Search Results (155)

Fluorescence correlation spectroscopy (FCS) measures spontaneous temporal fluorescence fluctuations within a femtolitre observation volume to extract, with single-molecule sensitivity, the concentration, mobility, oligomeric state, and interactions of proteins in living cells. This review traces how FCS and its spatiotemporal derivatives, implemented on different types of optical microscopes, have mapped protein trafficking across the three physically distinct environments a protein must navigate from the cell surface to its genomic targets. At the plasma membrane, FCS resolves nanodomains with millisecond confinement times and distinguishes cytoskeletal corralling from cholesterol-dependent trapping through the FCS diffusion law, revealing how receptor signalling is organised below the diffraction limit. In the cytoplasm, FCS quantifies how macromolecular crowding slows protein diffusion by a factor of 3–4 relative to water, drives anomalous sub-diffusion, and coexists with directed transport, while resolving the markedly slower dynamics of liquid–liquid phase-separated condensates. In the nucleus, FCS-derived pair correlation and brightness analyses show that chromatin acts as a size-selective filter where an inert protein dimer can take more than 10-fold longer than its monomer to traverse the same nuclear distance, and that this oligomeric-state-dependent gating governs the genomic access of transcription factors. Across all three compartments, a protein’s diffusive behaviour is not incidental to its function but is itself a direct readout of the physical organisation of its environment, establishing FCS as a uniquely quantitative bridge between molecular dynamics and cellular decision-making. Full article

Proteasome dysfunction has been implicated in the pathogenesis of many human diseases, and the proteasome system has emerged as a major therapeutic target for cancer treatment. Besides proteasome activity, the dynamics of proteasome localization provide another tier of mechanism for regulating proteasome function and cellular protein homeostasis. While proteasomes are highly enriched in the nucleus, they can dynamically reshuffle across cellular compartments in response to metabolic cues. This localization shift is coupled with autophagy as a major mechanism for cell survival under stress. Under certain metabolic stress, proteasomes can reorganize into distinct membraneless condensates, termed proteasome condensates. While the current understanding of the biological significance of proteasome condensates is limited, they may provide proteolytic control to meet metabolic needs under stress. This review highlights how distinct metabolic cues, such as carbon starvation, amino acid deficiency, and senescence, regulate divergent proteasome fates including proteasome subcellular translocation, autophagic degradation of proteasomes, and proteasome condensate formation. A better understanding of proteasome regulation in a spatiotemporal manner will help identify new therapeutic targets for diseases affected by proteasome dysfunction. Full article

The highly pathogenic H5 subtype avian influenza viruses (AIVs) pose persistent threats to the poultry industry and public health owing to their high lethality and pandemic potential. Migratory wild birds play a pivotal role in the global dissemination and genetic reassortment of the virus, serving as both natural reservoirs and long-distance vectors that drive its spatiotemporal spread. However, the extent and evolutionary drivers of antigenic divergence among H5 AIVs circulating in wild birds in East China remain poorly understood. Here, we aim to characterize the evolutionary dynamics and antigenic divergence of H5 AIVs isolated from wild birds in East China between 2013 and 2022. Whole-genome sequencing and phylogenetic analysis revealed that the isolates belonged to multiple clades, including 2.3.2.1 and 2.3.4.4, and encompassed the H5N1, H5N6, and H5N8 subtypes. Key amino acid site analysis showed that the glycosylation site patterns in the HA and NA proteins varied among clades, with some strains exhibiting gains or losses of glycosylation sites, while certain strains had acquired mutations associated with mammalian adaptation. Cross-hemagglutination inhibition (HI) assays combined with antigenic cartography demonstrated that the majority of the isolates were antigenically well-matched with the contemporaneous vaccine strains used in China, indicating that these vaccines effectively covered the predominant circulating antigenic variants at the time. Nevertheless, potential antigenic mismatches were still observed between some circulating strains and these vaccine strains. These findings suggest that wild birds in East China may contribute to the regional movement and diversification of H5 AIVs, highlighting the value of sustained surveillance for early warning and vaccine strain evaluation. Full article

c-Jun N-terminal kinase (JNK) is a highly conserved, stress-activated protein kinase that plays key roles in cellular development and cell fate. An extensive study over more than 30 years has identified roughly 100 substrates for this kinase including the transcription factor c-Jun and other cell fate effectors. These studies have shown that JNK activation is tightly regulated both spatially through recruitment to subcellular locations and temporally through specific activation dynamics. Ultimately, these two regulatory mechanisms contribute to JNK’s function as a major driver of cell fate and function. A growing field of live-cell imaging, biosensor development, and other novel approaches to manipulate kinase function and localization are now providing novel insights into JNK function at the single-cell level. The purpose of this review is to illustrate our historical understanding of the spatiotemporal functions of JNK signaling within cells as well as emerging studies within the field. Ultimately, we aim to provide insight into remaining knowledge gaps within the field and how emerging technologies may help address these questions. Full article

Direct cellular reprogramming, the conversion of one somatic cell type into another, represents a remarkable advancement in regenerative medicine. Its potential to transform fibrotic tissue into functional parenchyma underscores its therapeutic promise. However, several critical challenges remain unresolved, including limited reprogramming efficiency, the long-term functional stability of converted cells, their integration within pre-existing cellular circuits, and safety concerns related to transgene integration and immunological responses to reprogramming-based viral vectors. Approaches based on the exogenous administration of recombinant proteins and miRNAs have also emerged, though these rely on factors that are naturally prone to exhaustion and degradation, potentially restricting their efficacy. This review is divided into three main sections. The first part addresses direct cellular reprogramming in the context of other cell-based applications, outlining its main applications and current biological limitations. The second part examines how different biomaterials, ranging from hydrogel scaffolds to nanoparticles, can modulate direct cellular reprogramming by providing mechanical and topographical cues and by enabling tighter control over the concentration and spatiotemporal dynamics of reprogramming factors and viral vectors. The third part discusses key findings in biomaterial-assisted reprogramming strategies, highlighting emerging opportunities for clinically translatable approaches. The convergence of regenerative biology and biomaterials science may ultimately generate advanced gel-based and hybrid cellular reprogramming platforms for in vitro testing and, in situ applications, for promoting cell fate stabilization and facilitating the regeneration of damaged tissues and organs. Full article

Leaf architecture critically impacts crop yield. The Auxin Response Factor (ARF) family is a key regulator of leaf development, yet remains uncharacterized in the important legume crop chickpea (Cicer arietinum L.), which bears pinnate compound leaves. Here, we performed a genome-wide identification and analysis of ARF genes in chickpea. We identified 33 CaARF genes and resolved their phylogenetic structure through comparison with six other key dicot species. The analysis revealed a deeply conserved core set of ARF proteins across species, all sharing the N-terminal DNA-binding domain (DBD), with most the C-terminal PB1 domain, connected by a middle region (MR). We also uncovered instances of lineage-specific expansion, e.g., a chickpea-specific ARF clade, which is characterized by the absence of the C-terminal PB1 domain. Expression profiling using public transcriptome data and qRT-PCR revealed distinct spatiotemporal expression patterns for CaARF genes across tissues and during compound leaf development. Detailed in situ hybridization analysis for selected candidates, chosen based on phylogenetic proximity to known leaf-development-related ARFs in other species, localized their transcripts to specific regions within compound leaf primordia. Focusing on CaARF5, the closest ortholog of Arabidopsis MONOPTEROS/ARF5, we confirmed its nuclear localization and dynamic expression during chickpea leaf development. Functional complementation assays demonstrated that CaARF5 could restore developmental defects in the Arabidopsis mp mutant. Our study establishes an evolutionary and molecular framework for the chickpea ARF family, highlighting conserved features and species-specific innovations, and provides essential resources for future research on auxin-mediated leaf development and ARF-targeted legume breeding. Full article

RNA N⁶-methyladenosine (m⁶A) is a prevalent epitranscriptomic modification that governs plant growth, development, and environmental adaptation. This review synthesizes recent advances in understanding the molecular mechanisms and biological functions of m⁶A in plants. The m⁶A landscape is dynamically regulated by methyltransferases (writers), demethylases (erasers), and m⁶A-binding proteins (readers), which collectively influence mRNA stability, translation efficiency, alternative polyadenylation (APA), and chromatin crosstalk. Functionally, m⁶A integrates diverse developmental processes—including embryogenesis, organogenesis, flowering, fruit ripening, and leaf senescence—with abiotic stress responses such as salt, drought, cold, and heat. Notably, m⁶A modification exhibits remarkable species-, cultivar-, and tissue-specific plasticity, enabling precise spatiotemporal gene regulation. Recent breakthroughs have revealed bidirectional crosstalk between m⁶A and histone modifications, forming a multi-layered regulatory network, while emerging concepts including phase separation, RNA structure dynamics, and stress memory further expand the functional repertoire of m⁶A. Despite significant progress, plant epitranscriptomics remains mechanistically underexplored, with critical gaps persisting in our understanding of translation initiation mechanisms, upstream regulatory signals controlling writers/erasers activities, and the functional significance of individual m⁶A sites. This review provided systematic insights into the complexity and specificity of m⁶A regulation in plants, offering a theoretical foundation for future efforts to decipher and ultimately manipulate this epitranscriptional layer for crop improvement. Full article

Background: Modern oncology views immune system dysfunction as a key factor in carcinogenesis. The induction of immunogenic cell death (ICD), a form of regulated cell death capable of activating adaptive immunity, represents a promising therapeutic strategy. Damage-associated molecular patterns (DAMPs) play a central role in this process. This review aims to summarize current knowledge of DAMPs, their release mechanisms during ICD, their classification, and their prognostic and therapeutic significance in antitumor immunity. Methods: We systematically reviewed and synthesized literature published in Pubmed and Google Scholar on ICD and DAMPs, focusing on distinct forms of DAMPs which were categorized based on recognition mechanisms (five classes) and cellular origin (extracellular, mitochondrial, nuclear, and cytosolic). Key molecules, their receptors, downstream signaling pathways, and clinical associations were analyzed. Results: The spatiotemporally coordinated release of the pattern of DAMPs promotes dendritic cell maturation, antigen presentation, activation of cytotoxic T lymphocytes, and elimination of tumor cells. DAMPs can exhibit a dual role: they are able to induce sterile inflammation essential for antitumor immunity, but may also contribute to metastasis and chronic inflammation. Among all DAMPs, high-mobility group box 1 (HMGB1, a nuclear DAMP) and calreticulin (CRT, a cytosolic protein) demonstrate the greatest prognostic value. Other DAMPs (e.g., extracellular matrix components, uric acid) act as signal amplifiers during various forms of cell death. Conclusions: Understanding the spatiotemporal dynamics of DAMP release is critical for activating immune responses against malignant cells. Monitoring DAMPs may improve patient stratification, predict therapeutic responses, and enable personalized immunotherapeutic strategies. Further investigation of ICD mechanisms and DAMP release represents a fundamental basis for developing novel anticancer therapies. Full article

Nitrogen metabolism is fundamental to all organisms, with ammonium transporters (Amt) playing a pivotal role in transmembrane ammonium transport. Brachiopods, as “living fossils”, offer unique insights into the evolutionary adaptation of marine invertebrates. This study systematically identified and characterized the Amt gene family in the brachiopod Lingula anatina. Five canonical Amt genes were identified, with nonrandom chromosomal distribution and evidence of lineage-specific duplication events. Phylogenetic analysis revealed that these Amt proteins cluster into three well-supported clades, showing closer affinity to Caenorhabditis elegans, reflecting conserved ancestral features predating protostome radiation. Structural predictions showed that LanAmtA and LanAmtB retain the canonical 11-transmembrane helix (TMH) topology with an extracellular N-terminus, while LanAmtC features a unique 12-TMH architecture with an intracellular N-terminus, resembling certain vertebrate Amt-related proteins. Critical functional residues involved in ammonium selectivity and transport were preserved across all paralogs. Expression profiling revealed non-redundant spatiotemporal patterns: LanAmtA1 and LanAmtB2 dominate early embryogenesis, with LanAmtB2 becoming the major isoform in late developmental stages; LanAmtC exhibits constitutive high expression across adult tissues. Collectively, our findings demonstrate that the L. anatina Amt family expanded via local duplications, evolving structural stability, regulatory diversity, and functional specificity. This study provides a comprehensive molecular framework for understanding the evolutionary adaptation of nitrogen-handling mechanisms in basal lophotrochozoans and sheds light on how intertidal organisms cope with dynamic environmental conditions. Full article

Astrocytes undergo pronounced reactivity during traumatic brain injury (TBI); however, the temporal dynamics of this response and the signaling mechanisms regulating astrocyte proliferation remain incompletely defined. In this study, we characterized the spatiotemporal profile of astrocyte reactivity and proliferation in the hippocampus during TBI and investigated the involvement of mammalian target of rapamycin complex 1 (mTORC1) signaling in these processes. Using a mouse model of TBI, we found that injury triggered a rapid astrocytic response in the hippocampus, characterized by increased glial fibrillary acidic protein (GFAP) expression and morphological hypertrophy as early as 4 h post-injury. Astrocyte proliferation emerged subsequently, peaked during the acute phase (48 and 72 h), and declined to baseline levels at 7 days post-trauma, indicating a transient proliferative response during TBI. Concurrently, mTORC1 signaling was robustly activated in reactive astrocytes in the hippocampus and was specifically associated with proliferative reactive astrocytes during injury. Pharmacological inhibition of mTORC1 signaling with rapamycin significantly reduced reactive astrocyte proliferation during TBI without altering astrocytic hypertrophy. Together, these findings demonstrate that TBI induces a rapid but transient astrocyte activation and proliferation response in the hippocampus and that mTORC1 activation is required for the proliferation, but not the hypertrophic activation, of reactive astrocytes during traumatic brain injury. Full article

Background/Objectives: The coordination of flowering and dormancy represents a fundamental adaptive strategy for perennial plant survival. Recent studies have demonstrated that AIL1 homologs act as integrators of short-day signals, playing pivotal roles in seasonal growth cessation and dormancy regulation in poplar. Despite these advances, whether AIL1-mediated regulatory mechanisms are conserved in Platanus acerifolia, a species with distinct phylogenetic and ecological characteristics, remains an open question. Methods: In this study, two AIL1 homologs, PaAIL1a and PaAIL1b, were isolated from P. acerifolia. Their biological functions were systematically investigated through sequence analysis, spatiotemporal expression profiling, environmental stress treatments, yeast one-hybrid assays, and luciferase (LUC) transient expression assays. Results: PaAIL1s (PaAIL1a and PaAIL1b) exhibited ubiquitous expression across diverse tissues and organs, functioning as mediators of photoperiod and temperature signaling to orchestrate bud dormancy regulation. Interaction and activation assays placed these factors downstream of PaFUL proteins. While displaying functional redundancy in dormancy induction and floral development, the paralogs underwent distinct subfunctionalization: PaAIL1a specifically regulated flowering initiation and dormancy release, whereas PaAIL1b acted predominantly during the ecodormancy phase. Conclusions: These results establish PaAIL1 genes as critical integrators of environmental signals and developmental programs, governing seasonal growth dynamics in this species. Full article

Traumatic brain injury (TBI) initiates a cascade of molecular and cellular reactions leading to long-term disturbances of neuronal and glial homeostasis. One of the key mechanisms of secondary injury is a pathological increase in intracellular Ca²⁺ concentration. Parvalbumin (PV) plays an important role in the regulation of Ca²⁺ homeostasis in neurons. In turn, connexin 43 (Cx43) is the principal protein of astrocytic gap junctions (GJs), which ensure neuroglial communication. The spatiotemporal changes in these proteins and the mechanisms of their interaction after TBI remain insufficiently studied. In the present study, a comprehensive analysis of the expression, localization, and spatial organization of PV and Cx43 in the cerebral cortex following TBI was performed. In intact tissue, PV was localized predominantly in neurons, whereas Cx43 formed typical punctate structures of astrocytic GJs. Twenty-four hours after TBI, a sharp activation of PV with pronounced nuclear translocation was observed against the background of a catastrophic decrease in Cx43 expression, accompanied by a reduction in the number of NeuN⁺ neurons and signs of apoptosis. However, after 7 days, a mirror-opposite effect was detected, characterized by decreased PV expression and increased Cx43 levels with its aggregation into cluster-like structures, as well as partial restoration of NeuN immunoreactivity. In addition, molecular dynamics simulations demonstrated that the stability of the PV–Cx43 complex is determined by the presence of Ca²⁺ and physiological pH, whereas acidosis and Ca²⁺ overload destabilize their interaction. Taken together, these results reveal a phase-dependent mirror-opposite pattern of PV and Cx43 expression and localization and emphasize the key role of Ca²⁺- and pH-dependent neuroglial interactions in TBI. Full article

T-cell intracellular antigen 1 (TIA1) is a multifunctional RNA-binding protein (RBP) belonging to the RNA recognition motif (RRM) family. Under steady-state conditions, it is predominantly localized in the nucleus and highly expressed in the nervous system, where it regulates neuronal and glial functions. TIA1 modulates mRNA splicing, stability, and translation and promotes stress granule (SG) assembly under cellular stress. Recent studies indicate that the spatiotemporal dynamics of TIA1 in neurodegenerative contexts influence disease progression by regulating inflammatory responses, apoptosis, and related pathways. This review discusses the molecular structure and functions of TIA1, focusing on its expression in neurons and glia, as well as its implications in neurodegenerative disorders and stroke. The findings highlight TIA1 as a promising target for novel neuroprotective therapeutic strategies. Full article

Protein–protein interactions (PPIs) are fundamental regulators of cellular function and disease. Systematic mapping of the interactome is essential for identifying therapeutic targets and advancing drug design, a pursuit that has driven significant innovation to capture the spatiotemporal regulation of PPIs in vivo. This review summarizes this methodological revolution. We outline foundational, first-generation techniques—yeast two-hybrid and co-immunoprecipitation—which established frameworks for binary interaction mapping and static network generation, especially when integrated with mass spectrometry. The discussion then pivots to second-generation methods, including proximity-dependent labeling and advanced imaging, which enable the capture of PPIs within their native, dynamic cellular contexts. We provide a comparative analysis of these techniques, detailing their principles, strengths, and limitations. The review concludes with a practical framework for method selection and a perspective on emerging frontiers—such as spatial proteomics and single-cell interactomics—that are poised to further decode the evolving interactome. This concise overview serves as a strategic guide for specialists adopting new techniques and a broader audience integrating network-level data into their research. Full article

Inherited retinal degenerations, such as retinitis pigmentosa, are a leading cause of irreversible vision loss, yet broadly effective treatments remain elusive. Impaired cellular waste clearance via autophagy–lysosomal pathways have been implicated in photoreceptor death, but the spatiotemporal dynamics of these processes during degeneration remain poorly understood. Using the rd10 mouse model of retinitis pigmentosa, we characterised autophagy–lysosomal dysfunction at key stages of photoreceptor degeneration (postnatal day P17, P22, P35) through super-resolution imaging of RFP-EGFP-LC3 reporter mice, Western blot, and bulk RNA sequencing. Autophagosome and autolysosome numbers were significantly elevated across all photoreceptor compartments (inner/outer segments, outer nuclear layer, outer plexiform layer) at P17, prior to significant photoreceptor nuclei loss. Autophagosome and autolysosome size progressively increased from P22 onwards, suggesting accumulation of unprocessed intracellular waste. Molecular analyses revealed downregulation of mTOR protein, upregulation of autophagy-related genes, and increased lysosomal processes from P17. These histological and molecular findings are consistent with early autophagy induction followed by overwhelmed degradative capacity. Our findings identify autophagy–lysosomal change as an early event in photoreceptor loss in the rd10 model, revealing a critical therapeutic window for mutation-independent interventions targeting cellular clearance pathways in inherited retinal degenerations. Full article