Nanoscopic Profiling of Small Extracellular Vesicles via High‐Speed Atomic Force Microscopy (HS‐AFM) Videography

Journal of Extracellular Vesicles, Journal Year: 2025, Volume and Issue: 14(4)

Published: March 26, 2025

Small extracellular vesicles (sEVs), which carry lipids, proteins and RNAs from their parent cells, serve as biomarkers for specific cell types biological states. These vesicles, including exosomes microvesicles, facilitate intercellular communication by transferring cellular components between cells. Current methods, such ultracentrifugation Tim-4 affinity method, yield high-purity sEVs. However, despite small size, purified sEVs remain heterogeneous due to varied intracellular origins. In this technical note, we used high-speed atomic force microscopy (HS-AFM) in conjunction with exosome markers (IgGCD63 IgGCD81) explore the origins of at single-sEV resolution. Our results first revealed nanotopology HEK293T-derived under physiological conditions. Larger (diameter > 100 nm) exhibited greater height fluctuations compared smaller ≤ nm). Next, found that mouse-origin IgGCD63, rabbit-origin IgGcontrol IgGCD81, iconic 'Y' conformation, similar structural dynamics properties. Last, marker antibodies predominantly co-localised sEVd nm but not nm, demonstrating CD63-CD81-enriched sEV CD63-CD81-depleted subpopulations. summary, demonstrate nanoscopic profiling surface on using HS-AFM is feasible characterising distinct subpopulations a mixture.

Language: Английский

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Spatiotemporal dynamics of protamine–DNA condensation revealed by high-speed atomic force microscopy

Nucleic Acids Research, Journal Year: 2025, Volume and Issue: 53(6)

Published: March 20, 2025

Abstract Protamines (PRMs) play a crucial role in sperm chromatin condensation, replacing histones to form nucleo–PRM structures, specifically PRM–DNA complexes. Despite their importance reproduction, the detailed mechanisms underlying PRM-mediated DNA condensation have remained elusive. In this study, we employed high-speed atomic force microscopy (HS-AFM) directly visualize real-time binding dynamics of PRM under physiological conditions. Our HS-AFM observations reveal that insertion initiating formation coils. Further, observed heterogeneous spatial distribution PRM-induced looping. With continuous addition, progresses through series folding transitions, forming coiled-like structures evolve into clockwise spirals, rod-shaped intermediates, and ultimately toroid-like nanostructures. Based on these observations, propose CARD (Coil-Assembly-Rod-Doughnut) model describe stepwise process toroid during condensation. findings underscore versatility capturing spatiotemporal interactions provide critical insights molecular driving compaction. This study advances our understanding architecture offers framework for future research organization, reproductive biology, nucleic acid therapeutics.

Language: Английский

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Strategies for the Viral Exploitation of Nuclear Pore Transport Pathways

Viruses, Journal Year: 2025, Volume and Issue: 17(2), P. 151 - 151

Published: Jan. 23, 2025

Viruses frequently exploit the host’s nucleocytoplasmic trafficking machinery to facilitate their replication and evade immune defenses. By encoding specialized proteins other components, they strategically target host nuclear transport receptors (NTRs) nucleoporins within spiderweb-like inner channel of pore complex (NPC), enabling efficient access nucleus. This review explores intricate mechanisms governing import export viral with a focus on interplay between factors determinants that are essential for these processes. Given pivotal role shuttling in life cycle, we also examine therapeutic strategies aimed at disrupting pathways. includes evaluating efficacy pharmacological inhibitors impairing assessing potential as antiviral treatments. Furthermore, emphasize need continued research develop targeted therapies leverage vulnerabilities trafficking. Emerging high-resolution techniques, such advanced imaging computational modeling, transforming our understanding dynamic interactions viruses NPC. These cutting-edge tools driving progress identifying novel opportunities uncovering deeper insights into pathogenesis. highlights importance advancements paving way innovative strategies.

Language: Английский

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Zooming into Gene Activation: Estrogen Receptor α Dimerization and DNA Binding Visualized by High-Speed Atomic Force Microscopy

ACS Nano, Journal Year: 2025, Volume and Issue: unknown

Published: April 18, 2025

Estrogen receptor α (ERα) is pivotal in gene regulation, particularly estrogen-responsive cancers. However, the full-length molecular dynamic structure of ERα remains elusive. In this study, we employ high-speed atomic force microscopy (HS-AFM) to visualize interactions with estrogen response element (ERE) under both ligand-present and ligand-absent conditions. binds ERE even absence estrogen, although presence ligand significantly enhances binding precision stability. Our real-time, high-resolution HS-AFM imaging captures structural transitions from monomeric dimeric forms, elucidating mechanisms by which modulates DNA-binding specificity. Based on these findings, propose a ligand-induced dimerization (LID) model, wherein facilitates optimal loading onto DNA. These insights deepen our understanding hormone signaling cancer hold promise for development future therapeutic strategies targeting hormone-related malignancies.

Language: Английский

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Unraveling dynamics of nuclear pore and chromatin via HS-AFM

Anatomical Science International, Journal Year: 2025, Volume and Issue: unknown

Published: May 19, 2025

Abstract High-speed atomic force microscopy (HS-AFM) enables real-time visualization of biological processes with nanometer-level resolution. This review highlights how HS-AFM has been instrumental in uncovering the dynamic interplay between nuclear pore complexes (NPCs)—which regulate nucleocytoplasmic transport—and genome guardians, including DNA repair proteins and chromatin regulators. Structurally, NPCs resemble a multi-layered spider cobweb, serving as crucial molecular gatekeepers for maintaining cellular homeostasis, while guardians safeguard genomic integrity through organization. Through HS-AFM, researchers have gained unprecedented insights into NPC dynamics, revealing their adaptability during transport, reorganization, viral infection. It also elucidated interact NPCs, influencing organization at periphery regulating trafficking. These discoveries underscore critical role NPC-genome interactions stability, gene expression, broad implications diseases such cancer, infections, neurodegenerative disorders. In conclusion, transformed our ability to study landscape nanoscale, bridging gap structural biology functional genomics. By capturing dynamics chromatin, provides an essential tool unraveling mechanisms that govern transport regulation. Future advancements technology, higher temporal resolution, correlative imaging, AI-driven analysis, will further expand its potential biomedical research, paving way novel diagnostic therapeutic strategies.

Language: Английский

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A Preemptive Scan Speed Control Strategy Based on Topographic Data for Optimized Atomic Force Microscopy Imaging

Actuators, Journal Year: 2025, Volume and Issue: 14(6), P. 262 - 262

Published: May 26, 2025

Rapid advancement in the nanotechnology and semiconductor industries has driven demand for fast, precise measurement systems. Atomic force microscopy (AFM) is a standout metrology technique due to its high precision wide applicability. However, when operated at speeds, quality of AFM images often deteriorates, especially areas where sharp topographic features are present. This occurs because feedback speed Z-scanner cannot keep up with sample height changes during raster scanning. study presents simple variable scan control strategy improving imaging while maintaining image obtained low speeds. The proposed aims leverage similarity profiles between successive lines. information collected from previous line used assess surface complexity adjust following scan. system this algorithm was found reduce time needed one by over 50% compared fixed-speed scanning similar level accuracy. calculated mean square errors (MSEs) show that combination adjustments preemptive topography prediction successfully allowed us suppress potential oscillations adjustment process, thereby enhancing stability adaptive as well.

Language: Английский

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Nanoimaging of SARS-CoV-2 viral invasion toward the nucleus and genome

Cell Reports Physical Science, Journal Year: 2024, Volume and Issue: 5(9), P. 102111 - 102111

Published: July 24, 2024

The SARS-CoV-2 virus, responsible for the COVID-19 pandemic, has been linked to significant worldwide illness and death. Examining ultrastructure nanomechanical characteristics of viruses, from a physical standpoint, aids in categorizing their mechanical attributes, providing valuable information novel treatment approaches pinpointing susceptible regions that can guide precise medical interventions. This review presents structural virus particles, focusing on interaction with cells effects nuclear pore transit epigenetic modifications. We present latest progress utilizing high-speed atomic force microscope nanoscale observation its constituents. viruses utilize several components interact host's transport receptors nucleoporins complex influence genome modality. In this review, we also provide an updated summary how parts system these interactions change chromatin.

Language: Английский

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