Living cells continuously consume energy to sustain their structure, mechanics, and function. Energy-dependent nanometer-scale mechanical fluctuations, called nanomotion, provide a label-free physical readout of this activity in individual cells. When interpreted as dynamic signatures of cellular activity, these fluctuations form the basis of single-cell mechanodynamics. In this review, we examine how such fluctuations can be measured and interpreted across biomolecular, organellar, and cellular scales. We discuss established mechanodynamic sensing modalities, including atomic force microscope (AFM) cantilever sensing and optical nanomotion detection (ONMD), together with complementary approaches and AFM nanoendoscopy, which provides direct access to intracellular mechanical properties and establishes an experimental foundation for future organelle-level mechanodynamic measurements. We consider how mechanodynamic readouts are being applied in antimicrobial susceptibility testing (AST), mechanobiology, functional phenotyping, disease physiology, and life detection. Collectively, these developments support single-cell mechanodynamics as an emerging framework for linking structure, mechanics, metabolism, perturbation responses, and cellular function.
Due to the widespread use of ionizing radiation in medicine, industry, and military technologies, its adverse impact on biological systems has become a major concern. Radiation-induced alterations in structural and mechanical properties serve as biomarkers of ionizing radiation exposure in biological systems. Atomic force microscopy (AFM) enables the nanoscale quantification of these properties. This review discusses recent advances in the investigation of radiobiological effects in biological systems (deoxyribonucleic acid, collagen, biological nanoparticles, viruses, and cells) using AFM. It also highlights key knowledge gaps that can be addressed using AFM to improve our understanding of the mechanisms underlying complications following various types of radiation exposure, as well as promising directions for the development of AFM methods in radiobiology and radiation medicine.
Single-particle cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling atomic-resolution structure determination of purified macromolecular assemblies. Recent advances in in situ single-particle cryo-EM have extended this capability to near-atomic structural analysis directly within native cellular environments. However, major challenges remain because many cellular targets are low in abundance, structurally heterogeneous, and difficult to detect. In parallel, cryo-electron tomography (cryo-ET) combined with subtomogram averaging enables in situ visualization of macromolecular assemblies while preserving their three-dimensional cellular context, but limited throughput and resolution have constrained high-resolution analysis of rare or heterogeneous complexes. In this review, we discuss recent advances in sample preparation, data acquisition, image processing, and high-resolution refinement that improve the throughput, sensitivity, and resolution of in situ structural biology. We further highlight how integrating in situ single-particle cryo-EM with cryo-ET bridges cellular visualization and near-atomic structure determination, providing a scalable framework for investigating dynamic macromolecular assemblies directly in their native cellular context.
Understanding the multifaceted nature of neurodegenerative disease (NDDs) remains a significant challenge due to the complex intracellular /intercellular phenomena. This review explores the latest findings about the biochemical stress markers within the complex landscape of NDDs, with a specific focus on novel 3D cell cultures.Recent research highlights the importance of structural complexity and cellular crosstalk which lead to responses resembling those observed in the brain. However, issues such as vascularization and shear stress (which affect experimental reproducibility) remain.Here we emphasize the role of biochemical stressors in cellular interactions and communication relevant to NDDs, highlighting the emerging opportunities coming from exosomes/extracellular vesicles and noncoding RNAs as biomarkers, alongside the usefulness of brain organoids in terms of translational relevance.By bridging the gap between molecular mechanisms and organ dysfunction, 3D models emerge as essential tools for the discovery of novel theragnostic biomarkers and for the development of targeted therapeutic strategies.
Single-molecule spectroscopy is emerging as a powerful approach for elucidating the molecular structure and dynamics of biomolecular condensates. Unlike ensemble methods, it can resolve heterogeneous conformations, dynamics, interactions, concentrations, and transport properties across a broad range of length- and timescales, even for minute amounts of biomolecules. Recent applications, especially fluorescence-based methods such as single-molecule Förster resonance energy transfer, fluorescence correlation spectroscopy (FCS), fluorescence anisotropy, and nanosecond FCS, have revealed how proteins and nucleic acids behave within dense phases, and how molecular-scale dynamics relate to mesoscopic properties and biological function. Combined with molecular simulations, these measurements yield mechanistic insight into condensate organization, dynamics, and aging. We highlight recent advances, key applications, and promising directions for probing the properties of condensates with single-molecule spectroscopy.
Biomolecular condensation has emerged as a central mechanism of cellular organization, regulating fundamental processes from transcription to stress responses. Its dysregulation - often involving transitions from dynamic condensates to more solid or aggregated states - has been linked to human disease and thus represents a growing therapeutic opportunity. Yet, despite substantial progress, precisely relating protein sequence to condensate behavior, function, and dysregulation remains a largely unresolved challenge. Recent advances in high-throughput approaches are beginning to address this gap. By combining large-scale mutagenesis to fitness, fluorescence- or imaging-based selections, and deep sequencing, these methods enable systematic interrogation of different types of protein self-assembly across vast sequence spaces. However, most of the currently available assays measure indirect readouts such as solubility, stability, or cellular fitness and differ in the way they capture different parameters of the self-assembly process. Here, we review emerging high-throughput strategies to study protein condensation and aggregation at scale, emphasizing what they truly measure, their limitations, and how the cross-talk among these complementary approaches can provide a more accurate and mechanistic mapping of sequence-to-assembly relationships.
To maintain homeostasis of the human organism, intercellular interactions must occur with a high degree of synchrony and coordination. Traditionally, the nervous and endocrine systems have been considered the primary controllers of internal organ function. Only recently, substantial evidence has emerged indicating that the immune system—beyond preserving the body’s structural and functional integrity through defense against external and internal threats—is also capable of regulating, coordinating, and synchronizing the activity of tissues, organs, and systems of organs. This review examines noncanonical functions of the immune system that support its consideration as a key coordinator of internal organ physiology. We describe the characteristics of immune-mediated communication and the multiple signaling pathways that enable effective synchronization in the absence of a pronounced hierarchical structure. Examples illustrating how immune cells and biochemical factors coordinate physiological processes are highlighted.
Protein aggregation is a hallmark in several neurodegenerative diseases, in which proteins assemble into structurally diverse misfolded states, ranging from amorphous aggregates to amyloid fibrils. Several aggregation-prone proteins can undergo phase separation to form biomolecular condensates, creating distinct chemical environments compared to the surrounding dilute phase. These environments dictate protein conformations, interaction networks, and free energy landscapes, thereby modulating aggregation pathways. Notably, condensates exert dual and context-dependent effects: they can promote aggregation by stabilizing misfolded intermediates and facilitating assembly, or they can suppress aggregation by buffering interactions and retaining proteins. Here, we summarize the current literature and describe biomolecular condensates as key regulators of protein misfolding and aggregation and highlight the importance of the local milieu in determining aggregation outcomes.
B-form DNA is a structural icon in biology. Noncanonical DNA conformations are increasingly recognized as essential drivers of genomic phenomena. These atypical structures, including hairpins, G-quadruplexes, and multistranded junctions, are often transient and highly dynamic, making them difficult to characterize using traditional methods. Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as an indispensable tool for resolving these rapid conformational changes and elucidating their role in human disease. Here, we discuss the sub-millisecond folding dynamics of single-stranded DNA, the mechanisms of protein-induced DNA compaction, and the pathogenic strand-slippage of tandem repeats associated with neurodegenerative diseases. Finally, we highlight the challenges of translating in vitro findings to live cells and the potential for fluorophore labels to perturb the delicate energy landscape.
Label-free optical biosensing combined with machine learning enables live-cell analysis with high spatial and temporal resolution and can improve cell-state classification, response profiling, and estimation of biomechanically relevant variables. This review organizes works into single-modal and multimodal workflows. In single-modal analysis, representation-oriented approaches improve signal quality and provide data reconstruction or calibration before modeling, whereas inference-oriented approaches map optical data to phenotypes, adhesion behavior, or other biologically relevant variables. These roles are examined across surface-enhanced Raman spectroscopy (SERS), surface plasmon resonance/resonant waveguide grating (SPR/RWG), and digital holographic microscopy (DHM). Multimodal workflows are grouped into reference-based calibration, in which an auxiliary modality supervises a primary platform, and joint multimodal inference, in which complementary readouts are fused to estimate cell state robustly.
Cryo-electron tomography (cryo-ET) has emerged as a transformative technique for visualizing the native ultrastructure of eukaryotic parasites, from proteins to cellular architecture. Recent technical advances in sample preparation, data collection, and computational analysis have enabled unprecedented insights into structural cell biology of medically important pathogens including Toxoplasma gondii, Plasmodium falciparum, Trypanosoma brucei and Trypanosoma cruzi, Cryptosporidium parvum, and Microsporidia. This review highlights the range of resolutions and cellular structures accessible by cryo-ET, and the kinds of biological insights that may be obtained, using eukaryotic parasites as case studies. Lower-resolution data (20-30 Å) provide structural information on organelles, whole-cells, and cell-cell interactions, while at the higher-resolution end, near-atomic structures can be resolved in situ using subtomogram averaging, typically for large, abundant particles such as ribosomes. Combined with orthogonal techniques, cryo-ET is a powerful tool for studying the structural cell biology of parasites.
Structural biology is no longer confined to isolated macromolecules. Correlative light and electron microscopy workflows at cryogenic temperatures have been developed and optimised in cultured cell systems, opening the door for cellular structural biology. The next frontier is expanding these approaches to primary cells, tissues, biopsies, and patient material. The complexity of these samples means each presents unique challenges. Here, we review recent advances that enable the study of primary cells and complex samples. These examples demonstrate the diversity in available workflows and indicate that there is still no 'one solution for all'.
The crowded intracellular milieu shapes the thermodynamics and kinetics of biochemical reactions. RNA, an abundant and structurally versatile polymer, contributes to this crowding by acting both as a physical agent that restricts diffusion and enhances excluded volume, and as a sequence- and structure-specific scaffold driving multivalent RNA-RNA and RNA-protein interactions. These properties position RNA as a biologically active determinant of intracellular organization, distinct from inert synthetic crowders and purely structural scaffolds. This minireview examines how RNA shapes the formation, composition, and material properties of biomolecular condensates, highlighting the molecular grammar encoded in RNA sequence, length and valency, structure, and chemical modifications. We discuss how concentration-dependent, biphasic effects of RNA on condensate assembly can tip the balance between functional compartmentalization and pathological liquid-to-solid transitions implicated in neurodegenerative disease and cancer. Finally, we outline challenges in defining RNA-specific thresholds and translating structural insights into therapeutic strategies for mitigating aberrant RNA-mediated crowding.
Polymeric (p) immunoglobulins (Igs) and their secretory (S) forms are critical to vertebrate immunity. The pIgs and SIgs comprise a molecularly diverse family of antibodies that contain multiple Ig monomers, up to one joining chain (JC), and up to one secretory component. A subset of pIgs function in circulation whereas SIgs populate mucosal barriers. The pIgs and SIgs exhibit unique functions compared to monomeric Igs, yet their underlying molecular structures remained largely elusive until 2020 when cryo-electron microscopy revealed SIgA and SIgM to be remarkably asymmetric antibody assemblies. More recent reports have uncovered IgM and IgA complexes with host receptors and pathogenic virulence factors, species-specific structural differences, and conformational relationships between pIgs' structural cores and antigen binding fragments. Together, these findings highlight pIg and SIg conformational asymmetry as a key feature and establish a foundation to advance our understanding of pIg and SIg structure-function relationships critical for understanding immunity.
Biomolecular condensates organize the intracellular space by linking molecular interactions to mesoscale properties. While ensemble-averaged measurements provide insights into phase behavior, they obscure the intrinsic heterogeneity and asynchronous evolution of individual assemblies. Single-condensate fluorescence techniques have emerged as a transformative toolset, providing high-resolution access to the dynamics, internal organization, and material states of individual droplets. This review highlights recent advances in quantitative imaging, focusing on how high-throughput landscape mapping, fluctuation spectroscopy, and time-resolved environment sensing enable the characterization of condensates as distinct physical entities. By bridging molecular-scale fluctuations and emergent material behaviors, single-condensate approaches provide a vital framework for understanding functional adaptation and the pathological transitions associated with condensate aging.
G protein-coupled receptors (GPCRs) are key regulators of cellular signaling and major drug targets. Although X-ray crystallography and cryo-electron microscopy have provided high-resolution receptor structures, these static snapshots capture only a fraction of the conformational states underlying GPCR function. Molecular dynamics (MD) simulations complement experimental structures by enabling the mapping of receptor conformational landscapes and their relationship to functional outcomes. Large-scale simulation resources such as GPCRmd allow systematic exploration and comparison of GPCR motions across receptors and ligands. MD studies reveal transient conformational states, cryptic binding pockets, lipid- and water-mediated interactions, and allosteric communication networks that shape ligand recognition and the impact of signaling bias.
Biomolecules' conformational landscapes span multiple timescales and structural populations, challenging traditional structural biology methods that predominantly capture static states. Over the past three decades, Förster resonance energy transfer (FRET) has evolved from a qualitative molecular ruler into a quantitative framework for integrative structural biology. Advances in single-molecule FRET (smFRET), fluorophore modeling, molecular simulations, and experimental standardization have transformed FRET into a powerful approach for reconstructing dynamic structural ensembles. We didactically reviewed the various developments in FRET-guided structural modeling and discussed the next frontier of FRET-guided structural biology, where machine learning, AlphaFold-based structure prediction, and super-resolution microscopy are converging with smFRET to validate, refine, and visualize dynamic conformational ensembles directly in their native cellular context. Together, these advances position FRET-guided structural modeling as a central experimental framework for the next generation of dynamic structural biology.
RNA molecules populate complex structural landscapes that are continuously reshaped throughout the RNA lifecycle by equilibrium and non-equilibrium processes. Resolving these structural landscapes represents a central challenge in RNA biochemistry. We review recent advances in RNA chemical probing, sequencing, and computational deconvolution technologies that are revolutionizing our ability to measure the complexities of RNA folding in cells and the deep involvement of these complexities in RNA functional mechanisms. We highlight new methods for deconvolving structural ensembles, distinguishing isoform-specific architectures using long-read sequencing, capturing co-transcriptional folding intermediates in vivo, and measuring higher-order RNA structures while also underscoring remaining challenges. We conclude by outlining future directions in probe development, sequencing, and integrative modeling, and discuss how resolving RNA structural ensembles with increasingly high resolution will likely reveal new therapeutic opportunities to selectively target functional RNA heterogeneity.
Over the past decades, bacterial cell biology has revealed that bacteria possess complex subcellular structures and regulatory mechanisms once thought to be unique to eukaryotes, establishing them as powerful model systems for studying fundamental cellular principles. However, their small size makes bacterial subcellular structures difficult to image with optical microscopy. In-cell cryo-electron microscopy (cryo-EM) is a cutting-edge technique that enables high-resolution visualization of macromolecular complexes and the networks they form in their native context. In this review, we highlight the unique advantages of both cryo-electron tomography (cryo-ET) and in-cell single-particle analysis (in-cell SPA) in bacterial cell biology and summarize key advances from the past two years, including structural studies of macromolecular complexes and investigations of dynamic cellular processes, underscoring their growing importance and potential as a scaffold for multidisciplinary approaches.
