Tsukasa Oikawa

Epithelial cells undergo epithelial–mesenchymal transition (EMT) during migration and regain their epithelial phenotype in the post-migration phase (mesenchymal–epithelial transition [MET]). We established an experimental system that reproduces a three-compartment epithelial structure comprising the original epithelium, its EMT state, and its MET state. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane, migrated through 3.0 µm or larger micropores. The 3.0-µm-pored membrane induced an epithelial structure with three distinct states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain the three-compartment epithelial structure. Live imaging revealed that KCs moved in an oscillatory manner, with actin-rich filopodia-like structures extending into and out of the 3.0 µm micropores, while the cells migrated unidirectionally into larger micropores. Piezo1 and keratin 6 were identified as negative modulators of KC entry into and exit from the 3.0 µm micropores. These results demonstrate that non-cancerous epithelial cells migrate through confined spaces in an oscillatory manner, which might contribute to the formation of a three-compartment epithelial structure that recapitulates key aspects of wound healing.

Epithelial cells undergo epithelial–mesenchymal transition (EMT) during migration and regain their epithelial phenotype in the post-migration phase (mesenchymal–epithelial transition; MET). We established an experimental system that reproduces a three-compartment epithelial structure comprising the original epithelium, its EMT state, and its MET state. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-µm or larger micropores. The 3.0-µm-pored membrane induced an epithelial structure with three distinct states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain the three-compartment epithelial structure. Live imaging revealed that KCs moved in an oscillatory manner, with actin-rich filopodia-like structures extending into and out of the 3.0-µm micropores, while the cells migrated unidirectionally into larger micropores. Piezo1 and keratin 6 were identified as negative modulators of KC entry into and exit from the 3.0-µm micropores. These results demonstrate that non-cancerous epithelial cells migrate through confined spaces in an oscillatory manner, which might contribute to the formation of a three-compartment epithelial structure that recapitulates key aspects of wound healing.

Epithelial cells undergo epithelial–mesenchymal transition (EMT) during migration and regain their epithelial phenotype in the post-migration phase (mesenchymal–epithelial transition; MET). We established an experimental system that reproduces a three-compartment epithelial structure comprising the original epithelium, its EMT state, and its MET state. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-µm or larger micropores. The 3.0-µm-pored membrane induced an epithelial structure with three distinct states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain the three-compartment epithelial structure. Live imaging revealed that KCs moved in an oscillatory manner, with actin-rich filopodia-like structures extending into and out of the 3.0-µm micropores, while the cells migrated unidirectionally into larger micropores. Piezo1 and keratin 6 were identified as negative modulators of KC entry into and exit from the 3.0-µm micropores. These results demonstrate that non-cancerous epithelial cells migrate through confined spaces in an oscillatory manner, which might contribute to the formation of a three-compartment epithelial structure that recapitulates key aspects of wound healing.

Osteoclasts are multinucleated giant cells that are formed by the fusion of precursor cells. Cell–cell fusion is mediated by membrane protrusion driven by actin reorganization, but the role of membrane mechanics in this process is unknown. Utilizing live-cell imaging, optical tweezers, manipulation of membrane-to-cortex attachment (MCA), and genetic interference, we show that a decrease in plasma membrane (PM) tension is a mechanical prerequisite for osteoclast fusion. Upon RANKL-induced differentiation, ezrin expression in fusion progenitor cells is reduced, resulting in a decrease in MCA-dependent PM tension. A forced elevation of PM tension by reinforcing the MCA conversely suppresses cell–cell fusion. Mechanistically, reduced PM tension leads to membrane protrusive invadosome formation driven by membrane curvature-inducing/sensing BAR proteins, thereby promoting cell–cell fusion. These findings provide insights into the mechanism of cell–cell fusion under the control of membrane mechanics.

Mitochondrial functions range from catabolic to anabolic, which are tightly coordinated to meet cellular demands for proliferation and motility. MitoNEET is a mitochondrial outer membrane protein with a CDGSH domain and is involved in mitochondrial function. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose their epithelial characteristics and acquire mesenchymal traits, such as motility, which is a vital step for organism development and wound-healing. Cellular motility is associated with high ATP consumption owing to lamellipodia formation, which is supported by upregulated oxidative phosphorylation (OXPHOS) capacity. However, how mitoNEET is involved in the regulation of OXPHOS capacity and subsequent cellular motility remains unclear. Here we show that loss of mitoNEET regulation during EMT impairs both OXPHOS enhancement and cell motility in non-transformed NMuMG mouse mammary gland epithelial cells. We found that mitoNEET is downregulated during EMT, and that the aberrant expression of mitoNEET abolishes the upregulation of OXPHOS, leading to the inhibition of cell motility. Furthermore, we found that mitoNEET topology may be crucial for the regulation of the mitochondrial electron transfer chain, suggesting an additional regulatory pathway for OXPHOS capacity. Our results demonstrate that mitochondrial OXPHOS capacity during EMT is partly regulated by the dynamics of the outer membrane protein. We believe that our findings are the first step towards understanding the mechanisms by which mitochondrial outer membrane protein topology affects organelle functions.

H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here, we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 toward phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.

Histones are key molecules of epigenetic regulation and inheritance, and are thought to be chaperoned and transported into the nucleus appropriately prior to being integrated into nucleosomes. H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 towards phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. Induction of the TMEM255A gene by p53 linked p53 with CTDNEP1, in which TMEM255A stabilized CTDNEP1. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 molecules may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.

TP53 mutations in cancer cells often evoke cell invasiveness, whereas fibroblasts show invasiveness in the presence of intact TP53. AMAP1 (also called DDEF1 or ASAP1) is a downstream effector of ARF6 and is essential for the ARF6-driven cell-invasive phenotype. We found that AMAP1 levels are under the control of p53 (TP53 gene product) in epithelial cells but not in fibroblasts, and here addressed that molecular basis of the epithelial-specific function of p53 in suppressing invasiveness via targeting AMAP1. Using MDA-MB-231 cells expressing wild-type and p53 mutants, we identified miRNAs in which their expression is controlled by normal-p53. Among them, we identified miRNAs that target AMAP1 mRNA, and analyzed their expression levels and epigenetic statuses in epithelial cells and nonepithelial cells. We found that normal-p53 suppresses AMAP1 mRNA in cancer cells and normal epithelial cells, and that more than 30 miRNAs are induced by normal-p53. Among them, miR-96 and miR-182 were found to target the 3′-untranslated region of AMAP1 mRNA. Fibroblasts did not express these miRNAs at detectable levels. The ENCODE dataset demonstrated that the promoter region of the miR-183-96-182 cistron is enriched with H3K27 acetylation in epithelial cells, whereas this locus is enriched with H3K27 trimethylation in fibroblasts and other non-epithelial cells. miRNAs, such as miR-423, which are under the control of p53 but not associated with AMAP1 mRNA, demonstrated similar histone modifications at their gene loci in epithelial cells and fibroblasts, and were expressed in these cells. Histone modifications of certain miRNA loci, such as the miR-183-96-182 cistron, are different between epithelial cells and non-epithelial cells. Such epithelial-specific miRNA regulation appears to provide the molecular basis for the epithelial-specific function of p53 in suppressing ARF6-driven invasiveness.

In addition to its classical roles as a tumor suppressor, p53 has also been shown to act as a guardian of epithelial integrity by inducing the microRNAs that target transcriptional factors driving epithelial–mesenchymal transition. On the other hand, the ENCODE project demonstrated an enrichment of putative motifs for the binding of p53 in epithelial-specific enhancers, such as CDH1 (encoding E-cadherin) enhancers although its biological significance remained unknown. Recently, we identified two novel modes of epithelial integrity (i.e., maintenance of CDH1 expression): one involves the binding of p53 to a CDH1 enhancer region and the other does not. In the former, the binding of p53 is necessary to maintain permissive histone modifications around the CDH1 transcription start site, whereas in the latter, p53 does not bind to this region nor affect histone modifications. Furthermore, these mechanisms likely coexisted within the same tissue. Thus, the mechanisms involved in epithelial integrity appear to be much more complex than previously thought. In this review, we describe our findings, which may instigate further experimental scrutiny towards understanding the whole picture of epithelial integrity as well as the related complex asymmetrical functions of p53. Such understanding will be important not only for cancer biology but also for the safety of regenerative medicine.