Protein misfolding and amyloid nucleation through liquid–liquid phase separation

Chemical Society Reviews, Год журнала: 2024, Номер 53(10), С. 4976 - 5013

Опубликована: Янв. 1, 2024

Protein misfolding and amyloid aggregation, linked to neurodegenerative diseases, can result from liquid–liquid phase separation (LLPS) a subsequent liquid-to-solid transition. This represents LLPS as generic mechanism in nucleation.

Язык: Английский

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Single-molecule live-cell RNA imaging with CRISPR–Csm

Nature Biotechnology, Год журнала: 2025, Номер unknown

Опубликована: Фев. 18, 2025

Abstract Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution real time. However, single-molecule live-cell imaging unmodified endogenous has not yet been achieved a generalizable manner. Here, we present fluorescence situ hybridization (smLiveFISH), robust approach that combines programmable RNA-guided, RNA-targeting CRISPR–Csm complex with multiplexed guide RNAs for direct and efficient visualization range cell types, including primary cells. Using smLiveFISH, track native NOTCH2 MAP1B transcripts living identify two distinct localization mechanisms cotranslational translocation mRNA endoplasmic reticulum directional transport toward periphery. This method potential to unlock principles governing spatiotemporal organization health disease.

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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Metastable condensates suppress conversion to amyloid fibrils

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Март 3, 2024

Stress granules form via co-condensation of RNA binding proteins with prion-like low complexity domains (PLCDs) and molecules released by stress-induced polysomal runoff. Homotypic interactions among PLCDs can drive amyloid fibril formation this is enhanced ALS-associated mutations. We find that homotypic condensation versus are separable for A1-LCD, the PLCD hnRNPA1. These lead to condensates metastable fibrils globally stable. Metastable suppress formation, mutations enhance weakening condensate metastability. Mutations designed A1-LCD metastability restore wild-type behaviors stress in cells even when present. This suggests be suppressed enhancing through condensate-driving interactions.

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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Advanced surface passivation for high-sensitivity studies of biomolecular condensates

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(22)

Опубликована: Май 23, 2024

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of through biochemical reconstitution and microscopic detection these structures. Quantitative visualization assays biomolecular rely on surface passivation to minimize background artifacts due condensate adhesion. However, challenge undesired interactions between glass surfaces, which can alter material properties impair observational accuracy, remains a critical hurdle. Here, we introduce efficient, broadly applicable, simple method employing self-assembly surfactant Pluronic F127 (PF127). The greatly reduces nonspecific binding across range systems for both phase-separated droplets dilute phase. Additionally, by integrating PF127 with Biotin-NeutrAvidin system, achieve controlled multipoint attachment surfaces. This not only preserves but also facilitates long-time fluorescence recovery after photobleaching imaging high-precision single-molecule analyses. Using this method, explored dynamics polySIM molecules within polySUMO/polySIM at level. Our observations suggest potential heterogeneity distribution available polySIM-binding sites condensates.

Язык: Английский

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Single-molecule live-cell RNA imaging with CRISPR-Csm

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июль 16, 2024

High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors individual molecules in single cells. However, single-molecule live-cell unmodified endogenous has not yet been achieved. Here, we present fluorescence

Язык: Английский

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Advancing Biosensing through Super-Resolution Fluorescence Microscopy

Biosensors and Bioelectronics, Год журнала: 2025, Номер unknown, С. 117374 - 117374

Опубликована: Март 1, 2025

Язык: Английский

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Interaction networks within biomolecular condensates feature topological cliques near the interface

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Март 27, 2025

Biomolecular condensates are typically maintained by networks of molecular interactions, with canonical examples including those formed prion-like low complexity domains (LCDs) proteins. Single-component LCD have been predicted to exhibit small-world network topologies and spatial inhomogeneities in protein compaction. Here, we systematically characterize underlying investigate the relationship between single molecule properties topologies. We employ a chemically specific coarse-grained model probe generalize our findings varying sequence hydrophobicity via generic that describes "hydrophobic-polar" (HP) polymers. For both systems, find sustained featuring "hubs" "cliques". Molecular hubs high betweenness centrality localize near centers adopt more elongated conformations. In contrast, cliques-densely interacting molecules form locally fully connected subgraphs-are bridged tend condensate interface. Interestingly, power-law relationships structure dynamics individual centrality, which connectivity. Thus, work demonstrates connectivity can be from single-molecule properties. Furthermore, cliques longer lifetimes their constituent remain spatially constrained, suggesting role shaping interface material

Язык: Английский

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Probing the Formation and Liquid-to-Solid Transition of FUS Condensates via the Lifetimes of Fluorescent Proteins

The Journal of Physical Chemistry Letters, Год журнала: 2025, Номер unknown, С. 3553 - 3561

Опубликована: Март 31, 2025

Liquid–liquid phase separation (LLPS) of biomolecules is a fundamental cellular process that essential for maintaining homeostasis and facilitating biochemical activities. On the other hand, aberrant alters condensate fluidity causes transition from liquid-like condensates to solid-like condensates, which may lead formation pathological aggregations often observed in neurodegenerative diseases. Condensate usually assessed by fluorescence recovery after photobleaching. Here, we reveal lifetimes several fluorescent proteins are sensitive LLPS liquid-to-solid transition. Furthermore, identify key residues regulate sensitivity toward separation. Thus, apply lifetime imaging microscopy (FLIM) visualize living cells, demonstrating FLIM nondestructive method tracking changes real time.

Язык: Английский

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