Keiko Kawauchi

Chemotherapeutic agents targeting ribosome biogenesis induce profound reorganization of nucleolar architecture, yet how the tumor suppressor p53 governs these structural responses remains unclear. Here, we show that loss of p53 leads to NF-κB-dependent disappearance of nucleolar caps induced by doxorubicin (DOXO). Under these conditions, fibrillarin (FBL), which is normally confined to the nucleolus, relocates to the nucleoplasm and forms foci that partially associate with G-quadruplex (G4) structures, non-canonical nucleic acid secondary structures enriched at transcriptionally active genomic regions. To examine whether this redistribution is linked to transcriptional changes, we integrated publicly available transcriptomic datasets and identified genes that were upregulated in p53-deficient cells under DOXO treatment and downregulated upon FBL depletion. Given that casein kinase 2 alpha (CK2α) is a nuclear binding partner of FBL, we further analyzed CK2α-dependent gene programs. This analysis revealed that a fraction of FBL-responsive genes overlapped with CK2α-dependent signatures and were enriched for promoter-proximal G4 structures. Among candidate regulators, the G4-binding transcription factor MAZ emerged as a potential mediator linking nucleoplasmic FBL and CK2α to G4-associated transcriptional regulation. Together, our findings identify a mechanism linking loss of p53 to G4-associated transcriptional reprogramming through nucleolar architectural disruption mediated by an FBL–CK2α–MAZ axis during DOXO treatment.

Integrating the latest insights from mechanobiology into tissue engineering could lead to innovative technologies. Here we show a method to effectively elicit the regenerative response of transplanted cells by utilizing mechanical stress generated in vivo. The essential feature of our method is that it does not use specific ligands for the vital mechanosensor integrins to mechanically activate them. In our method, azide groups are introduced into the integrin, and the hydrogel is modified with cyclooctyne (DBCO) groups. Thus, bioorthogonal click reaction between the azide groups and the DBCO groups forms direct, stable, irreversible covalent bonds between the cellular integrin and the hydrogel. We demonstrate that the integrin–hydrogel linkage is in ON state regardless of the intensity of the stress, the cell cycle, or the extracellular environment, so that mechanical stress is rapidly and reliably transmitted to the nucleus through the linkage in vivo, resulting in regenerative response of the transplanted cells.

Freestanding cell‐sheets are valuable bio‐materials for use in regenerative medicine and tissue engineering. Because cell‐sheets experience various mechanical stimulations during handling, it is important to understand the responses of cells to these stimulations. Here, we demonstrate changes in the localization of various proteins during the stretching of fibroblast cell‐sheets. These proteins are known to be involved in mechano‐sensing. Upon stretching, actin filaments appear parallel to the stretching direction. At cell‐cell junctions, β‐catenin forms clusters that co‐localize with accumulated vinculin and zyxin as well as the actin filaments. The p130 Crk‐associated substrate, known to be present in focal adhesions, is also recruited to these clusters and phosphorylated. Our results suggest that mechano‐sensing machinery is formed at cell‐cell junctions when the cell‐sheets are stretched.

Background/Objectives: Actin plays a crucial role not only in the cytoplasm, but also in the nucleus, influencing various cellular behaviors, including cell migration and gene expression. Recent studies reveal that nuclear actin dynamics is altered by cellular stresses, such as DNA damage; however, the effect of heat shock on nuclear actin dynamics, particularly in the nucleolus, remains unclear. This study aims to elucidate the contribution of nucleolar actin to cellular responses under heat shock conditions. Methods: Nuclear actin dynamics in response to heat shock were investigated using nAC-GFP, a GFP-tagged actin chromobody, to visualize nuclear actin in HeLa cells. Bioinformatic analyses were also performed. Results: Heat shock induced the reversible assembly of nAC-GFP in the nucleolus, with disassembly occurring upon recovery in a heat shock protein (Hsp) 70-dependent manner. Because the nucleolus, formed via liquid–liquid phase separation (LLPS), sequesters misfolded proteins under heat shock to prevent irreversible aggregation, we hypothesized that nucleolar actin-binding proteins might also be sequestered in a similar manner. Using several databases, we identified 47 actin-binding proteins localized in the nucleolus and determined the proportion of intrinsically disordered regions (IDRs) known to promote LLPS. Our analysis revealed that many of these 47 proteins exhibited high levels of IDRs. Conclusions: The findings from our bioinformatics analysis and further cellular studies may help elucidate new roles for actin in the heat shock response.

Freestanding cell-sheets are valuable bio-materials for use in regenerative medicine and tissue engineering. Because cell-sheets experience various mechanical stimulations during handling, it is important to understand the responses of cells to these stimulations. Here, we demonstrate changes in the localization of various proteins during the stretching of fibroblast cell-sheets. These proteins are known to be involved in mechano-sensing. Upon stretching, actin filaments appear parallel to the stretching direction. At cell-cell junctions, β-catenin forms clusters that co-localize with accumulated vinculin and zyxin as well as the actin filaments. The p130 Crk-associated substrate, known to be present in focal adhesions, is also recruited to these clusters and phosphorylated. Our results suggest that mechano-sensing machinery is formed at cell-cell junctions when the cell-sheets are stretched.

Shikonin is extracted from the roots of Lithospermum erythrorhizon, and shikonin extracts have been shown to have inhibitory effects on several bacteria. However, shikonin extracts are difficult to formulate because of their poor water solubility. In the present study, we prepared a shikonin dispersion, which was solubilized by the inclusion of β-1,3-1,6 glucan, and analysed the inhibitory effects of this dispersion on Streptococcus mutans and non-mutans streptococci. The shikonin dispersion showed pronounced anti-S. mutans activity, and inhibited growth of and biofilm formation by this bacterium. The shikonin dispersion also showed antimicrobial and antiproliferative effects against non-mutans streptococci. In addition, a clinical trial was conducted in which 20 subjects were asked to brush their teeth for 1 week using either shikonin dispersion-containing or non-containing toothpaste, respectively. The shikonin-containing toothpaste decreased the number of S. mutans in the oral cavity, while no such effect was observed after the use of the shikonin-free toothpaste. These results suggest that shikonin dispersion has an inhibitory effect on S. mutans and non-mutans streptococci, and toothpaste containing shikonin dispersion may be effective in preventing dental caries.

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-typep53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

Hydroxyapatite adsorbs various substances, but little is known about the effects on oral bacteria of adsorption onto hydroxyapatite derived from scallop shells. In the present study, we analyzed the effects of adsorption of Streptococcus mutans onto scallop-derived hydroxyapatite. When scallop-derived hydroxyapatite was mixed with S. mutans, a high proportion of the bacterial cells adsorbed onto the hydroxyapatite in a time-dependent manner. An RNA sequencing analysis of S. mutans adsorbed onto hydroxyapatite showed that the upregulation of genes resulted in abnormalities in pathways involved in glycogen and histidine metabolism and biosynthesis compared with cells in the absence of hydroxyapatite. S. mutans adsorbed onto hydroxyapatite was not killed, but the growth of the bacteria was inhibited. Electron microscopy showed morphological changes in S. mutans cells adsorbed onto hydroxyapatite. Our results suggest that hydroxyapatite derived from scallop shells showed a high adsorption ability for S. mutans. This hydroxyapatite also caused changes in gene expression related to the metabolic and biosynthetic processes, including the glycogen and histidine of S. mutans, which may result in a morphological change in the surface layer and the inhibition of the growth of the bacteria.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF‐7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal‐related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy‐treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK‐dependent paraptosis.