| 1. |
＠Kenta Shigetomi, Yumiko Ono, Kenji Matsuzawa, Junichi Ikenouchi, Cholesterol-rich domain formation mediated by ZO proteins is essential for tight junction formation, Proceedings of the National Academy of Sciences, https://doi.org/10.1073/pnas.2217561120, 2023.02. |
| 2. |
Ryo Shiomi, Kenta Shigetomi, Tetsuichiro Inai, Masami Sakai, Junichi Ikenouchi, CaMKII regulates the strength of the epithelial barrier., Scientific reports, 10.1038/srep13262, 5, 13262-13262, 2015.08, Epithelial cells define the boundary between the outside and the inside of our body by constructing the diffusion barrier. Tight junctions (TJs) of epithelial cells function as barriers against invasion of harmful microorganisms into the human body and free diffusion of water or ions from the body. Therefore, formation of TJs has to be strictly controlled in epithelial cells. However, the molecular mechanisms governing this regulation are largely unknown. In this study, we identified Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a regulator of the barrier function of TJs. CaMKII inhibition led to enlargement of TJ-areas and up-regulation of the barrier function. CaMKII inhibition induced excess TJ formation in part by the activation of AMP-activated protein kinase (AMPK) and subsequent phosphorylation of claudin-1. As up-regulation of epithelial barriers is essential for the prevention of chronic inflammatory diseases, the identification of CaMKII as a modulator of TJ function paves the way for the development of new drugs to treat these diseases.. |
| 3. |
Tamako Nishimura, Shoko Ito, Hiroko Saito, Sylvain Hiver, Kenta Shigetomi, Junichi Ikenouchi, Masatoshi Takeichi, DAAM1 stabilizes epithelial junctions by restraining WAVE complex-dependent lateral membrane motility., The Journal of cell biology, 215, 4, 559-573, 2016.11, Epithelial junctions comprise two subdomains, the apical junctional complex (AJC) and the adjacent lateral membrane contacts (LCs), that span the majority of the junction. The AJC is lined with circumferential actin cables, whereas the LCs are associated with less-organized actin filaments whose roles are elusive. We found that DAAM1, a formin family actin regulator, accumulated at the LCs, and its depletion caused dispersion of actin filaments at these sites while hardly affecting circumferential actin cables. DAAM1 loss enhanced the motility of LC-forming membranes, leading to their invasion of neighboring cell layers, as well as disruption of polarized epithelial layers. We found that components of the WAVE complex and its downstream targets were required for the elevation of LC motility caused by DAAM1 loss. These findings suggest that the LC membranes are motile by nature because of the WAVE complex, but DAAM1-mediated actin regulation normally restrains this motility, thereby stabilizing epithelial architecture, and that DAAM1 loss evokes invasive abilities of epithelial cells.. |
| 4. |
Masanao Kinoshita, Hikaru Ano, Michio Murata, Kenta Shigetomi, Junichi Ikenouchi, Nobuaki Matsumori, Emphatic visualization of sphingomyelin-rich domains by inter-lipid FRET imaging using fluorescent sphingomyelins., Scientific reports, 10.1038/s41598-017-16361-x, 7, 1, 16801-16801, 2017.12, Imaging the distribution of sphingomyelin (SM) in membranes is an important issue in lipid-raft research. Recently we developed novel fluorescent SM analogs that exhibit partition and dynamic behaviors similar to native SM, and succeeded in visualizing lateral domain-segregation between SM-rich liquid-ordered (Lo) and SM-poor liquid-disordered (Ld) domains. However, because the fluorescent contrast between these two domains depends directly on their partition ratio for the fluorescent SMs, domain-separation becomes indeterminate when the distribution difference is not great enough. In this study, we propose the use of inter-lipid Förster resonance energy transfer (FRET) imaging between fluorescent SMs to enhance the contrast of the two domains in cases in which the inter-domain difference in SM distribution is inadequate for conventional monochromic imaging. Our results demonstrate that inter-lipid FRET intensity was significantly higher in the Lo domain than in the Ld domain, resulting in a clear and distinguishable contrast between the two domains even in poorly phase-separated giant unilamellar vesicles. In addition, we show that inter-lipid FRET imaging is useful for selective visualization of highly condensed assemblies and/or clusters of SM molecules in living cell membranes. Thus, the inter-lipid FRET imaging technique can selectively emphasize the SM-condensed domains in both artificial and biological membranes.. |
| 5. |
Kenta Shigetomi, Junichi Ikenouchi, Regulation of the epithelial barrier by post-translational modifications of tight junction membrane proteins., Journal of biochemistry, 10.1093/jb/mvx077, 163, 4, 265-272, 2018.04, Body and organ surfaces in multicellular organisms are covered with a sheet of epithelial cells. The tight junction (TJ) is an adhesive structure that seals the gap between epithelial cells and functions as a selective barrier to prevent the entry of antigens and pathogenic microbes from the extracellular environment. Several transmembrane proteins that constitute the TJ (claudin, occludin, tricellulin and angulin) have been identified. As over-expression of these proteins does not enlarge TJs or enhance epithelial barrier function, it remains unclear how TJ membrane proteins are regulated to modulate the amount of TJ and the strength of the epithelial barrier. In this review, we discuss the post-translational modifications of TJ membrane proteins and their physiological significance from the viewpoint of the dynamic regulation of the epithelial barrier.. |
| 6. |
Kenta Shigetomi, Yumiko Ono, Tetsuichiro Inai, Junichi Ikenouchi, Adherens junctions influence tight junction formation via changes in membrane lipid composition., The Journal of cell biology, 10.1083/jcb.201711042, 217, 7, 2373-2381, 2018.07, Tight junctions (TJs) are essential cell adhesion structures that act as a barrier to separate the internal milieu from the external environment in multicellular organisms. Although their major constituents have been identified, it is unknown how the formation of TJs is regulated. TJ formation depends on the preceding formation of adherens junctions (AJs) in epithelial cells; however, the underlying mechanism remains to be elucidated. In this study, loss of AJs in α-catenin-knockout (KO) EpH4 epithelial cells altered the lipid composition of the plasma membrane (PM) and led to endocytosis of claudins, a major component of TJs. Sphingomyelin with long-chain fatty acids and cholesterol were enriched in the TJ-containing PM fraction. Depletion of cholesterol abolished the formation of TJs. Conversely, addition of cholesterol restored TJ formation in α-catenin-KO cells. Collectively, we propose that AJs mediate the formation of TJs by increasing the level of cholesterol in the PM.. |
| 7. |
Kenta Shigetomi, Junichi Ikenouchi, Cell Adhesion Structures in Epithelial Cells Are Formed in Dynamic and Cooperative Ways., BioEssays : news and reviews in molecular, cellular and developmental biology, 10.1002/bies.201800227, 41, 7, e1800227, 2019.07, There are many morphologically distinct membrane structures with different functions at the surface of epithelial cells. Among these, adherens junctions (AJ) and tight junctions (TJ) are responsible for the mechanical linkage of epithelial cells and epithelial barrier function, respectively. In the process of new cell-cell adhesion formation between two epithelial cells, such as after wounding, AJ form first and then TJ form on the apical side of AJ. This process is very complicated because AJ formation triggers drastic changes in the organization of actin cytoskeleton, the activity of Rho family of small GTPases, and the lipid composition of the plasma membrane, all of which are required for subsequent TJ formation. In this review, the authors focus on the relationship between AJ and TJ as a representative example of specialization of plasma membrane regions and introduce recent findings on how AJ formation promotes the subsequent formation of TJ.. |
| 8. |
Kenji Matsuzawa, Hayato Ohga, Kenta Shigetomi, Tomohiro Shiiya, Masanori Hirashima, Junichi Ikenouchi, MAGIs regulate aPKC to enable balanced distribution of intercellular tension for epithelial sheet homeostasis., Communications biology, 10.1038/s42003-021-01874-z, 4, 1, 337-337, 2021.03, Constriction of the apical plasma membrane is a hallmark of epithelial cells that underlies cell shape changes in tissue morphogenesis and maintenance of tissue integrity in homeostasis. Contractile force is exerted by a cortical actomyosin network that is anchored to the plasma membrane by the apical junctional complexes (AJC). In this study, we present evidence that MAGI proteins, structural components of AJC whose function remained unclear, regulate apical constriction of epithelial cells through the Par polarity proteins. We reveal that MAGIs are required to uniformly distribute Partitioning defective-3 (Par-3) at AJC of cells throughout the epithelial monolayer. MAGIs recruit ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein 2 (ASPP2) to AJC, which modulates Par-3-aPKC to antagonize ROCK-driven contractility. By coupling the adhesion machinery to the polarity proteins to regulate cellular contractility, we propose that MAGIs play essential and central roles in maintaining steady state intercellular tension throughout the epithelial cell sheet.. |
| 9. |
Kenta Shigetomi, Yumiko Ono, Tetsuichiro Inai, Junichi Ikenouchi, Adherens junctions influence tight junction formation via changes in membrane lipid composition, Journal of Cell Biology, 10.1083/jcb.201711042, 217, 7, 2373-2381, 2018.07, Tight junctions (TJs) are essential cell adhesion structures that act as a barrier to separate the internal milieu from the external environment in multicellular organisms. Although their major constituents have been identified, it is unknown how the formation of TJs is regulated. TJ formation depends on the preceding formation of adherens junctions (AJs) in epithelial cells; however, the underlying mechanism remains to be elucidated. In this study, loss of AJs in α-catenin-knockout (KO) EpH4 epithelial cells altered the lipid composition of the plasma membrane (PM) and led to endocytosis of claudins, a major component of TJs. Sphingomyelin with long-chain fatty acids and cholesterol were enriched in the TJ-containing PM fraction. Depletion of cholesterol abolished the formation of TJs. Conversely, addition of cholesterol restored TJ formation in α-catenin-KO cells. Collectively, we propose that AJs mediate the formation of TJs by increasing the level of cholesterol in the PM.. |
