RESUMEN
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.
Asunto(s)
Fosfoproteínas , Uniones Estrechas , Proteínas de la Zonula Occludens/metabolismo , Uniones Estrechas/metabolismo , Fosfoproteínas/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo , Claudinas/genética , Claudinas/metabolismoRESUMEN
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.
Asunto(s)
Anexina A2 , Animales , Ratones , Anexina A2/genética , Apoptosis , Células Epiteliales , Epitelio , Especies Reactivas de OxígenoRESUMEN
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.
Asunto(s)
Uniones Adherentes/fisiología , Adhesión Celular/fisiología , Membrana Celular/fisiología , Uniones Estrechas/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Claudinas/genética , Claudinas/metabolismo , Células Epiteliales/fisiología , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones NoqueadosRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Membrana Celular/química , Proteínas del Citoesqueleto/metabolismo , Retroalimentación Fisiológica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/genéticaRESUMEN
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.
Asunto(s)
Caveolina 1/genética , Transformación Celular Neoplásica/patología , Células Epiteliales/patología , Proteínas de Microfilamentos/genética , Neoplasias/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Butadienos/farmacología , Caveolas/metabolismo , Caveolina 1/metabolismo , Línea Celular , Cromonas/farmacología , Contactina 1/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Perros , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Filaminas/metabolismo , Sistema de Señalización de MAP Quinasas , Células de Riñón Canino Madin Darby , Proteínas de Microfilamentos/metabolismo , Morfolinas/farmacología , Miosina Tipo II/metabolismo , Nitrilos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/patología , Embrión de Mamíferos/patología , Fibroblastos/patología , Microdominios de Membrana/patología , Mitocondrias/patología , Membranas Mitocondriales/patología , Presenilina-1/fisiología , Presenilina-2/fisiología , Enfermedad de Alzheimer/metabolismo , Animales , Western Blotting , Células Cultivadas , Embrión de Mamíferos/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Fibroblastos/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Presenilina-1/antagonistas & inhibidores , Presenilina-2/antagonistas & inhibidores , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones SubcelularesRESUMEN
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.
Asunto(s)
Proteína 2 con Dominio MARVEL/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Células CACO-2 , Células Epiteliales/metabolismo , HumanosRESUMEN
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 ß (PIP5Kß). Thus, clustering of PIP5Kß 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.
Asunto(s)
Microvellosidades/metabolismo , Esfingomielinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Sialoglicoproteínas/metabolismoRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina , Actinas , Citoplasma/metabolismo , Citosol/metabolismo , Membrana Celular/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismoRESUMEN
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.
Asunto(s)
Colorantes Fluorescentes , Microscopía Fluorescente , Colorantes Fluorescentes/química , Humanos , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Membrana Celular/metabolismo , Membrana Celular/químicaRESUMEN
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). When epithelial 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.
Asunto(s)
Polaridad Celular , Esfingomielinas/metabolismo , Uniones Estrechas/metabolismo , Línea Celular , Membrana Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , HumanosRESUMEN
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.
Asunto(s)
Células Epiteliales/metabolismo , Espacio Extracelular/metabolismo , Receptores de LDL/metabolismo , Uniones Estrechas/metabolismo , Animales , Línea Celular Tumoral , Perros , Células Epiteliales/química , Espacio Extracelular/química , Espacio Extracelular/genética , Humanos , Proteína 2 con Dominio MARVEL , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína , Receptores de LDL/química , Receptores de LDL/genética , Uniones Estrechas/química , Uniones Estrechas/genéticaRESUMEN
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.
Asunto(s)
Uniones Adherentes/genética , Células Epiteliales/fisiología , Uniones Adherentes/fisiología , Polaridad CelularRESUMEN
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 "fibroblastic" 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.
Asunto(s)
Uniones Adherentes/metabolismo , Polaridad Celular/fisiología , Células Epiteliales/citología , Proteínas de la Membrana/fisiología , Fosfoproteínas/fisiología , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Uniones Adherentes/ultraestructura , Animales , Línea Celular , Células Epiteliales/ultraestructura , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Neuropéptidos/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1 , Proteína de la Zonula Occludens-2 , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1RESUMEN
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.
Asunto(s)
Uniones Adherentes/química , Cadherinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , alfa Catenina/metabolismo , Animales , Adhesión Celular , Técnicas de Cultivo de Célula , Células Epiteliales , Fibroblastos , Péptidos y Proteínas de Señalización Intracelular/análisis , Ratones , Fosforilación , Unión ProteicaRESUMEN
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.
Asunto(s)
Actinas , Calcio , Humanos , Actinas/genética , Actinas/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismoRESUMEN
Epithelial cells are constantly exposed to osmotic stress. The influx of water molecules into the cell in a hypo-osmotic environment increases plasma membrane tension as it rapidly expands. Therefore, the plasma membrane must be supplied with membrane lipids since expansion beyond its elastic limit will cause the cell to rupture. However, the molecular mechanism to maintain a constant plasma membrane tension is not known. In this study, we found that the apical membrane selectively expands when epithelial cells are exposed to hypo-osmotic stress. This requires the activation of mTORC2, which enhances the transport of secretory vesicles containing sphingomyelin, the major lipid of the apical membrane. We further show that the mTORC2-Rab35 axis plays an essential role in the defense against hypotonic stress by promoting the degradation of the actin cortex through the up-regulation of PI(4,5)P2 metabolism, which facilitates the apical tethering of sphingomyelin-loaded vesicles to relieve plasma membrane tension.
Asunto(s)
Esfingomielinas , Muerte Celular , Membrana Celular/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Presión Osmótica/fisiología , Esfingomielinas/metabolismoRESUMEN
The epithelial cell sheet functions as a barrier to prevent invasion of pathogens. It is necessary to eliminate intercellular gaps not only at bicellular junctions, but also at tricellular contacts, where three cells meet, to maintain epithelial barrier function. To that end, tight junctions between adjacent cells must associate as closely as possible, particularly at tricellular contacts. Tricellulin is an integral component of tricellular tight junctions (tTJs), but the molecular mechanism of its contribution to the epithelial barrier function remains unclear. In this study, we revealed that tricellulin contributes to barrier formation by regulating actomyosin organization at tricellular junctions. Furthermore, we identified α-catenin, which is thought to function only at adherens junctions, as a novel binding partner of tricellulin. α-catenin bridges tricellulin attachment to the bicellular actin cables that are anchored end-on at tricellular junctions. Thus, tricellulin mobilizes actomyosin contractility to close the lateral gap between the TJ strands of the three proximate cells that converge on tricellular junctions.
Asunto(s)
Actomiosina/metabolismo , Células Epiteliales/metabolismo , Proteína 2 con Dominio MARVEL/metabolismo , Uniones Estrechas/metabolismo , Actinas/metabolismo , Animales , Perros , Ratones , Unión Proteica , Vinculina/metabolismo , alfa Catenina/metabolismoRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Citoesqueleto de Actina , Actinas/metabolismo , Animales , Señalización del Calcio/fisiología , Línea Celular , Línea Celular Tumoral , Proteínas del Citoesqueleto/antagonistas & inhibidores , Células HEK293 , Humanos , Proteínas de la Membrana/fisiologíaRESUMEN
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.