Cytoplasmic zoning in membrane blebs.

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.

Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells.

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.

Tricellulin secures the epithelial barrier at tricellular junctions by interacting with actomyosin.

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.

A Clockwork Bleb: cytoskeleton, calcium, and cytoplasmic fluidity.

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.

MAGIs regulate aPKC to enable balanced distribution of intercellular tension for epithelial sheet homeostasis.

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.

STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing.

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.

Coordinated changes in cell membrane and cytoplasm during maturation of apoptotic bleb.

Apoptotic cells form membrane blebs, but little is known about how the formation and dynamics of membrane blebs are regulated. The size of blebs gradually increases during the progression of apoptosis, eventually forming large extracellular vesicles called apoptotic bodies that have immune-modulating activities. In this study, we investigated the molecular mechanism involved in the differentiation of blebs into apoptotic blebs by comparing the dynamics of the bleb formed during cell migration and the bleb formed during apoptosis. We revealed that the enhanced activity of ROCK1 is required for the formation of small blebs in the early phase of apoptosis, which leads to the physical disruption of nuclear membrane and the degradation of Lamin A. In the late phase of apoptosis, the loss of asymmetry in phospholipids distribution caused the enlargement of blebs, which enabled translocation of damage-associated molecular patterns to the bleb cytoplasm and maturation of functional apoptotic blebs. Thus, changes in cell membrane dynamics are closely linked to cytoplasmic changes during apoptotic bleb formation.

Cell Adhesion Structures in Epithelial Cells Are Formed in Dynamic and Cooperative Ways.

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.

Roles of membrane lipids in the organization of epithelial cells: Old and new problems.

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.

α-Catenin Controls the Anisotropy of Force Distribution at Cell-Cell Junctions during Collective Cell Migration.

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.

Adherens junctions influence tight junction formation via changes in membrane lipid composition.

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.

Regulation of the epithelial barrier by post-translational modifications of tight junction membrane proteins.

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.

DAAM1 stabilizes epithelial junctions by restraining WAVE complex-dependent lateral membrane motility.

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.

CaMKII regulates the strength of the epithelial barrier.

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.

Targeting cholesterol in a liquid-disordered environment by theonellamides modulates cell membrane order and cell shape.

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 3ß-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.

EPLIN is a crucial regulator for extrusion of RasV12-transformed cells.

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.

Tricellulin regulates junctional tension of epithelial cells at tricellular contacts through Cdc42.

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.

Lipid polarity is maintained in absence of tight junctions.

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.

LSR defines cell corners for tricellular tight junction formation in epithelial cells.

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.

Phosphorylation state regulates the localization of Scribble at adherens junctions and its association with E-cadherin-catenin complexes.

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.