Fluorescent Solvatochromic Probes for Long-Term Imaging of Lipid Order in Living Cells.

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.

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.

Cholesterol-rich domain formation mediated by ZO proteins is essential for tight junction formation.

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.

mTORC2 suppresses cell death induced by hypo-osmotic stress by promoting sphingomyelin transport.

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.

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.

Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation.

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 Ca2+ 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 Ca2+ 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.

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.

Emphatic visualization of sphingomyelin-rich domains by inter-lipid FRET imaging using fluorescent sphingomyelins.

Imaging the distribution of sphingomyelin (SM) in membranes is an important issue in lipid-raft research. Recently we developed novel fluorescent SM analogs that exhibit partition and dynamic behaviors similar to native SM, and succeeded in visualizing lateral domain-segregation between SM-rich liquid-ordered (Lo) and SM-poor liquid-disordered (Ld) domains. However, because the fluorescent contrast between these two domains depends directly on their partition ratio for the fluorescent SMs, domain-separation becomes indeterminate when the distribution difference is not great enough. In this study, we propose the use of inter-lipid Förster resonance energy transfer (FRET) imaging between fluorescent SMs to enhance the contrast of the two domains in cases in which the inter-domain difference in SM distribution is inadequate for conventional monochromic imaging. Our results demonstrate that inter-lipid FRET intensity was significantly higher in the Lo domain than in the Ld domain, resulting in a clear and distinguishable contrast between the two domains even in poorly phase-separated giant unilamellar vesicles. In addition, we show that inter-lipid FRET imaging is useful for selective visualization of highly condensed assemblies and/or clusters of SM molecules in living cell membranes. Thus, the inter-lipid FRET imaging technique can selectively emphasize the SM-condensed domains in both artificial and biological membranes.

Membrane bleb: A seesaw game of two small GTPases.

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.

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.