ABSTRACT The translocase of the outer mitochondrial membrane (TOM) complex is the entry gate for mitochondrial proteins synthesized in the cytosol. Previous biochemical studies indicated that the TOM complex functions as a trimer and exists in a dynamic equilibrium with a Tom22‐lacking dimer in intact mitochondria. In contrast, recent cryo‐electron microscopy (cryo‐EM) studies revealed near‐atomic structures of the Tom22‐containing core dimer. However, the relationship between the core dimer and the functional trimer remains unclear. Here, we analyzed the oligomeric organization and structural properties of purified yeast TOM complexes using high‐speed atomic force microscopy (HS‐AFM). Purified yeast TOM complexes predominantly adopted a trimeric organization but underwent deformation and dissociation during repeated HS‐AFM scanning, leaving dimeric and monomeric particles. Trimeric particles exhibited spherical shapes consistent with previous crosslinking models, whereas dissociated dimers closely resembled TOM core‐dimer structures determined by cryo‐EM. HS‐AFM also revealed the peripheral localization of the Tom20 receptor and enabled direct visualization of substrate–TOM interactions, with TOM association accompanied by reduced substrate mobility. Together, these findings provide quantitative and structural evidence for a predominantly trimeric organization of the purified TOM complex and suggest that the trimer contains a stable core‐dimer unit with a more loosely associated third Tom40‐containing unit.
ABSTRACT Real‐time visualization of specific mRNA expression in living cells is essential for understanding diverse biological processes; however, many existing approaches require modifications to target genes. In this study, we developed a programmable RNA‐mediated bimolecular fluorescence complementation (RmBiFC) system by fusing split Venus fluorescent protein fragments with the Pumilio homology domain as a modular RNA‐binding domain. Using murine leukemia virus mRNA as a model target, we systematically optimized the orientation of the probes and demonstrated robust detection of cells expressing target mRNA. This system enables live‐cell imaging of target mRNA‐expressing cells without modifying the target gene. This provides a valuable means of tracking cells expressing wild‐type genes, which is challenging to achieve using conventional reporter‐based approaches. The RmBiFC system represents a versatile and non‐invasive platform with broad potential for identifying specific cell populations based on their transcriptomic profiles at the cellular level.
Mechanical cues influence cell behavior and fate and are frequently accompanied by changes in nuclear shape; however, how epithelial nuclei accommodate such deformations remains incompletely understood. Here, we investigated the formation and regulation of nuclear wrinkles (NWs), inward folds of the nuclear envelope, in human epithelial cells. Using quantitative confocal imaging in 2.5D spheroid cultures and controlled 2D monolayers, we found that NWs formed frequently in MCF10A cells but rarely in hTERT-RPE1 cells, indicating pronounced cell-type specificity. NW frequency increased with cell density and was tightly associated with coordinated geometric changes consistent with nuclear rounding. Disruption of F-actin organization, but not microtubules, robustly induced NW formation, and acute cell rounding triggered by trypsinization was sufficient to induce widespread wrinkling across multiple cell types. Live-cell imaging revealed that NWs are dynamic and reversible at low cell density but become stabilized under sustained confinement. NW formation occurred without detectable nuclear envelope rupture, DNA damage, or stress-associated histone phosphorylation. Quantitative analysis supports a passive geometric model in which redistribution of excess nuclear surface area accommodates nuclear shape remodeling, allowing epithelial nuclei to buffer mechanical constraints while preserving nuclear integrity. ### Competing Interest Statement The authors have declared no competing interest. Japan Society for the Promotion of Science, https://ror.org/00hhkn466, JP21H04764, JP24H02325, JP26H02362 Japan Agency for Medical Research and Development, JP24ama121020, 24jf0126008h0001
Statins, inhibitors of HMG-CoA reductase, exert immunomodulatory effects beyond lipid lowering, yet how they regulate T helper (Th) cell differentiation remains poorly understood. We show that fluvastatin administration to C57BL/6 mice for 7 days suppressed interferon-γ and elevated interleukin-4 production by splenic CD4+ T cells, shifting the immune balance toward Th2 without altering splenocyte numbers or major immune cell subset proportions. This effect was not shared by rosuvastatin, suggesting that lipophilicity-dependent cellular uptake influences immunomodulatory potency. In vitro experiments confirmed a direct Th2-promoting action of fluvastatin on CD4+ T cells. Basophils were excluded as mediating cells, as fluvastatin suppressed rather than enhanced basophil interleukin-4 production. Continuous fluvastatin exposure during granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven dendritic cell (DC) differentiation altered the phenotype of the resulting DCs and markedly enhanced their Th2-polarizing capacity in co-cultures with antigen-specific CD4+ T cells. These functional changes were accompanied by alterations in DC surface molecule expression, although their causal contribution remains to be established. These findings indicate that continuous fluvastatin exposure during DC differentiation is associated with an altered DC phenotype and enhanced Th2-polarizing capacity.
Epithelial-mesenchymal transition (EMT) is crucial for initiation of cancer metastasis. EMT-induced epithelial cancer cells gain stemness characteristics, which enhance cancer malignancy and chemoresistance. Therefore, it is important to clarify the function of EMT-related genes for development of therapeutics for cancer metastasis. Here, we showed that Signal-transducing adaptor protein-2 (STAP-2) inhibits EMT in cancer cells through repressing SMAD-mediated ZEB1 expression, which is a master transcription factor of EMT. STAP-2 inhibits the interaction between SMAD3 and SMAD4, resulting in the downregulation of SMAD2/3/4 complex formation and TGF-β signal-induced gene expression. STAP-2 expression was repressed by SMAD2/3-mediated TGF-β signals and activated by OVOL1, which is one of the EMT repressor transcription factors. Our study showed that STAP-2 is a novel EMT repressor gene which represses ZEB1 expression and motility of cancer cells.
