn alternative framework for fluorescence correlation spectroscopy
Fluorescence correlation spectroscopy is widely used for in vivo and in vitro applications, yet extracting information from experiments still requires long acquisition times. Here, the authors exploit Bayesian non-parametrics to directly analyze the output of confocal fluorescence experiments thereby probing physical processes on much faster timescales.
- Sina Jazani
- , Ioannis Sgouralis
- & Steve Pressé
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Article
| Open AccessSingle-molecule localization microscopy and tracking with red-shifted states of conventional BODIPY conjugates in living cells
Single-molecule localization microscopy (SMLM) requires the use of fluorophores with specific sets of properties. Here the authors employ conventional BODIPY dyes as SMLM fluorophores by making use of rarely reported red-shifted ground state BODIPY dimers to image fatty acids, lipid droplets and lysosomes at single-molecule resolution.
- Santosh Adhikari
- , Joe Moscatelli
- & Elias M. Puchner
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| Open AccessInitial state of DNA-Dye complex sets the stage for protein induced fluorescence modulation
Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. Here, authors provide a perspective on understanding the general phenomenon of induced fluorescence modulation
- Fahad Rashid
- , Vlad-Stefan Raducanu
- & Samir M. Hamdan
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| Open AccessHigh precision FRET studies reveal reversible transitions in nucleosomes between microseconds and minutes
Nucleosomes compact the genome and regulate access to specific DNA sequences. Here the authors employ single-molecule FRET studies to characterize nucleosome dynamics at different salt concentrations and dissect nucleosome disassembly into elementary steps.
- Alexander Gansen
- , Suren Felekyan
- & Jörg Langowski
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Article
| Open AccessCell shape-independent FtsZ dynamics in synthetically remodeled bacterial cells
The FtsZ protein assembles into a structure known as ‘Z-ring’ at midcell for bacterial cell division. Here, Söderström et al. show that Z-ring assembly and dynamics in E. coli cells with unnatural shapes, such as squares and hearts, are generally similar to those observed in cells with normal shape.
- Bill Söderström
- , Alexander Badrutdinov
- & Ulf Skoglund
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Article
| Open AccessIdentifying weak interdomain interactions that stabilize the supertertiary structure of the N-terminal tandem PDZ domains of PSD-95
Biologically relevant weak and transient interdomain interactions within proteins are difficult to analyze. Here, the authors combine multiscale molecular dynamics simulations and high-precision FRET experiments to characterize interactions between the tandem PDZ domains of PSD-95, revealing previously hidden conformational states.
- Inna S. Yanez Orozco
- , Frank A. Mindlin
- & Hugo Sanabria
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Article
| Open AccessIntermembrane crosstalk drives inner-membrane protein organization in Escherichia coli
Outer membrane proteins (OMPs) in Gram-negative bacteria have restricted lateral mobility. Here, Rassam et al. show that the bacteriocin ColE9, via its interactions with OMPs, imposes this restricted mobility on the inner membrane proteins of the Tol-Pal complex.
- Patrice Rassam
- , Kathleen R. Long
- & Colin Kleanthous
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Article
| Open AccessNDP52 activates nuclear myosin VI to enhance RNA polymerase II transcription
Myosin VI (MVI) is known to interact with RNA Polymerase II and to play non-cytoplasmic roles in cells. Here, the authors provide evidence that the transcription co-activator NDP52 regulates MVI binding to DNA and that MVI interacts with nuclear receptors to drive gene expression.
- Natalia Fili
- , Yukti Hari-Gupta
- & Christopher P. Toseland
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Article
| Open AccessPolymorphic regenerated silk fibers assembled through bioinspired spinning
Natural silk fibers are produced using a simple and green approach compared to alternative synthetic methods. Here, the authors show a bioinspired approach to spin regenerated silk fibers using anisotropic liquid crystals and dry spinning, resulting in remarkably robust fibers.
- Shengjie Ling
- , Zhao Qin
- & Markus J. Buehler
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| Open AccessDetecting stoichiometry of macromolecular complexes in live cells using FRET
Measuring thein vivo stoichiometry of protein-protein interactions is challenging. Here the authors take a FRET-based approach, quantifying stoichiometry based on ratiometric comparison of donor and acceptor fluorescence, and apply their method to report on a Ca2+-induced switch in calmodulin binding to Ca2+ion channels.
- Manu Ben-Johny
- , Daniel N. Yue
- & David T. Yue
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Article
| Open AccessStructural roles of guide RNAs in the nuclease activity of Cas9 endonuclease
In bacteria, CRISPR-Cas9 identifies and cleaves the target DNA with the assistance of a tracrRNA and a crRNA. Here the authors use single-molecule spectroscopy to investigate conformational changes and show that the tracrRNA keeps Cas9 in a functionally active form.
- Youngbin Lim
- , So Young Bak
- & Seong Keun Kim
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Article
| Open AccessBoundaries steer the contraction of active gels
The actomyosin cytoskeleton consists of a contractile array but how it becomes organized is not clear. Here the authors reconstitute a controllable contractile system to show that force balances at boundaries determine contraction dynamics, and spatial anisotropy leads to self-organization or aligned contractile fibres.
- Matthias Schuppler
- , Felix C. Keber
- & Andreas R. Bausch
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| Open AccessMechanics of epithelial closure over non-adherent environments
Closure of epithelial gaps such as wounds is thought to involve contraction of an actomyosin ‘purse-string’. By creating non-adherent gaps to exclude contributions of adhesive protrusion, the authors find that large-scale tension, more than purse-string contraction, mediates closure.
- Sri Ram Krishna Vedula
- , Grégoire Peyret
- & Benoit Ladoux
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Article
| Open AccessSeparating NADH and NADPH fluorescence in live cells and tissues using FLIM
NAD and NADP play fundamentally different roles in cellular metabolism, and yet these pyridine nucleotides cannot be distinguished spectroscopically in living cells. Blacker et al.demonstrate that fluorescence lifetime imaging can be used to quantify NADPH/NADH balance in cultured cells and in the mammalian cochlea.
- Thomas S. Blacker
- , Zoe F. Mann
- & Michael R. Duchen