Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion.

Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

Discovery of anti-inflammatory physiological peptides that promote tissue repair by reinforcing epithelial barrier formation.

Epithelial barriers that prevent dehydration and pathogen invasion are established by tight junctions (TJs), and their disruption leads to various inflammatory diseases and tissue destruction. However, a therapeutic strategy to overcome TJ disruption in diseases has not been established because of the lack of clinically applicable TJ-inducing molecules. Here, we found TJ-inducing peptides (JIPs) in mice and humans that corresponded to 35 to 42 residue peptides of the C terminus of alpha 1-antitrypsin (A1AT), an acute-phase anti-inflammatory protein. JIPs were inserted into the plasma membrane of epithelial cells, which promoted TJ formation by directly activating the heterotrimeric G protein G13. In a mouse intestinal epithelial injury model established by dextran sodium sulfate, mouse or human JIP administration restored TJ integrity and strongly prevented colitis. Our study has revealed TJ-inducing anti-inflammatory physiological peptides that play a critical role in tissue repair and proposes a previously unidentified therapeutic strategy for TJ-disrupted diseases.

The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts.

Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif-containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.

Pregnenolone functions in centriole cohesion during mitosis.

Cell division is controlled by a multitude of protein enzymes, but little is known about roles of metabolites in this mechanism. Here, we show that pregnenolone (P5), a steroid that is produced from cholesterol by the steroidogenic enzyme Cyp11a1, has an essential role in centriole cohesion during mitosis. During prometa-metaphase, P5 is accumulated around the spindle poles. Depletion of P5 induces multipolar spindles that result from premature centriole disengagement, which are rescued by ectopic introduction of P5, but not its downstream metabolites, into the cells. Premature centriole disengagement, induced by loss of P5, is not a result of precocious activation of separase, a key factor for the centriole disengagement in anaphase. Rather, P5 directly binds to the N-terminal coiled-coil domain of short-form of shugoshin 1 (sSgo1), a protector for centriole cohesion and recruits it to spindle poles in mitosis. Our results thus reveal a steroid-mediated centriole protection mechanism.

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.

Molecular organization of tricellular tight junctions.

When the apicolateral border of epithelial cells is compared with a polygon, its sides correspond to the apical junctional complex, where cell adhesion molecules assemble from the plasma membranes of two adjacent cells. On the other hand, its vertices correspond to tricellular contacts, where the corners of three cells meet. Vertebrate tricellular contacts have specialized structures of tight junctions, termed tricellular tight junctions (tTJs). tTJs were identified by electron microscopic observations more than 40 years ago, but have been largely forgotten in epithelial cell biology since then. The identification of tricellulin and angulin family proteins as tTJ-associated membrane proteins has enabled us to study tTJs in terms of not only the paracellular barrier function but also unknown characteristics of epithelial cell corners via molecular biological approaches.

JNK1/2-dependent phosphorylation of angulin-1/LSR is required for the exclusive localization of angulin-1/LSR and tricellulin at tricellular contacts in EpH4 epithelial sheet.

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks.

Analysis of the 'angulin' proteins LSR, ILDR1 and ILDR2--tricellulin recruitment, epithelial barrier function and implication in deafness pathogenesis.

Tricellular tight junctions (tTJs) seal the extracellular space at tricellular contacts (TCs), where the corners of three epithelial cells meet. To date, the transmembrane proteins tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) are known to be molecular components of tTJs. LSR recruits tricellulin to tTJs, and both proteins are required for the full barrier function of epithelial cellular sheets. In the present study, we show that two LSR-related proteins, immunoglobulin-like domain-containing receptor (ILDR) 1 and ILDR2, are also localized at TCs and recruit tricellulin. At least one of LSR, ILDR1 and ILDR2 was expressed in most of the epithelial tissues in mice. The expressions of LSR, ILDR1 and ILDR2 were generally complementary to each other, although LSR and ILDR1 were co-expressed in some epithelia. ILDR1 was required for the establishment of a strong barrier of the epithelium, similar to LSR, when introduced into cultured epithelial cells, whereas ILDR2 provided a much weaker barrier. We further analyzed human ILDR1, mutations in which cause a familial deafness, DFNB42, and found that most DFNB42-associated ILDR1 mutant proteins were defective in recruitment of tricellulin. We also found that tricellulin mutant proteins associated with another familial deafness, DFNB49, were not recruited to TCs by ILDR1. These findings show the heterogeneity of the molecular organization of tTJs in terms of the content of LSR, ILDR1 or ILDR2, and suggest that ILDR1-mediated recruitment of tricellulin to TCs is required for hearing. Given their common localization at epithelial cell corners and recruitment of tricellulin, we propose to designate LSR, ILDR1 and ILDR2 as angulin family proteins.

Lipolysis-stimulated lipoprotein receptor: a novel membrane protein of tricellular tight junctions.

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions that restrict the free diffusion of solutes at the extracellular space of tricellular contacts. Their presence at cell corners, situated in the angles between three adjacent epithelial cells, was identified early by electron microscopy, but despite their potential importance, tTJs have been generally ignored in epithelial cell biology. Tricellulin was the first molecular component of tTJs shown to be involved in their formation and in epithelial barrier function. However, the precise molecular organization and function of tTJs are still largely unknown. Recently, we identified the lipolysis-stimulated lipoprotein receptor (LSR) as a tTJ-associated membrane protein. LSR recruits tricellulin to tTJs, suggesting that the LSR-tricellulin system plays a key role in tTJ formation. In this paper, we summarize the identification and characterization of LSR as a molecular component of tTJs.

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.

The E3 ubiquitin ligase LNX1p80 promotes the removal of claudins from tight junctions in MDCK cells.

The structural continuity of tight junctions (TJs) is consistently maintained even when epithelial cells divide and move within the cellular sheet. This process is associated with dynamic remodeling of TJs by coordinated internalization and generation of claudin-based TJ strands, but the molecular mechanism behind the regulated turnover of TJs remains largely unknown. In this study, we identified the p80 isoform of the E3 ubiquitin ligase ligand of Numb-protein X1 (LNX1p80) as a protein binding to claudin-1. Interestingly, the concentration of claudins in TJs was remarkably reduced when LNX1p80 was overexpressed in MDCK cells, and there was a reduction not only in the number of TJ strands but also in the amount of detergent-insoluble claudins. We also found that LNX1p80 promoted polyubiquitylation of claudins. This ubiquitylation is dependent on its RING-finger domain and is not mediated by Lys48 of ubiquitin, which is used for protein degradation by the proteasome. Furthermore, LNX1p80 was often colocalized with claudins in vesicular structures containing markers for late endosomes and lysosomes. These findings suggest that LNX1p80 is involved in the ubiquitylation, endocytosis and lysosomal degradation of claudins, and that the turnover of TJs is regulated by ubiquitylation.

Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508.

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRDeltaF508, by specifically promoting ubiquitylation of CFTRDeltaF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRDeltaF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRDeltaF508 and catalyzes further polyubiquitylation of CFTRDeltaF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRDeltaF508.

MKKS is a centrosome-shuttling protein degraded by disease-causing mutations via CHIP-mediated ubiquitination.

McKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin-proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.

Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation.

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be refolded or degraded to maintain the homeostasis of the ER. Components of both productive folding and ER-associated degradation (ERAD) mechanisms are known to be up-regulated by the unfolded protein response (UPR). We describe two novel components of mammalian ERAD, Derlin-2 and -3, which show weak homology to Der1p, a transmembrane protein involved in yeast ERAD. Both Derlin-2 and -3 are up-regulated by the UPR, and at least Derlin-2 is a target of the IRE1 branch of the response, which is known to up-regulate ER degradation enhancing alpha-mannosidase-like protein (EDEM) and EDEM2, receptor-like molecules for misfolded glycoprotein. Overexpression of Derlin-2 or -3 accelerated degradation of misfolded glycoprotein, whereas their knockdown blocked degradation. Derlin-2 and -3 are associated with EDEM and p97, a cytosolic ATPase responsible for extraction of ERAD substrates. These findings indicate that Derlin-2 and -3 provide the missing link between EDEM and p97 in the process of degrading misfolded glycoproteins.

EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin.

Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin.