“Forcing” new interpretations of molecular tension sensor studies

Cell Reports Methods, Journal Year: 2024, Volume and Issue: 4(7), P. 100821 - 100821

Published: July 1, 2024

Molecular tension sensors are central tools for mechanobiology studies but have limitations in interpretation. Reporting Cell Reports Methods, Shoyer et al. discover that fluorescent protein photoswitching concert with sensor extension may expand the use and interpretation of common force-sensing tools. Mechanical forces emerged as critical stimuli a surprising diversity key cellular processes including cell migration, T activation, stem differentiation.1Zuela-Sopilniak N. Lammerding J. Can't handle stress? Mechanobiology disease.Trends Mol. Med. 2022; 28: 710-725Abstract Full Text PDF PubMed Scopus (21) Google Scholar As such, there has been significant interest decoding pathways responsible converting mechanical into responses (Figure 1). It is increasingly appreciated this correspondence stepwise process initiated by application force to specific (mechanotransmission) that, response, undergoes conformational changes result modifications its structure, dynamics, function whole (mechanosensing). These conformation then biochemically interpreted (mechanotransduction), often through engagement or assembly transducer proteins, drive downstream alterations gene expression (mechanoresponse). Recently, wide variety technologies—collectively known molecular (MTSs)2Huang Y. Chen T. X. Zhang Liu S. Lu M. C. Ding Yang al.Decoding Biomechanical Cues Based on DNA Sensors.Small. 2024; 20e2310330Crossref (0) Scholar,3Fischer L.S. Rangarajan Sadhanasatish Grashoff Force Measurement Tension Sensors.Annu. Rev. Biophys. 2021; 50: 595-616Crossref (33) Scholar—have developed better characterize quantify these cultured cells. technologies range from surface-immobilized capable digitally quantifying overall output genetically encoded integrated force-related proteins interest. One first MTSs was 2010 al.4Grashoff Hoffman B.D. Brenner M.D. Zhou R. Parsons M.T. McLean M.A. Sligar S.G. C.S. Ha Schwartz Measuring across vinculin reveals regulation focal adhesion dynamics.Nature. 2010; 466: 263-266Crossref (1151) enabled detection piconewton communicated individual within This groundbreaking technology essentially acted blueprint successors, which most composed well-characterized force-responsive spring-like element some kind reporter communicating spring thus transduction. The readouts fluorescence occur applied extended, though other such bioluminescence resonance energy transfer (BRET)5Aird E.J. Tompkins K.J. Ramirez M.P. Gordon W.R. Enhanced Sensor Bioluminescence Resonance Energy Transfer (BRET).ACS Sens. 2020; 5: 34-39https://doi.org/10.1021/acssensors.9b00796Crossref (24) also reported. can be achieved observation Förster (FRET) between donor acceptor (FPs) flank element. When stretched, FPs physically separated one another, yields change their FRET ratio proportional distance them used inform requirements change. Importantly, majority employ types only observing very step sensing—mechanotransmission—because extends instantaneously response force, leading an analog signal lacks information concerning duration loading rate, limiting our understanding sensor-embedded undergoing. There remains untapped source would how initial sensing event ligand-receptor pair sustained allows clustering cascading mediate formation biologically important adhesions immune synapses. In current issue al.6Shoyer T.C. Collins K.L. Ham T.R. Blanchard A.T. Malavade J.N. Johns B.A. West J.L. Detection switching cellulo.Cell Methods. 4100815Google present elegant approach aims elucidate rich escapes traditional while establishing framework standardization enhance reproducibility enable accurate comparisons. described above, report However, it recently discovered alter conformation,7Ganim Z. Rief Mechanically single-molecule GFP unfolding refolding.Proc. Natl. Acad. Sci. USA. 2017; 114: 11052-11056Crossref (42) decrease potentially confounding results. With mind, models predict precise characteristics—such rate force—required FPs. were simulate what FRET-based MTS data look like when signals undergoing switching. simulated efficiency (E, termini spring) stochasticity (S, each FP detected relative theoretical amount) distinct trends depending if an, which, experiencing E versus S plotted. To test simulations, (a "spring" spider silk protein) flanked two FRET-capable FPs, mTFP1 mVenus, designed bind actin filaments evaluate forces. From this, both measured matched (albeit slight variability attributed complexity) results (mVenus) at lower (mTFP-1) higher difference stability mVenus (mTFP-1 > m-Venus) seen dynamic simulations. abundant lamellar network near periphery cell, suggesting region greatest (in either magnitude, duration, metric) filaments. further ensure finding reflecting measurement, disrupted Latrunculin A, resulted loss signatures. presents feat, comprises readout cellulo mechanosensing does not require secondary binding8Zhong B.L. Vachharajani V.T. Dunn A.R. Facile cells STReTCh.Cell Rep. 2100278Google demonstrates compatible suggests added benefit well, existing reanalyzed reveal highlight previous inserted re-assessed. indicated robust regions previously identified transmitting gradients9LaCroix A.S. Lynch A.D. Berginski M.E. Tunable extension-based control loading.Elife. 2018; 7e33927https://doi.org/10.7554/eLife.33927Crossref (63) corresponded gradients Intriguingly, point mutants prevent catch bond (bonds strengthen applied) analyzed, decreased completely lost mutant. bonds necessary they regulate magnitude. should noted currently proposed framework, cannot disentangled other. future work cataloging propensity broader luciferases identify pairs decouple parameters. Nevertheless, much demonstration MTSs, major milestone toward steps mechanotransmission achieved. A second yet equally take away cautionary tale skew Factors orientation reported reason incongruency different measurements same protein. summary, deeper mechanosensitivity exploitation role underlying biology disease expanding elevate level biochemical will "force" forward. authors declare no competing interests. celluloShoyer al.Cell MethodsJuly 9, 2024In BriefShoyer develop probe affect inside living leverage study mechanosensitive processes. Fluorescent manipulated perturbations force-activate substrate stiffness, consistent sensitivity dynamics. Full-Text Open Access

Language: Английский

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In-Silico Analyses of Molecular Force Sensors for Mechanical Characterization of Biological Systems

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 22, 2024

ABSTRACT Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed tools for situ measurements. Here we use all-atom steered dynamics simulations to predict study the relationship between design parameters mechanical properties three types of commonly used cellular research: two peptide-and one DNA-based. The peptide-based consist a pair fluorescent proteins, which can undergo Förster resonance energy transfer (FRET), linked by spider silk (GPGGA) n or synthetic (GGSGGS) disordered regions. DNA-based sensor consists fluorophore-labeled strands DNA that be unzipped sheared upon application with FRET signal readout dissociation. We simulated nine sensors, each kind. After equilibration, flexible peptide linkers different lengths were stretched applying their N-and C-terminal Cα atoms opposite directions. Similarly, equilibrated pulled on phosphate atom terminal guanine strand selected other direction. These performed at constant velocity (0.01 nm/ns – 10 nm/ns) (10 pN 500 pN) all versions sensors. Our results show how response these depends length, sequence, configuration loading rate. Mechanistic insights gained from analyses indicate interpretation experimental should consider influence transient formation secondary structure overstretching predictions guide optimal fluorophore choice facilitate rational new protein, DNA, hybrid systems, devices. STATEMENT OF SIGNIFICANCE Biomolecular structures involved various processes, including muscle function perception, generate, convey, respond forces. In-vivo accurate measurement is challenging but needed understand function. present comprehensive computational analysis report pico-Newton level biomolecular systems. atom-level simulation provide mechanistic insight data interpretation, selection parameters, development tailored specific applications environments.

Language: Английский

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Dimeric-molecular beacon based intramolecular strand displacement amplification enables robust analysis of miRNA

Talanta, Journal Year: 2024, Volume and Issue: 280, P. 126778 - 126778

Published: Aug. 25, 2024

Language: Английский

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Aptamer‐based Immune Checkpoint Inhibition for Cancer Immunotherapy

ChemBioChem, Journal Year: 2024, Volume and Issue: 26(1)

Published: Oct. 17, 2024

Abstract Cancer has long been a significant threat to human life and health. The advent of immune checkpoint blockade strategies reversed cancer‐induced suppression, advanced the development immunotherapy, offered new hope in fight against cancer. Aptamers, which possess same specificity affinity as antibodies, are advantageous due their synthetic accessibility ease modification, providing novel insights for research. In this review, we outline key aptamers currently developed checkpoints such CTLA‐4, PD‐1, PD−L1 Siglec‐15. We explore potential therapeutic strategies, including functionalizing or engineering covalent binding, valency control, nanostructure assembly, well investigating molecular mechanisms glycosylated protein functions cell‐cell interactions. Finally, future applications immunotherapy discussed.

Language: Английский

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