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The cytoplasm in mammalian cells is considered homogeneous. In this study, we report that the cytoplasmic fluidity is regulated in the blebbing cells; the cytoplasm of rapidly expanding membrane blebs is more disordered than the cytoplasm of retracting blebs. The increase of cytoplasmic fluidity in the expanding bleb is caused by a sharp rise in the calcium concentration. The STIM-Orai1 pathway regulates this rapid and restricted increase of calcium in the expanding blebs. Conversely, activated ERM protein binds to Orai1 to inhibit the store-operated calcium entry in retracting blebs, which results in decreased in cytoplasmic calcium, rapid reassembly of the actin cortex.

DOI： 10.1038/s41467-020-20826-5

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DOI： 10.1038/srep13262

Language：English   Publishing type：Research paper (scientific journal)  

Cellular architectures require regulated mechanisms to correctly localize the appropriate plasma membrane lipids and proteins. Microvilli are dynamic filamentous-actin-based protrusions of the plasma membrane that are found in the apical membrane of epithelial cells. However, it remains poorly understood how their formation is regulated. In the present study, we found that sphingomyelin clustering underlies the formation of microvilli. Clustering of sphingomyelin is required for the co-clustering of the sialomucin membrane protein podocalyxin-1 at microvilli. Podocalyxin-1 recruits ezrin/radixin/moesin (ERM)-binding phosphoprotein-50 (EBP50; also known as NHERF1), which recruits ERM proteins and phosphatidylinositol 4-phosphate 5-kinase β (PIP5Kβ). Thus, clustering of PIP5Kβ leads to local accumulation of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], which enhances the accumulation of ERM family proteins and induces the formation of microvilli. The present study revealed novel interactions between sphingomyelin and the cytoskeletal proteins from which microvilli are formed, and it clarified the physiological importance of the chemical properties of sphingomyelin that facilitate cluster formation.

DOI： 10.1242/jcs.122325.

Language：English   Publisher：Scientific Reports  

Epithelia are tightly connected cellular sheets, that shield our body from the external environment. They are continuously maintained by a pooled population of undifferentiated cells through differentiation. However, the maintenance mechanisms remain incompletely understood due to the difficulty of experimentally observing the differentiation process. To address this issue, we developed a culture method for long-term expansion of primary mammary basal cells with a set of compounds, that includes undifferentiated cells. An effective differentiation method regarding Claudin expression was also developed by simply removing compounds. To verify this differentiation-switching technique, we obtained microarray data comparing each differentiation state. Subsequent cellular analysis confirmed key transcription factors in each state: (1) EGR1 in undifferentiated basal cells is important for suppressing Claudin expression through maintaining the epithelial-mesenchymal transition (EMT) transcription factor TWIST1, (2) ELF3 in differentiated cells is important for actin organization and subsequent Claudin localization at the cell-cell border, that corresponds to the amount of GRHL3, an actin organizer. Their relevance was also observed in tissues and organoids. In summary, we present an effective tool for verifying molecular mechanisms that determine Claudin status in normal basal cell differentiation, that would be beneficial in epithelial cell biology, cancer biology, physiology, and regeneration research.

DOI： 10.1038/s41598-025-95463-3

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Language：English   Publisher：Journal of the American Chemical Society  

Fluorescence thermometry has emerged as a significant area of research in the field of remote temperature sensing with high accuracy. However, the development of noninvasive and reliable small organic fluorescence thermometers (FTs) remains challenging. In this study, we developed a high-sensitivity, high-resolution small organic ratiometric FT with a solvatochromic dye and analyzed its temperature response mechanism. π-Extended fluorene-based D−π-A-type small solvatochromic dyes (FπAc, FπF, and FπVC) were synthesized and characterized. A significant solvatochromic shift of >200 nm was observed between n-hexane and tetrahydrofuran (THF) for both FπF and FπVC, with emission in THF reaching the red region (701 nm, 828 nm). This substantial solvatochromic shift was accompanied by a concurrent decrease in the fluorescence quantum yield and is attributable to accelerated internal conversion. The fluorescence spectra of FπF in THF and FπVC in diethyl ether exhibited blue shifts and increased fluorescence intensities with a decrease in polarity at increased temperatures. Our observations revealed an absolute sensitivity of 21%/°C and a relative sensitivity of 3.0%/°C at the maximum. These results represent the highest sensitivity and resolution reported for single fluorophore small organic ratiometric FTs dispersed in solution. The positive temperature coefficient of the fluorescence intensity was attributed to control of the nonradiative decay pathway by solvatochromism. The temperature responsiveness of FπF could be detected in living HeLa cells by ratiometric confocal microscopy. Using the proposed strategy to develop FTs, we plan to build a library of FTs that will cover various environments of interest in both simple and complex systems.

DOI： 10.1021/jacs.5c01173

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Language：English   Publisher：Oxford University Press  

Language：English   Publisher：Microscopy (Oxford, England)  

Cell membrane structures are supramolecular complexes that require the ordered assembly of membrane proteins and lipids. The morphology of various cell adhesion structures in multicellular organisms, such as those between epithelial cells, neural synapses and immune synapses, was initially described through electron microscopic analyses. Subsequent studies aimed to catalog their constituent proteins, which encompass transmembrane cell adhesion molecules, cytoskeletal proteins and scaffolding proteins that bind the two components. However, the diversity of plasma membrane lipids and their significance in the organization of cell adhesion structures were underappreciated until recently. It is now understood that phase separation of lipids and liquid-liquid phase separation of proteins are important driving forces for such self-assembly. In this review, we summarized recent findings on the role of lipids as scaffolds for supramolecular complexes using tight junctions in epithelial cells as an example.

DOI： 10.1093/jmicro/dfae039

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Advanced Science  

High-resolution spatio-temporal monitoring of the cell membrane lipid order provides visual insights into the complex and sophisticated systems that control cellular physiological functions. Solvatochromic fluorescent probes are highly promising noninvasive visualization tools for identifying the ordering of the microenvironment of plasma membrane microdomains. However, conventional probes, although capable of structural analysis, lack the necessary long-term photostability required for live imaging at the cellular level. Here, an ultra-high-light-resistant solvatochromic fluorescence probe, 2-N,N-diethylamino-7-(4-methoxycarbonylphenyl)-9,9-dimethylfluorene (FπCM) is reported, which enables live lipid order imaging of cell division. This probe and its derivatives exhibit sufficient fluorescence wavelengths, brightness, polarity responsiveness, low phototoxicity, and remarkable photostability under physiological conditions compared to conventional solvatochromic probes. Therefore, these probes have the potential to overcome the limitations of fluorescence microscopy, particularly those associated with photobleaching. FπCM probes can serve as valuable tools for elucidating mechanisms of cellular processes at the bio-membrane level.

DOI： 10.1002/advs.202309721

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Language：English   Publisher：Journal of Biochemistry  

Blebs are membrane structures formed by the detachment of the plasma membrane from the underlying actin cytoskeleton. It is now clear that a wide variety of cells, including cancer cells, actively form blebs for cell migration and cell survival. The expansion of blebs has been regarded as the passive ballooning of the plasma membrane by an abrupt increase in intracellular pressure. However, recent studies revealed the importance of ‘cytoplasmic zoning’, i.e. local changes in the hydrodynamic properties and the ionic and protein content of the cytoplasm. In this review, we summarize the current understanding of the molecular mechanisms behind cytoplasmic zoning and its role in bleb expansion.

DOI： 10.1093/jb/mvad084

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Language：English   Publisher：Proceedings of the National Academy of Sciences of the United States of America  

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

DOI： 10.1073/pnas.2307118120

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DOI： 10.1073/pnas.2307118120.

Publisher：Optics Continuum  

A multiplex CARS imaging system, equipped with an EM-CCD camera, was developed to improve the sensitivity of backward CARS imaging in biological analysis using an inverted microscope. The signal-to-noise ratio was improved by a factor of ca. 3 compared to a conventional CCD mode through the use of EM gain. When imaging epithelial cells in the backward CARS configuration, intracellular organelles such as lipid droplets and nuclei were spectroscopically identified with an exposure time of only 100 ms/pixel.

DOI： 10.1364/OPTCON.497869

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Language：Japanese   Publisher：日本脂質生化学会  

上皮細胞に存在するタイトジャンクション(TJ)と呼ばれる構造は、上皮細胞のバリア機能を担う。TJは、細胞接着分子クローディンが、裏打ちタンパク質ZOと結合を介して形質膜上で集積することで形成されるという仮説が広く受け入れられてきた。この仮説は、アドヘレンスジャンクション(AJ)と呼ばれるカドヘリンを介した別の接着機構からの類推に基づいており、その妥当性は検証されていない。私たちは、以前にTJ領域にコレステロールが高度に集積していることを報告した(Shigetomi et al.J.Cell Biol.,2018)。本研究では、クローディンの集積において、ZOタンパク質との結合ではなく、コレステロールに富む膜ドメインとの相互作用が重要であることを見出した。クローディンの発現を全て消失させた細胞において、ZOタンパク質と結合しないクローディン変異体を発現させた場合でも依然としてTJが形成される一方で、コレステロールに富む膜ドメインとの相互作用に必要なパルミトイル化修飾を消失させたクローディン変異体はTJを形成できないことが明らかになった。以上の結果から、TJの形成機構は、AJの形成機構とは異なり、コレステロールの集積したドメインがTJ形成において重要な役割を果たしていること、また、ZOタンパク質が細胞接着領域にコレステロールを集積させる機能を有していることが明らかになった。(著者抄録)

Language：English   Publisher：Proceedings of the National Academy of Sciences of the United States of America  

Tight junctions (TJs) are cell-adhesion structures responsible for the epithelial barrier. We reported that accumulation of cholesterol at the apical junctions is required for TJ formation [K. Shigetomi, Y. Ono, T. Inai, J. Ikenouchi, J. Cell Biol. 217, 2373–2381 (2018)]. However, it is unclear how cholesterol accumulates and informs TJ formation—and whether cholesterol enrichment precedes or follows the assembly of claudins in the first place. Here, we established an epithelial cell line (claudin-null cells) that lacks TJs by knocking out claudins. Despite the lack of TJs, cholesterol normally accumulated in the vicinity of the apical junctions. Assembly of claudins at TJs is thought to require binding to zonula occludens (ZO) proteins; however, a claudin mutant that cannot bind to ZO proteins still formed TJ strands. ZO proteins were however necessary for cholesterol accumulation at the apical junctions through their effect on the junctional actomyosin cytoskeleton. We propose that ZO proteins not only function as scaffolds for claudins but also promote TJ formation of cholesterol-rich membrane domains at apical junctions.

DOI： 10.1073/pnas.2217561120

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Language：English   Publisher：FEBS Journal  

When the plasma membrane (PM) detaches from the underlying actin cortex, the PM expands according to intracellular pressure and a spherical membrane protrusion called a bleb is formed. This bleb retracts when the actin cortex is reassembled underneath the PM. Whereas this phenomenon seems simple at first glance, there are many interesting, unresolved cell biological questions in each process. For example, what is the membrane source to enlarge the surface area of the PM during rapid bleb expansion? What signals induce actin reassembly for bleb retraction, and how is cytoplasmic fluidity regulated to allow rapid membrane deformation during bleb expansion? Furthermore, emerging evidence indicates that cancer cells use blebs for invasion, but little is known about how molecules that are involved in bleb formation, expansion, and retraction are coordinated for directional amoeboid migration. In this review, we discuss the molecular mechanisms involved in the regulation of blebs, which have been revealed by various experimental systems.

DOI： 10.1111/febs.16220

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1091/mbc.E19-12-0691

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1002/bies.201800227

Language：English   Publishing type：Research paper (scientific journal)  

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca
²⁺
channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca
²⁺
influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

DOI： 10.1038/s41467-018-04436-w

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1083/jcb.201711042

Language：English   Publishing type：Research paper (scientific journal)  

Adherens junctions (AJs) control epithelial cell behavior, such as collective movement and morphological changes, during development and in disease. However, the molecular mechanism of AJ remodeling remains incompletely understood. Here, we report that the conformational activation of α-catenin is the key event in the dynamic regulation of AJ remodeling. α-catenin activates RhoA to increase actomyosin contractility at cell-cell junctions. This leads to the stabilization of activated α-catenin, in part through the recruitment of the actin-binding proteins, vinculin and afadin. In this way, α-catenin regulates force sensing, as well as force transmission, through a Rho-mediated feedback mechanism. We further show that this is important for stable directional alignment of multiple cells during collective cell movement by both experimental observation and mathematical modeling. Taken together, our findings demonstrate that α-catenin controls the establishment of anisotropic force distribution at cell junctions to enable cooperative movement of the epithelial cell sheet. Collective cell movement requires multicellular coordination. Matsuzawa et al. show that anisotropic distribution of activated α-catenin is necessary to establish directional coordination of collectively migrating epithelial cells. α-catenin activation achieves this by modifying lateral cell adhesions through protein recruitment and activation of F-actin polymerization signaling.

DOI： 10.1016/j.celrep.2018.05.070

Language：English   Publishing type：Research paper (scientific journal)  

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 posttranslational modifications of TJ membrane proteins and their physiological significance from the viewpoint of the dynamic regulation of the epithelial barrier.

DOI： 10.1093/jb/mvx077

Language：English   Publishing type：Research paper (scientific journal)  

Epithelial cells have characteristic membrane domains. Identification of membrane proteins playing an important role in these membrane domains has progressed and numerous studies have been performed on the functional analysis of these membrane proteins. On the other hand, the precise roles of membrane lipids in the organization of these membrane domains are largely unknown. Historically, the concept of lipid raft arose from the analysis of lipid composition of the apical membrane, and it can be said that epithelial cells are an optimal experimental model for elucidating the functions of lipids. In this review, I discuss the role of lipids in the formation of epithelial polarity and in the formation of cell membrane structures of epithelial cells such as microvilli in the apical domain, cell-cell adhesion apparatus in the lateral domain and cell-matrix adhesion in the basal domain.

DOI： 10.1080/21688370.2018.1502531

Language：English   Publishing type：Research paper (scientific journal)  

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 (L_o) and SM-poor liquid-disordered (L_d) 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 L_o domain than in the L_d 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.

DOI： 10.1038/s41598-017-16361-x

Language：English   Publishing type：Research paper (scientific journal)  

The plasma membrane is generally associated with underling actin cytoskeleton. When the plasma membrane detaches from actin filaments, it is expanded by the intracellular pressure and the spherical membrane protrusion which lacks underlying actin cortex, termed bleb, is formed. Bleb is widely used for migration across species; however, the molecular mechanism underlying membrane blebbing remains largely unknown. Our recent study revealed that 2 small GTPases, Rnd3 and RhoA, are important regulators of membrane blebbing. In the expanding blebs, Rnd3 is recruited to the plasma membrane and inhibits RhoA activity by activating RhoGAP. On the other hand, RhoA is activated at the retracting membrane and removes Rnd3 from plasma membrane by the activity of ROCK (Rho-associated protein kinase). ROCK is also important for the rapid reassembly of actin cortex and retraction of membrane blebs by activating Ezrin. We propose that a Rnd3 and RhoA cycle underlies the core machinery of continuous membrane blebbing.

DOI： 10.1080/21541248.2016.1199266

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/15384101.2016.1204860

Language：English   Publishing type：Research paper (scientific journal)  

The actin cytoskeleton usually lies beneath the plasma membrane. When the membrane-associated actin cytoskeleton is transiently disrupted or the intracellular pressure is increased, the plasma membrane detaches from the cortex and protrudes. Such protruded membrane regions are called blebs. However, the molecular mechanisms underlying membrane blebbing are poorly understood. This study revealed that epidermal growth factor receptor kinase substrate 8 (Eps8) and ezrin are important regulators of rapid actin reassembly for the initiation and retraction of protruded blebs. Live-cell imaging of membrane blebbing revealed that local reassembly of actin filaments occurred at Eps8- and activated ezrin-positive foci of membrane blebs. Furthermore, we found that a RhoA-ROCK-Rnd3 feedback loop determined the local reassembly sites of the actin cortex during membrane blebbing.

DOI： 10.1073/pnas.1600968113

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1083/jcb.201603107

Language：English   Publishing type：Research paper (scientific journal)  

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 Ca²⁺ /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.

DOI： 10.1038/srep13262

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/j.chembiol.2015.04.011.

Language：English   Publishing type：Research paper (scientific journal)  

Roles of lipids in the cell membrane are poorly understood. This is partially due to the lack of methodologies, for example, tool chemicals that bind to specific membrane lipids and modulate membrane function. Theonellamides (TNMs), marine sponge-derived peptides, recognize 3β-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells through as yet unknown mechanisms. Here, we show that TNMs recognize cholesterol-containing liquid-disordered domains and induce phase separation in model lipid membranes. Modulation of membrane order was also observed in living cells following treatment with TNM-A, in which cells shrank considerably in a cholesterol-, cytoskeleton-, and energy-dependent manner. These findings present a previously unrecognized mode of action of membrane-targeting natural products. Meanwhile, we demonstrated the importance of membrane order, which is maintained by cholesterol, for proper cell morphogenesis.

DOI： 10.1016/j.chembiol.2015.04.011

Language：English  

At the initial stage of carcinogenesis, a mutation occurs in a single cell within a normal epithelial layer. We have previously shown that RasV12-transformed cells are apically extruded from the epithelium when surrounded by normal cells. However, the molecular mechanisms underlying this phenomenon remain elusive. Here, we demonstrate that Cav-1-containing microdomains and EPLIN (also known as LIMA1) are accumulated in RasV12-transformed cells that are surrounded by normal cells. We also show that knockdown of Cav-1 or EPLIN suppresses apical extrusion of RasV12-transformed cells, suggesting their positive role in the elimination of transformed cells from epithelia. EPLIN functions upstream of Cav-1 and affects its enrichment in RasV12-transformed cells that are surrounded by normal cells. Furthermore, EPLIN regulates non-cell-autonomous activation of myosin-II and protein kinase A (PKA) in RasV12-transformed cells. In addition, EPLIN substantially affects the accumulation of filamin A, a vital player in epithelial defense against cancer (EDAC), in the neighboring normal cells, and vice versa. These results indicate that EPLIN is a crucial regulator of the interaction between normal and transformed epithelial cells.

DOI： 10.1242/jcs.163113

Language：English   Publishing type：Research paper (scientific journal)  

At the initial stage of carcinogenesis, a mutation occurs in a single cell within a normal epithelial layer. We have previously shown that RasV12-transformed cells are apically extruded from the epithelium when surrounded by normal cells. However, the molecular mechanisms underlying this phenomenon remain elusive. Here, we demonstrate that Cav-1-containing microdomains and EPLIN (also known as LIMA1) are accumulated in RasV12-transformed cells that are surrounded by normal cells. We also show that knockdown of Cav-1 or EPLIN suppresses apical extrusion of RasV12-transformed cells, suggesting their positive role in the elimination of transformed cells from epithelia. EPLIN functions upstream of Cav-1 and affects its enrichment in RasV12-transformed cells that are surrounded by normal cells. Furthermore, EPLIN regulates non-cell-autonomous activation of myosin-II and protein kinase A (PKA) in RasV12-transformed cells. In addition, EPLIN substantially affects the accumulation of filamin A, a vital player in epithelial defense against cancer (EDAC), in the neighboring normal cells, and vice versa. These results indicate that EPLIN is a crucial regulator of the interaction between normal and transformed epithelial cells.

DOI： 10.1242/jcs.163113

Language：English  

When the surface view of each epithelial cell is compared with a polygon, its sides correspond to cell-cell junctions, whereas its vertices correspond to tricellular contacts, whose roles in epithelial cell morphogenesis have not been well studied. Here, we show that tricellulin (also known as MARVELD2), which is localized at tricellular contacts, regulates F-actin organization through Cdc42. Tricellulin-knockdown epithelial cells exhibit irregular polygonal shapes with curved cell borders and impaired organization of F-actin fibers around tricellular contacts during cell-cell junction formation. The N-terminal cytoplasmic domain of tricellulin binds to the Cdc42 guanine-nucleotide-exchange factor (GEF) Tuba (also known as DNMBP and ARHGEF36), and activates Cdc42. A tricellulin mutant that lacks the ability to bind Tuba cannot rescue the curved cell border phenotype of tricellulin-knockdown cells. These findings indicate that tricellular contacts play crucial roles in regulating the actomyosin-mediated apical junctional complex tension through the tricellulin-Tuba-Cdc42 system.

DOI： 10.1242/jcs.150607

Language：English   Publishing type：Research paper (scientific journal)  

When the surface view of each epithelial cell is compared with a polygon, its sides correspond to cell-cell junctions, whereas its vertices correspond to tricellular contacts, whose roles in epithelial cell morphogenesis have not been well studied. Here, we show that tricellulin (also known as MARVELD2), which is localized at tricellular contacts, regulates F-actin organization through Cdc42. Tricellulin-knockdown epithelial cells exhibit irregular polygonal shapes with curved cell borders and impaired organization of Factin fibers around tricellular contacts during cell-cell junction formation. The N-terminal cytoplasmic domain of tricellulin binds to the Cdc42 guanine-nucleotide-exchange factor (GEF) Tuba (also known as DNMBP and ARHGEF36), and activates Cdc42. A tricellulin mutant that lacks the ability to bind Tuba cannot rescue the curved cell border phenotype of tricellulin-knockdown cells. These findings indicate that tricellular contacts play crucial roles in regulating the actomyosin-mediated apical junctional complex tension through the tricellulin-Tuba-Cdc42 system.

DOI： 10.1242/jcs.150607

Language：English   Publishing type：Research paper (scientific journal)  

Cellular architectures require regulated mechanisms to correctly localize the appropriate plasma membrane lipids and proteins. Microvilli are dynamic filamentous-actin-based protrusions of the plasma membrane that are found in the apical membrane of epithelial cells. However, it remains poorly understood how their formation is regulated. In the present study, we found that sphingomyelin clustering underlies the formation of microvilli. Clustering of sphingomyelin is required for the co-clustering of the sialomucin membrane protein podocalyxin-1 at microvilli. Podocalyxin-1 recruits ezrin/radixin/moesin (ERM)-binding phosphoprotein-50 (EBP50; also known as NHERF1), which recruits ERM proteins and phosphatidylinositol 4-phosphate 5-kinase b (PIP5Kβ). Thus, clustering of PIP5Kβ leads to local accumulation of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], which enhances the accumulation of ERM family proteins and induces the formation of microvilli. The present study revealed novel interactions between sphingomyelin and the cytoskeletal proteins from which microvilli are formed, and it clarified the physiological importance of the chemical properties of sphingomyelin that facilitate cluster formation.

DOI： 10.1242/jcs.122325

Language：English   Publishing type：Research paper (scientific journal)  

Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and-2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER-mitochondrial communication-as measured by cholesteryl ester and phospholipid synthesis, respectively-are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER-mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.

DOI： 10.1038/emboj.2012.202

Language：English   Publishing type：Research paper (scientific journal)  

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). Whenepithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.

DOI： 10.1074/jbc.M111.327064

Language：English   Publishing type：Research paper (scientific journal)  

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin-catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin-catenin system and may control cadherin-mediated cell-cell adhesion. (C) 2010 Elsevier Inc. All rights reserved

DOI： 10.1016/j.yexcr.2010.12.004

Other Link： http://www.sciencedirect.com/science/article/pii/S0014482710005628

Language：English   Publishing type：Research paper (scientific journal)  

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin-catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin-catenin system and may control cadherin-mediated cell-cell adhesion.

DOI： 10.1016/j.yexcr.2010.12.004

Language：English   Publishing type：Research paper (scientific journal)  

Epithelial cell contacts consist of not only bicellular contacts but also tricellular contacts, where the corners of three cells meet. At tricellular contacts, tight junctions (TJs) generate specialized structures termed tricellular TJs (tTJs) to seal the intercellular space. Tricellulin is the only known molecular component of tTJs and is involved in the formation of tTJs, as well as in the normal epithelial barrier function. However, the detailed molecular mechanism of how tTJs are formed and maintained remains elusive. Using a localization-based expression cloning method, we identified a novel tTJ-associated protein known as lipolysis-stimulated lipoprotein receptor (LSR). Upon LSR knockdown in epithelial cells, tTJ formation was affected and the epithelial barrier function was diminished. Tricellulin accumulation at the tricellular contacts was also diminished in these cells. By contrast, LSR still accumulated at the tricellular contacts upon tricellulin knockdown. Analyses of deletion mutants revealed that the cytoplasmic domain of LSR was responsible for the recruitment of tricellulin. On the basis of these observations, we propose that LSR defines tricellular contacts in epithelial cellular sheets by acting as a landmark to recruit tricellulin for tTJ formation.

DOI： 10.1242/jcs.072058

Language：English   Publishing type：Research paper (scientific journal)  

The Par-3/Par-6/aPKC/Cdc42 complex regulates the conversion of primordial adherens junctions (AJs) into belt-like AJs and the formation of linear actin cables during epithelial polarization. However, the mechanisms by which this complex functions are not well elucidated. In the present study, we found that activation of Arf6 is spatiotemporally regulated as a downstream signaling pathway of the Par protein complex. When primordial AJs are formed, Par-3 recruits a scaffolding protein, termed the FERM domain containing 4A (FRMD4A). FRMD4A connects Par-3 and the Arf6 guanine-nucleotide exchange factor (GEF), cytohesin-1. We propose that the Par-3/FRMD4A/cytohesin-1 complex ensures accurate activation of Arf6, a central player in actin cytoskeleton dynamics and membrane trafficking, during junctional remodeling and epithelial polarization.

DOI： 10.1073/pnas.0908423107

Language：English   Publishing type：Research paper (scientific journal)  

The tricellular tight junction (tTJ) forms at the convergence of bicellular tight junctions (bTJs) where three epithelial cells meet in polarized epithelia, and it is required for the maintenance of the transepithelial barrier. Tricellulin is a four transmembrane domain protein recently identified as the first marker of tTJ, but little is known about how tricellulin is localized at tTJs. As for the molecular mechanism of association of tricellulin with tight junctions (TJs), we found that tricellulin was incorporated into claudin-based TJs independently of binding to zona occludens-1. Unexpectedly, exogenous expression of tricellulin increased cross-links of TJ strands in the plasma membrane. As for the molecular mechanisms for localization of tricellulin at tricellular junctions, we found that knockdown of occludin caused mislo-calization of tricellulin to bTJs, implying that occludin supports tricellular localization of tricellulin by excluding tricellulin from bTJs.

DOI： 10.1091/mbc.E08-05-0530

Language：English   Publishing type：Research paper (scientific journal)  

β-Catenin functions as a transcriptional regulator in Wnt signaling. Its function is regulated by a specific destruction system. Plakoglobin is a close homologue of β-catenin in mammalian cells and is regulated in a similar fashion. When β-catenin or plakoglobin is exogenously expressed in cells, endogenous β-catenin is stabilized, which complicates estimation of the transcriptional activities of exogenously expressed proteins. To facilitate the design of experiments aimed at investigating the transcriptional activities of β-catenin and plakoglobin, we utilized F9 cells in which we knocked out endogenous β-catenin and/or plakoglobin by gene deletion and exogenously expressed wild-type and mutant β-catenin and/or plakoglobin. We show that C-terminally deleted β-catenin, but not plakoglobin, has a strong dominant-negative eifect on transcription without altering the nuclear accumulation of β-catenin. Moreover, we show that Wnt-3a activation of LEF/T-cell factor (TCF)-dependent transcription depends on β-catenin but not on plakoglobin. Using chimeras of β-catenin and plakoglobin, we demonstrate that plakoglobin has the potential to function in transcriptional regulation but is not responsible for Wnt-3a signaling in F9 cells. Our data show that preferential nuclear accumulation of β-catenin is not necessarily linked to its transcriptional activity. We also clearly demonstrate that plakoglobin is insufficient for LEF/TCF-dependent transcriptional activation by Wnt-3a in F9 cells.

DOI： 10.1128/MCB.02375-06

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

The molecular mechanisms of how primordial adherens junctions (AJs) evolve into spatially separated belt-like AJs and tight junctions (TJs) during epithelial polarization are not well understood. Previously, we reported the establishment of ZO-1/ZO-2-deficient cultured epithelial cells (1[ko]/2[kd] cells), which lacked TJs completely. In the present study, we found that the formation of belt-like AJs was significantly delayed in 1(ko)/2(kd) cells during epithelial polarization. The activation of Rac1 upon primordial AJ formation is severely impaired in 1(ko)/2(kd) cells. Our data indicate that ZO-1 plays crucial roles not only in TJ formation, but also in the conversion from "fi broblastic" AJs to belt-like "polarized epithelial" AJs through Rac1 activation. Furthermore, to examine whether ZO-1 itself mediate belt-like AJ and TJ formation, respectively, we performed a mutational analysis of ZO-1. The requirement for ZO-1 differs between belt-like AJ and TJ formation. We propose that ZO-1 is directly involved in the establishment of two distinct junctional domains, belt-like AJs and TJs, during epithelial polarization.

DOI： 10.1083/jcb.200612080

Language：English   Publishing type：Research paper (scientific journal)  

A fundamental question in cell and developmental biology is how epithelial cells construct the diffusion barrier allowing them to separate different body compartments. Formation of tight junction (TJ) strands, which are crucial for this barrier, involves the polymerization of claudins, TJ adhesion molecules, in temporal and spatial manners. ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, yet their functional roles are poorly understood. We established cultured epithelial cells (1(ko)/2(kd)) in which the expression of ZO-1/ZO-2 was suppressed by homologous recombination and RNA interference, respectively. These cells were well polarized, except for a complete lack of TJs. When exogenously expressed in 1(ko)/2(kd) cells, ZO-1 and ZO-2 were recruited to junctional areas where claudins were polymerized, but truncated ZO-1 (NZO-1) containing only domains PDZ1-3 was not. When NZO-1 was forcibly recruited to lateral membranes and dimerized, claudins were dramatically polymerized. These findings indicate that ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized.

DOI： 10.1016/j.cell.2006.06.043

Language：English   Publishing type：Research paper (scientific journal)  

For epithelia to function as barriers, the intercellular space must be sealed. Sealing two adjacent cells at bicellular tight junctions (bTJs) is well described with the discovery of the claudins. Yet, there are still barrier weak points at tricellular contacts, where three cells join together. In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts. When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized. These findings indicate the critical function of tricellulin for formation of the epithelial barrier.

DOI： 10.1083/jcb.200510043

Language：English   Publishing type：Research paper (scientific journal)  

Epithelium formation is a common event in animal morphogenesis. It has been reported that F9 cells differentiate into visceral endoderm-like epithelial cells when cell aggregates are cultured in the presence of retinoic acid. The present investigation set out to determine whether this in vitro model could be used under monolayer culture conditions, which is suitable for a detailed analysis of epithelial differentiation. We performed comparative gene expression analyses of F9 cells grown under aggregate and monolayer culture conditions prior to and following treatment with retinoic acid. Under these conditions, induction in the expression of differentiation marker genes was confirmed, even in monolayer cultures. Junctional complex and apical membrane formation, both of which are characteristic of epithelial cells, were also observed under monolayer culture conditions. Because of the merit of monolayer culture condition, we found that apical membrane and junctional complex formation are strictly regulated during epithelial differentiation. It was also revealed that F9 cells differentiated into epithelial cells predominantly on the fourth and fifth day following retinoic acid induction. These results showed that a monolayer culture of F9 cells represents a viable in vitro model that can be employed to elucidate mechanisms pertaining to epithelium formation.

DOI： 10.1111/j.1365-2443.2005.00899.x

Language：English   Publishing type：Research paper (scientific journal)  

Snail is a transcription repressor that plays a central role in the epithelium-mesenchyme transition (EMT), by which epithelial cells lose their polarity. Claudins and occludin are integral membrane proteins localized at tight junctions, which are responsible for establishing and maintaining epithelial cell polarity. We examined the relationship between Snail and the promoter activity of claudins and occludin. When Snail was overexpressed in cultured mouse epithelial cells, EMT was induced with concomitant repression of the expression of claudins and occludin not only at the protein but also at the mRNA level. We then isolated the promoters of genes encoding claudins and occludin, in which multiple E-boxes were identified. Transfection experiments with various promoter constructs as well as electrophoretic mobility assays revealed that Snail binds directly to the E-boxes of the promoters of claudin/occludin genes, resulting in complete repression of their promoter activity. Because the gene encoding E-cadherin was also reported to be repressed by Snail, we concluded that EMT was associated with the simultaneous repression of the genes encoding E-cadherin and claudins/occludin (i.e. the expression of adherens and tight junction adhesion molecules, respectively).

DOI： 10.1242/jcs.00389

Language：English   Publishing type：Research paper (scientific journal)  

In a large collection of human embryos (the Kyoto Collection of Human Embryos, Kyoto University), we encountered five cases with abnormal dilatation of the neural tube at the lumbosacral level. In these examples, the central canal was enlarged, and the roof plate of the neural tube was extremely thin and expanded. The mesenchymal tissue was scarce or lacking between the roof plate and the surface ectoderm. This type of anomaly was assumed to be formed after neural tube closure and may be an early form of spina bifida. In two of the cases, some abnormal cells were found ectopically between the thin roof plate and the surface ectoderm. Morphologically, these cells resembled those forming spinal ganglia and could be of the neural crest origin. Since neural crest cells are pluripotent and can differentiate into a variety of tissues, such ectopic cells might undergo abnormal differentiation into teratomatous tumors and/or lipomas, which are frequently associated with spina bifida. We also discuss the definition of spina bifida and the classification of neural tube defects from the embryological and pathogenic viewpoints and propose a new classification of neural tube defects.

DOI： 10.1007/s00401-001-0465-9

Event date： 2023.7

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：名古屋   Country：Japan  

Event date： 2023.6

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：奈良   Country：Japan  

Event date： 2022.11 - 2022.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：千葉   Country：Japan  

Event date： 2022.9

Language：English   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2021.12

Language：English   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2021.9

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.6 - 2021.7

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：Japan  

Event date： 2020.12

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Online   Country：Japan  

Event date： 2020.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：Online   Country：Japan  

Event date： 2020.8

Language：English   Presentation type：Oral presentation (general)  

Venue：Online   Country：Japan  

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2013.11

Language：English   Presentation type：Oral presentation (general)  

Venue：Toulouse   Country：France  

Language：Japanese  

Venue：神戸   Country：Japan  

細胞膜を構成している膜タンパク質や脂質は膜上で区画化され存在している。細胞膜には区画化を担うDiffusion Barrierが存在すると考えられている。特に明確な細胞膜構造を示すDiffusion Barrierの例として、上皮細胞のタイトジャンクションや神経細胞のaxon initial segment等が挙げられる。これらは、いずれも細胞の極性の形成・維持や細胞の機能発現において必要不可欠な構造である。しかしながらDiffusion Barrierの分子的実体はほとんど明らかになっていない。これまでの研究により、Diffusion Barrierの形成に、細胞膜を裏打ちするアクチンフィラメントが関与していることが示唆されている。細胞内部の構造であるアクチンフィラメントが、どのようにして細胞膜の脂質分子の動態を制御しうるかについては明らかになっていない。本研究において、我々はアクチンフィラメントを架橋し網目構造を形成させるタンパク質フィラミンの発現を消失させた細胞を用いて、細胞膜の直下に存在するアクチンフィラメントの有無が細胞膜脂質の分布や動態にどのような影響をもたらすかについて解析を行った。本発表では、現在までの観察結果とそれらを踏まえた考察について紹介する。

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Language：Japanese  

Language：Japanese  

Language：English  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：English  

Language：English  

Language：Japanese  

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review