Single-Molecule Displacement Mapping Unveils Sign-Asymmetric Protein Charge Effects on Intraorganellar Diffusion

Nano Letters, Год журнала: 2023, Номер 23(5), С. 1711 - 1716

Опубликована: Фев. 20, 2023

Using single-molecule displacement/diffusivity mapping (SM

Язык: Английский

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Single-Molecule Spectroscopy and Super-Resolution Mapping of Physicochemical Parameters in Living Cells

Annual Review of Physical Chemistry, Год журнала: 2024, Номер 75(1), С. 163 - 183

Опубликована: Фев. 16, 2024

By superlocalizing the positions of millions single molecules over many camera frames, a class super-resolution fluorescence microscopy methods known as single-molecule localization (SMLM) has revolutionized how we understand subcellular structures past decade. In this review, highlight emerging studies that transcend outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at levels. encoding/decoding high-dimensional information-such emission excitation spectra, motion, polarization, lifetime, beyond-for every molecule, mass accumulating these measurements for molecules, such multidimensional multifunctional approaches open new windows into intracellular architectures dynamics, well their underlying biophysical rules, far beyond diffraction limit.

Язык: Английский

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Single-Molecule Displacement Mapping Indicates Unhindered Intracellular Diffusion of Small (≲1 kDa) Solutes

Journal of the American Chemical Society, Год журнала: 2023, Номер 145(15), С. 8510 - 8516

Опубликована: Апрель 7, 2023

While fundamentally important, the intracellular diffusion of small (≲1 kDa) solutes has been difficult to elucidate due challenges in both labeling and measurement. Here we quantify spatially map translational patterns mammalian cells by integrating several recent advances. In particular, executing tandem stroboscopic illumination pulses down 400 μs separation, extend single-molecule displacement/diffusivity mapping (SMdM), a super-resolution quantification tool, with high coefficients

Язык: Английский

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Multidimensional Super-Resolution Microscopy Unveils Nanoscale Surface Aggregates in the Aging of FUS Condensates

Journal of the American Chemical Society, Год журнала: 2023, Номер 145(44), С. 24240 - 24248

Опубликована: Окт. 2, 2023

The intracellular liquid–liquid phase separation (LLPS) of biomolecules gives rise to condensates that act as membrane-less organelles with vital functions. FUS, an RNA-binding protein, natively forms through LLPS and further provides a model system for the often disease-linked liquid-to-solid transition biomolecular during aging. However, mechanism such maturation processes, well structural physical properties system, remains unclear, partly attributable difficulties in resolving internal structures micrometer-sized diffraction-limited optical microscopy. Harnessing set multidimensional super-resolution microscopy tools uniquely map out local physicochemical parameters single-molecule spectroscopy, here, we uncover nanoscale heterogeneities FUS elucidate their evolution over Through spectrally resolved localization (SR-SMLM) solvatochromic dye, unveil distinct hydrophobic nanodomains at condensate surface. SMLM fluorogenic amyloid probe, identify these aggregates. displacement/diffusivity mapping (SMdM), show nanoaggregates drastically impede diffusion. Notably, upon aging or mechanical shears, progressively expand on surface, thus leading growing low-diffusivity shell while leaving interior diffusion-permitting. Together, beyond uncovering fascinating arrangements mechanisms single-component condensates, demonstrated synergy approaches this study opens new paths understanding systems nanoscale.

Язык: Английский

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Single molecule–driven nanomotors reveal the dynamic-disordered chemomechanical transduction of active enzymes

Science Advances, Год журнала: 2025, Номер 11(5)

Опубликована: Янв. 31, 2025

Enzymes facilitate the conversion of chemical energy into mechanical work during biochemical reactions, thereby regulating dynamic metabolic activity living systems. However, directly observing release facilitated by fluctuating individual enzymes remains a challenge, leading to contentious debate regarding underlying reasons for this phenomenon. Here, we aim overcome challenge developing an oscillating nanomotor powered single-molecule enzyme, which allows real-time tracking transduction in enzymatic reactions. Through analysis shifts free profiles within nanomotors, our results unveil not only heterogeneous patterns enzyme molecules but also disorder particular over extended monitoring periods. By exploring six distinct types provide direct evidence supporting argument that reaction enthalpy governs release. This approach has implications understanding mechanism catalysis and highly efficient nanomotors.

Язык: Английский

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Single-Molecule Diffusivity Quantification Unveils Ubiquitous Net Charge-Driven Protein–Protein Interaction

Journal of the American Chemical Society, Год журнала: 2024, Номер 146(15), С. 10973 - 10978

Опубликована: Апрель 5, 2024

Recent microscopy and nuclear magnetic resonance (NMR) studies have noticed substantial suppression of intracellular diffusion for positively charged proteins, suggesting an overlooked role electrostatic attraction in nonspecific protein interactions a predominantly negatively environment. Utilizing single-molecule detection statistics, here, we quantify aqueous solutions how diffusion, the limit low diffuser concentration to avoid aggregate/coacervate formation, is modulated by differently interactor proteins over wide ranges. We thus report substantially suppressed when oppositely interactors are added at parts per million levels, yet unvaried diffusivities same-charge beyond 1%. The attraction-driven sensitive net charge states, as probed varying solution pH ionic strength or chemically modifying robust across different diffuser–interactor pairs. By converting measured diameters, further show that excess interactors, molecule effectively drags along just one monolayer where stop. unveil ubiquitous, charge-driven protein–protein shed new light on mechanism charge-based living cells.

Язык: Английский

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Perspective: a stirring role for metabolism in cells

Molecular Systems Biology, Год журнала: 2022, Номер 18(4)

Опубликована: Апрель 1, 2022

Язык: Английский

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Size-Dependent Suppression of Molecular Diffusivity in Expandable Hydrogels: A Single-Molecule Study

The Journal of Physical Chemistry B, Год журнала: 2023, Номер 127(14), С. 3333 - 3339

Опубликована: Апрель 3, 2023

By repurposing the recently popularized expansion microscopy to control meshwork size of hydrogels, we examine size-dependent suppression molecular diffusivity in resultant tuned hydrogel nanomatrices over a wide range polymer fractions ∼0.14-7 wt %. With our developed single-molecule displacement/diffusivity mapping (SMdM) methods, thus show that with fixed size, larger molecules exhibit more impeded diffusion and that, for same molecule, is progressively suppressed as reduced; this effect prominent molecules. Moreover, meshwork-induced obstruction uncoupled from due increased solution viscosities. Thus, two mechanisms, respectively, being diffuser-size-dependent independent, may separately scale down produce final slowdown complex systems like cell.

Язык: Английский

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Single-Molecule Tracking of Reagent Diffusion during Chemical Reactions

Journal of the American Chemical Society, Год журнала: 2023, Номер 145(19), С. 10512 - 10521

Опубликована: Апрель 20, 2023

Recent experiments have shown that the diffusion of reagent molecules is inconsistent with what Stokes-Einstein equation predicts during a chemical reaction. Here, we used single-molecule tracking to observe reactive click and Diels-Alder (DA) reactions. We found coefficient reagents remained unchanged within experimental uncertainty upon DA Yet, faster than predicted reaction when concentration catalyst exceed threshold. A stepwise analysis suggested fast scenario due but not involvement tracer itself. The present results provide evidence on faster-than-expected CuAAC in specific conditions propose new insights into understanding this unexpected behavior.

Язык: Английский

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Machine-learning-powered extraction of molecular diffusivity from single-molecule images for super-resolution mapping

Communications Biology, Год журнала: 2023, Номер 6(1)

Опубликована: Март 28, 2023

While critical to biological processes, molecular diffusion is difficult quantify, and spatial mapping of local diffusivity even more challenging. Here we report a machine-learning-enabled approach, pixels-to-diffusivity (Pix2D), directly extract the coefficient D from single-molecule images, consequently enable super-resolved mapping. Working with images recorded at fixed framerate under typical localization microscopy (SMLM) conditions, Pix2D exploits often undesired yet evident motion blur, i.e., convolution trajectory during frame recording time diffraction-limited point spread function (PSF) microscope. Whereas stochastic nature imprints diverse trajectories different molecules diffusing same given D, construct convolutional neural network (CNN) model that takes stack as input evaluates D-value output. We thus validate robust evaluation simulated data, experimental data successfully characterize differences for supported lipid bilayers compositions resolve gel fluidic phases nanoscale.

Язык: Английский

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