The dynamic properties of biomolecules are important to the biogenesis of essential building blocks, the maintenance of biological homeostasis, and the cellular responses to external stimuli critical for survival. Structural studies define biomolecular functions, and their dynamic behavior provides mechanistic insights into these processes. Real-time tracing of dynamic biomolecular structural changes and interactions is highly desirable. Current methodologies such as cryo-EM and X-ray crystallography could provide detailed structural information; however, the images are rather static. Insufficient temporal resolution precludes the observation of crucial transient intermediates. Furthermore, sample preparation and non-physiological imaging environments can compromise the native state of biomolecules. On the other hand, other tools, such as FRET and NMR, could detect dynamic changes of targets at high temporal resolution but lack real-time observation. Atomic force microscopy (AFM) enables nanoimaging and biophysical characterization of biomolecules but is limited by slow scanning speeds and strong tapping forces. Later, high-speed AFM (HS-AFM) with high spatiotemporal resolution and gentle tapping forces, emerged as an ideal nanoimaging approach for studying the functions of delicate biological samples. Building on these developments, we and others have contributed to expand HS-AFM toward biomedical applications, including the direct visualization of disease-relevant organelles and nanostructures under near-physiological conditions. In this review, we examine HS-AFM applications in biomedical science, emphasizing real-time nanoimaging of structural dynamics across biological systems relevant to infectious diseases, infertility, cancer, and neurodegeneration. We also critically discuss the technical limitations of HS-AFM and mitigation strategies.

Abstract Fulvestrant is a selective estrogen receptor downregulator (SERD) that antagonizes estrogen receptor α (ERα) and also promotes receptor degradation. However, how it affects full-length ERα dynamics at the single-molecule level remains unclear. Here, we used high-speed atomic force microscopy (HS-AFM) to directly visualize the dynamic behavior of full-length ERα under fulvestrant-bound, ligand-free, and other ligand-bound conditions. Among the four conditions, fulvestrant-bound ERα alone showed a reduced apparent molecular volume and increased surface mobility, behaving distinctly from the ligand-free, E2-bound, and 4-OHT-bound states. These results indicate that fulvestrant not only drives ERα inactivation and degradation but also is associated with changes in the observed molecular morphology and dynamics of full-length ERα. Our study provides direct single-molecule observation of altered molecular behavior under fulvestrant-bound conditions, which we interpret as reflecting changes in the conformational ensemble of full-length ERα.

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

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

Nanobiomedicine promises to revolutionize life quality and expectancy of patients with cognitive impairment and cancer malignancies, via unraveling key molecular processes related to their onset useful as biomarkers of disease to develop and improve the efficacy of therapies. However, it is still a challenge understanding and identifying these molecular mechanisms as biomarkers of disease, because of their high-level of polymorphism and nanoscale dimensions. Here, it provides a review work linking the potential and capabilities of atomic force microscopy (AFM) technologies in unraveling beyond imaging the common and hidden properties of transient and nanosized molecular processes in cancer and neurodegeneration. This study highlights the most prominent operational modes of AFM to achieve morphological, mechanical, and chemical characterization of the molecular processes leading to these diseases. Finally, it outlines the advantages of AFM compared with other techniques to guide newcomers and stakeholders toward potential future avenues opened by AFM methods in nanobiomedicine.

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

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

Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.

The size of the nuclear pore should, in principle, prevent HIV-1 entry. However, HIV-1 capsid is able to gain nuclear pore entry. In a recent issue of Nature, Fu et al. and Dickson et al. demonstrate that the HIV-1 capsid mimics the nuclear transport protein karyopherins to access host nuclei.

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

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, FtsZ—a prokaryotic homologue of the eukaryotic protein tubulin—polymerizes into treadmilling filaments that further organize into a cytoskeletal ring. In vitro, FtsZ filaments can form dynamic chiral assemblies. However, how the active and passive properties of individual filaments relate to these large-scale self-organized structures remains poorly understood. Here we connect single-filament properties with the mesoscopic scale by combining minimal active matter simulations and biochemical reconstitution experiments. We show that the density and flexibility of active chiral filaments define their global order. At intermediate densities, curved, flexible filaments organize into chiral rings and polar bands. An effectively nematic organization dominates for high densities and for straight, mutant filaments with increased rigidity. Our predicted phase diagram quantitatively captures these features, demonstrating how the flexibility, density and chirality of the active filaments affect their collective behaviour. Our findings shed light on the fundamental properties of active chiral matter and explain how treadmilling FtsZ filaments organize during bacterial cell division.

Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb–spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.

Biomolecular interactions underpin most processes inside the cell. Hence, a precise and quantitative understanding of molecular association and dissociation events is crucial, not only from a fundamental perspective, but also for the rational design of biomolecular platforms for state-of-the-art biomedical and industrial applications. In this context, atomic force microscopy (AFM) appears as an invaluable experimental technique, allowing the measurement of the mechanical strength of biomolecular complexes to provide a quantitative characterization of their interaction properties from a single molecule perspective. In the present review, the most recent methodological advances in this field are presented with special focus on bioconjugation, immobilization and AFM tip functionalization, dynamic force spectroscopy measurements, molecular recognition imaging and theoretical modeling. We expect this work to significantly aid in grasping the principles of AFM-based force spectroscopy (AFM-FS) technique and provide the necessary tools to acquaint the type of data that can be achieved from this type of experiments. Furthermore, a critical assessment is done with other nanotechnology techniques to better visualize the future prospects of AFM-FS.

Nuclear pore complexes (NPCs) are the central apparatus of nucleocytoplasmic transport. Disease-specific alterations of NPCs contribute to the pathogenesis of many cancers; however, the roles of NPCs in glioblastoma (GBM) are unknown. In this study, we report genomic amplification of NUP107, a component of NPCs, in GBM and show that NUP107 is overexpressed simultaneously with MDM2, a critical E3 ligase that mediates p53 degradation. Depletion of NUP107 inhibits the growth of GBM cell lines through p53 protein stabilization. Mechanistically, NPCs establish a p53 degradation platform via an export pathway coupled with 26S proteasome tethering. NUP107 is the keystone for NPC assembly; the loss of NUP107 affects the integrity of the NPC structure, and thus the proportion of 26S proteasome in the vicinity of nuclear pores significantly decreases. Together, our findings establish roles of NPCs in transport surveillance and provide insights into p53 inactivation in GBM.

Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.

Open reading frame 6 (ORF6), the accessory protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that suppresses host type-I interferon signaling, possesses amyloidogenic sequences. ORF6 amyloidogenic peptides self-assemble to produce cytotoxic amyloid fibrils. Currently, the molecular properties of the ORF6 remain elusive. Here, we investigate the structural dynamics of the full-length ORF6 protein in a near-physiological environment using high-speed atomic force microscopy. ORF6 oligomers were ellipsoidal and readily assembled into ORF6 protofilaments in either a circular or a linear pattern. The formation of ORF6 protofilaments was enhanced at higher temperatures or on a lipid substrate. ORF6 filaments were sensitive to aliphatic alcohols, urea, and SDS, indicating that the filaments were predominantly maintained by hydrophobic interactions. In summary, ORF6 self-assembly could be necessary to sequester host factors and causes collateral damage to cells via amyloid aggregates. Nanoscopic imaging unveiled the innate molecular behavior of ORF6 and provides insight into drug repurposing to treat amyloid-related coronavirus disease 2019 complications.

Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.