Cell structure and function最新文献

In higher eukaryotic cells, genomic DNA is packaged into dynamic chromatin domains whose physical behavior is coupled to DNA transactions such as transcription and DNA repair. Although chromatin organization is altered in cancer, how oncogenic signals modulate chromatin dynamics over time remains unclear. To address this issue, we established a doxycycline-inducible carcinogenesis model in hTERT-immortalized human RPE-1 cells expressing HPV16 E6/E7, MYC(T58A), and KRAS(G12V) (EMR cells) and investigated chromatin behavior during oncogene-driven transformation. Upon induction, EMR cells displayed accelerated proliferation, loss of contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice. Using time-resolved single-nucleosome imaging to track local chromatin dynamics over days to weeks of oncogene induction, we found that local nucleosome motion was unchanged at 1-3 days, significantly increased at 5-7 days, and returned to parental levels by 4 weeks, despite sustained oncogene expression and stable malignant growth. To explore the basis of this transient increase, we quantified DNA damage, histone marks, and transcription. γH2AX foci were elevated in EMR cells, but ATM/ATR inhibition had only minor effects on local chromatin motion, indicating that the DNA damage response is not the principal driver. By contrast, H3/H4 acetylation and nascent RNA synthesis were upregulated specifically during the early window of enhanced dynamics, whereas the heterochromatin mark H3K9me3 decreased, consistent with transient chromatin loosening associated with increased transcription. These findings reveal a biphasic change in local chromatin dynamics during human oncogene-driven transformation and provide a physical and temporal framework for understanding how oncogenic pathways reorganize chromatin.Key words: cancer, oncogenesis, single-nucleosome imaging, chromatin dynamics.

Multiplex live imaging enables simultaneous visualization of multiple signaling pathways in living cells, offering real-time insights into complex cellular networks. This methodology is essential in research fields such as cancer biology, where signaling activities exhibit heterogeneity, feedback regulation, crosstalk, and dynamic changes during pathological progression and the acquisition of therapeutic resistance. While conventional biochemical assays advanced our understanding of signaling signatures through static or population-level analyses, they lack the temporal resolution required to capture dynamic events at single-cell resolution.Recent methodological innovations have expanded multiplex live imaging through several strategies. Spectral multiplexing exploits broadened fluorescent protein palettes and optimized biosensor combinations, sometimes coupled with intracellular multiplexing methods that distinguish signals by targeting fluorescence to subcellular compartments. Intercellular multiplexing distributes reporters across cell populations, and temporal multiplexing leverages optical switching to separate signals over time. Additional modalities such as fluorescence anisotropy, fluorescence lifetime, and Raman imaging provide orthogonal readouts. Furthermore, computational approaches reinforce multiplex strategies by improved spectral unmixing, often complemented by deep learning-based algorithms. Collectively, these advances enable simultaneous tracking of multiple signaling pathways within single cells, revealing how diverse inputs are integrated into cellular responses.Here we review current strategies for multiplex live imaging, especially highlighting its applications to cancer signaling networks. Progress in fluorescent biosensor development, imaging technologies, and computational analysis will further promote the exploration of dynamic cellular regulations in basic research and translational medicine.Key words: multiplex live imaging, fluorescent biosensors, signal dynamics, image analysis, cancer heterogeneity.

Interleukin-1 receptor type 2 (IL-1R2) functions as a decoy receptor that suppresses IL-1-induced inflammatory signaling. Both membrane-bound IL-1R2 (WT IL-1R2) and its soluble form (sIL-1R2) bind interleukin-1α (IL-1α) at the cell surface or in the extracellular space, thereby inhibiting downstream signaling. However, the anti-inflammatory role of IL-1R2 varies depending on the cellular context and receptor structure. In this study, we generated two IL-1R2 deletion mutants-ΔTM, lacking the transmembrane domain, and ΔTMCP, lacking both the transmembrane and cytoplasmic domains-and compared their functions with those of WT IL-1R2 in HeLa cells. Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay were used to assess receptor expression, IL-1α binding, and IL-1β-induced interleukin-8 (IL-8) production, respectively. Both ΔTM and ΔTMCP were secreted more efficiently than WT IL-1R2. WT IL-1R2 exhibited weak intracellular interaction with IL-1α, whereas the deletion mutants showed minimal binding. WT IL-1R2 most effectively suppressed IL-1α extracellular release; however, ΔTM and ΔTMCP also reduced secretion. Notably, both deletion mutants suppressed IL-1β-induced IL-8 production more effectively than WT IL-1R2, indicating enhanced extracellular decoy activity. These findings demonstrate that structural modifications of IL-1R2 influence its function as a decoy receptor, and the enhanced inhibitory effects of the deletion mutants on IL-1 signaling provide new insight into the anti-inflammatory potential of soluble IL-1R2 in non-immune cells.Key words: Interleukin-1, Interleukin-1 receptor type 2, decoy receptor, transmembrane, soluble interleukin-1 receptor type 2.

Podocytes are terminally differentiated renal epithelial cells that play a crucial role in kidney filtration. Given this essential function, podocyte dysfunction results in kidney diseases known as podocytopathies. Previous studies have demonstrated that maintaining the activation-deactivation balance of mechanistic target of rapamycin complex 1 (mTORC1) is vital for podocyte function. Podocyte-specific knockout (KO) mouse models revealed that abnormal mTORC1 activation leads to severe podocytopathy. Therefore, elucidating the mechanism underlying mTORC1 activation in podocytes may contribute to the development of treatments for certain podocytopathies. In our previous study, we showed that macropinocytosis-large-scale endocytosis-is involved in the molecular mechanism of mTORC1 activation in podocytes. Growth factor (GF) stimulation induces circular dorsal ruffles (CDRs), which are large membrane protrusions on the dorsal surface of podocytes. CDRs serve as precursors to macropinocytosis, generating vesicles called macropinosomes, which transport extracellular nutrients to lysosomes, thereby activating mTORC1. These findings suggest that CDRs-derived macropinosomes modulate the mTORC1 pathway. In the present study, we investigated the molecular mechanism underlying macropinosome formation in podocytes, focusing on flotillin-1 (Flot1), a protein enriched in lipid microdomains. Imaging analysis revealed the localization of Flot1 at CDRs, and Flot1 depletion reduced macropinosome formation. Biochemical analysis further demonstrated impaired GF-stimulated mTORC1 activation in Flot1-KO cells, which exhibited slower growth than control cells. Notably, immuno-staining analysis showed that Flot1 is expressed specifically in podocytes but not in other renal cells. These findings indicate that Flot1 participates in the formation of CDRs-derived macropinosomes and contributes to macropinosome-dependent mTORC1 activation in podocytes.Key words: Flot1, circular dorsal ruffles, macropinocytosis, mTORC1, podocytes.

A primary cilium is a hair-like organelle that protrudes from the cell surface in many cell types. Growing evidence indicates that extracellular vesicles are released from primary cilia, and research is increasingly focused on defining the functions of these cilia-derived extracellular vesicles (EVs). EVs are known to modulate the behavior of various cancer cells, and structural and functional abnormalities in primary cilia have been reported in multiple cancer types. We previously demonstrated that PANC-1 cells, a human pancreatic ductal adenocarcinoma cell line, acquire enhanced primary cilia formation after surviving solitary culture conditions, and that their cilia contribute to tumor-like cell mass formation. Here, we explored part of the underlying mechanism of this phenotype by investigating the contribution of EVs released from the primary cilia of PANC-1 cells. PANC-1 clones generated by limiting dilution exhibited enhanced ciliogenesis and distinct ciliary morphologies compared with parental cells. These clones also released higher levels of cilia-derived EVs, including an expanded population of freely floating EVs within the culture environment. Biochemical analyses further showed that this increase was selective for primary cilia-derived EVs rather than reflecting a global rise in total EV production. Functionally, EV fractions enriched in cilia-derived EVs suppressed parental PANC-1 cell migration, altered cell morphology, and promoted cell aggregation, mimicking key behavioral traits of solitary condition-surviving PANC-1 clones. Together, these findings identify enhanced release of primary cilia-derived EVs as a distinct feature of PANC-1 cells adapted to solitary growth and suggest their potential involvement in the malignant and metastatic behaviors of pancreatic cancer.Key words: primary cilia, PDAC, extracellular vesicles, cell migration, cell aggregation.

Adherens junctions (AJs) mediate cell-cell adhesion and mechanical coupling in epithelial tissues. During AJ formation, punctate AJs (punctum adherens; PA) initially appear and subsequently transition into linear AJs or zonula adherens (ZAs). The mechanosensitive interaction of α-catenin with its binding partners-actin filaments and vinculin-is thought to act as a key switch that stabilizes AJs under tension. However, the physiological role of α-catenin's force sensitivity during the early stages of AJ formation remains unclear. Here, we analyzed α-catenin mutants with altered force sensitivity: Insensitive mutant L344P lacking vinculin binding, and Hypersensitive mutant L378P binding vinculin constitutively. Using calcium-switch assays combined with fluorescence and electron microscopy, we found that cells expressing insensitive α-catenin exhibited persistent, elongated PA-like structures corresponding to lateral associations of cellular protrusions from opposing cells, accompanied by delayed ZA formation. In contrast, cells expressing the hypersensitive mutant rapidly formed ZAs, possibly bypassing the PA stage. Similar phenotypes were observed in vinculin-knockout cells, indicating that the defects in Insensitive mutants result from the lack of vinculin recruitment to α-catenin. Based on these findings, we propose a model in which clusters of the cadherin-catenin complex (CCC) along actin filaments on opposing protrusions serve as initial adhesion sites. As protrusions shorten through actomyosin contraction, CCC clusters move toward the protrusion tips along actin filaments, where stretched α-catenin recruits vinculin to reinforce the adhesion, leading to PA formation. Thus, α-catenin's force sensitivity is crucial for smooth and timely AJ assembly, ensuring proper epithelial morphogenesis by coupling intercellular adhesion with cytoskeletal tension.Key words: α-catenin, vinculin, adherens junction, actin filament, force sensitivity.

Hypocalcemia and hypomagnesemia frequently occur under pathological conditions such as Crohn's disease or during diuretic treatment. However, how the combined deficiency of Ca²⁺ and Mg²⁺ affects cellular physiology has remained unclear. In this study, we focused on this issue and found that Ca²⁺/Mg²⁺ deprivation is a potent driver of stress granule (SG) formation. When SG formation was inhibited by G3BP1/2 knockdown, Ca²⁺/Mg²⁺ deprivation caused a further decrease in intracellular Mg²⁺ levels and an increase in cell death, indicating that SGs function to mitigate Mg²⁺ loss and protect cells from death under cation-deficient conditions. Furthermore, we found that the expression of the Mg²⁺ transporter MAGT1 is upregulated in an SG-dependent manner, and that MAGT1 knockdown further decreases intracellular Mg²⁺ levels and increases cell death. Collectively, our results demonstrate that SG formation acts as an adaptive mechanism to maintain Mg²⁺ homeostasis during Ca²⁺/Mg²⁺ deficiency.Key words: stress granule, MAGT1, magnesium, calcium.

S100A11 is a small calcium-binding protein that has been studied in the context of growth regulation and membrane repair. However, it has recently been linked to the disassembly of focal adhesions. This new role of S100A11 has been linked to calcium influx through the stretch-activated channel Piezo1. In this review, we look at what's currently known about S100A11's structural features, interactome, and functional roles. We focus on how it responds to mechanical stress and becomes recruited to focal adhesions. We also look into its role in the disassembly of these adhesions and consider potential mechanisms. To place its activity in context, we compare S100A11 with other members of the S100 family members and discuss its contribution to calcium-dependent cytoskeletal regulation and extracellular signaling. We examine the effects of S100A11 activity in cancer metastasis, wound healing, and fibrosis. Finally, we evaluate potential ways to modulate S100A11 function for prospective therapeutic intervention. Collectively, this review projects S100A11 as a mechanosensitive calcium effector at the intersection of adhesion biology and mechanotransduction.Key words: S100A11, focal adhesions, mechanosensing, Piezo1, cytoskeleton, cell migration, cancer.

Afadin and ZO-1 are actin-binding scaffold proteins localized at cell-cell junctions. Although these proteins contain multiple protein-binding motifs for various junctional proteins, their binding partners within cells are strictly regulated. Here, we investigated the mutual interactions among afadin, ZO-1, and actin filaments using cells lacking cellular junctions derived from EL and F9 non-epithelial cells. In EL-derived cells, afadin and ZO-1 independently colocalized with various types of actin filaments. In F9-derived cells, afadin and ZO-1 colocalized as aggregates. Gene disruption analyses revealed that afadin and ZO-1 independently form aggregates in the absence of cadherin-catenin complex. Nectin-2, an afadin-binding membrane protein, was detected in afadin aggregates but not in ZO-1 aggregates, suggesting the existence of a membrane protein that binds to ZO-1. We identified this protein as JAM-C. A comparison between α-catenin-deficient and β-catenin-deficient F9 cells suggested that the extracellular domain of E-cadherin interferes with afadin and ZO-1 aggregate formation. Furthermore, gene disruption of nectin-2 suggested that JAM-C-bound ZO-1, rather than unbound ZO-1, preferentially interacts with afadin. Together, these findings indicate that interactions among afadin, ZO-1, and actin filaments are strictly regulated by various cellular contexts.Key words: afadin, ZO-1, actin, F9 cell, L cell.

During early embryogenesis, gastrulation occurs within a specific region of the pluripotent epiblast, where cells undergo significant changes in their context. The induction of these cellular transformations in particular cell populations suggests the involvement of non-diffusible factors that activate signaling pathways in a spatiotemporal manner. Syntaxin4 (Stx4), a type IV membrane protein that functions as an intravesicular fusion mediator, often translocates across membranes to perform a latent extracellular role in locally regulating cellular behaviors. Through the culture of mouse embryonic egg cylinders isolated from E6.0 embryos and embryonic stem cells (ESCs), we demonstrate that the membrane translocation of Stx4 may play a crucial role in this early stage of development. Using membrane-impermeable antagonistic peptides against extracellular Stx4, along with several small-molecule inhibitors and activators, we found that cells with extracellular Stx4 deactivate focal adhesion kinase (FAK), which then impacts AKT/PI3K signaling and results in increased expression of P-cadherin, ultimately inducing the expression of the gastrulation marker brachyury. Activation of this signaling pathway also triggers Rho/ROCK signaling in ESCs, leading to morphological changes. These findings offer important insights into gastrulation by shedding light on the molecular mechanisms that initiate the spatiotemporal changes in the uniform pluripotent cell sheet.Key words: gastrulation, FAK, P-cadherin, Rho/ROCK, membrane flip.