Cell structure and function最新文献

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.

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.

Phospholipids are major components of biological membranes. They play an essential role in intracellular signaling and organelle dynamics; however, the availability of suitable lipid-specific probes is limited, which has hindered studies on their spatial distribution and functional dynamics in living cells. Previously, we demonstrated that octadecyl rhodamine B chloride (R18) is transported to the endoplasmic reticulum via nonvesicular membrane transport. In this study, we showed that R18 is internalized in a phosphatidylethanolamine (PE)-dependent manner in vivo. The internalization of R18 in Saccharomyces cerevisiae is blocked in PE-deficient mutants, but restored by ethanolamine supplementation, which suggests strict PE dependence. Moreover, R18 delivered to vacuoles through autophagy was not terminally retained, but underwent Pep4- and Atg15-dependent export from the vacuoles. The exported R18 was then redirected to endosomes following prolonged autophagy. These results suggest that R18 may serve as an indicator of PE dynamics and vacuole-endosome lipid transport, which contributes to lipid homeostasis inside vacuoles.Key words: autophagy, in vivo lipid dynamics, octadecyl rhodamine B (R18), phospholipase, phospholipid, vacuole, yeast.

Quantitative morphological analysis is crucial for understanding cellular processes. While 3D Z-stack imaging offers high-resolution data, the complexity of 3D structures makes direct interpretation and manual annotation challenging and time-consuming, especially for large datasets. Maximum Intensity Projection (MIP) is a common strategy to create more interpretable 2D representations, but this inevitably leads to artificial overlaps between structures, significantly hindering accurate automated segmentation of individual instances by conventional methods or standard deep learning tools. To address this critical challenge in 2D projection analysis, we developed DeMemSeg, a deep learning pipeline based on Mask R-CNN, specifically designed to segment overlapping membrane structures, called prospore membranes (PSMs) during yeast sporulation. DeMemSeg was trained on a custom-annotated dataset, leveraging a systematic image processing workflow. Our optimized model accurately identifies and delineates individual, overlapping PSMs, achieving segmentation performance and derived morphological measurements that are statistically indistinguishable from expert manual annotation. Notably, DeMemSeg successfully generalized to segment PSMs from unseen data acquired from gip1Δ mutant cells, capturing the distinct morphological defects in PSMs. DeMemSeg thus provides a robust, automated solution for objective quantitative analysis of complex, overlapping membrane morphologies directly from widely used 2D MIP images, offering a practical tool and adaptable workflow to advance cell biology research.Key words: deep learning-based segmentation, microscopy image processing, cellular morphology, yeast sporulation, membrane structure.

As the outermost organ, the skin is particularly susceptible to physical damage. Keratinocytes are a major component of the epidermis, and their migration plays a crucial role in skin wound healing. Supersulfides contribute to energy production to sustain the life activities of organisms and are anticipated to play a role in various physiological functions; however, minimal studies have investigated their presence and functions in the skin. This study aimed to determine the presence of supersulfides in the skin and investigate their effect on keratinocyte migration. Using sulfane sulfur probe 4 (SSP4), a fluorescent probe that detects sulfane sulfur, the presence of supersulfides in both skin tissue and keratinocytes was revealed. Moreover, the primary supersulfide biosynthetic enzyme, cysteinyl-tRNA synthetase 2 (CARS2), was expressed at both the tissue and cellular levels. CARS2 expression and SSP4 fluorescence intensity in keratinocytes increased during wound healing, suggesting that supersulfide is involved in the regulation of cell migration. Knockdown of CARS2 suppressed keratinocyte migration and markedly downregulated gene expression of various chemokines. Protein expression analysis revealed that supersulfides regulate E-cadherin and matrix metalloproteinase (MMP)-9 via extracellular signal-regulated kinase (ERK) and protein kinase B (Akt). Furthermore, Na₂S₄ treatment of keratinocytes with CARS2 knockdown restored cell migration. We propose that supersulfide in the skin represents a novel mechanism of re-epithelialization and may serve as a therapeutic target for skin wounds.Key words: supersulfide, cysteinyl-tRNA synthetase 2, keratinocyte, cell migration, wound healing.

For the biogenesis and maintenance of cilia, bidirectional protein trafficking within cilia is crucial, and is conducted by intraflagellar transport (IFT) trains containing the IFT-A and IFT-B complexes that are powered by dynein-2 and kinesin-II motors. We have recently shown that before the assembly of anterograde IFT trains, the IFT-A, IFT-B, and dynein-2 complexes are independently recruited to the mother centriole/basal body. The IFT-B complex, which consists of 16 subunits, can be divided into the IFT-B1 and IFT-B2 subcomplexes, and IFT-B1 can be further divided into the IFT-B1a and IFT-B1b subgroups. Here we investigated how the IFT-B complex is assembled and recruited to the mother centriole for ciliogenesis. Analyses using cells with knockouts of individual IFT-B subunits, and analyses of proteins coimmunoprecipitated with EGFP-fused IFT-B2, IFT-B1b, and IFT-B1a subunits expressed in these knockout cells demonstrated the following: (i) although IFT-B2 is dispensable for the linkage between IFT-B1b and IFT-B1a, it is essential for their localization to the mother centriole; (ii) IFT-B1b is essential both for bridging IFT-B2 and IFT-B1a, and for their localization to the mother centriole; (iii) IFT-B1a is not required for the linkage between IFT-B2 and IFT-B1b nor for their localization to the mother centriole; and (iv) all IFT-B components (IFT-B2, IFT-B1b, and IFT-B1a) are essential for ciliogenesis. Thus, although ciliogenesis is not a prerequisite for the recruitment of the IFT-B complex to the mother centriole, the linkage between IFT-B2 and IFT-B1b is crucial for the mother centriole localization of the IFT-B complex for ciliogenesis.Key words: cilia, ciliogenesis, distal appendages, IFT-B complex, mother centriole.