Establishment Of Apical-basal Polarity Research Articles

Low-input lipidomics reveals lipid metabolism remodelling during early mammalian embryo development.

Lipids are indispensable for energy storage, membrane structure and cell signalling. However, dynamic changes in various categories of endogenous lipids in mammalian early embryonic development have not been systematically characterized. Here we comprehensively investigated the dynamic lipid landscape during mouse and human early embryo development. Lipid signatures of different developmental stages are distinct, particularly for the phospholipid classes. We highlight that the high degree of phospholipid unsaturation is a conserved feature as embryos develop to the blastocyst stage. Moreover, we show that lipid desaturases such as SCD1 are required for in vitro blastocyst development and blastocyst implantation. One of the mechanisms is through the regulation of unsaturated fatty-acid-mediated fluidity of the plasma membrane and apical proteins and the establishment of apical-basal polarity during development of the eight-cell embryo to the blastocyst. Overall, our study provides an invaluable resource about the remodelling of the endogenous lipidome in mammalian preimplantation embryo development and mechanistic insights into the regulation of embryogenesis and implantation by lipid unsaturation.

Scribble and Discs-large direct initial assembly and positioning of adherens junctions during the establishment of apical-basal polarity.

Apical-basal polarity is a fundamental property of animal tissues. Drosophila embryos provide an outstanding model for defining mechanisms that initiate and maintain polarity. Polarity is initiated during cellularization, when cell-cell adherens junctions are positioned at the future boundary of apical and basolateral domains. Polarity maintenance then involves complementary and antagonistic interplay between apical and basal polarity complexes. The Scribble/Dlg module is well-known for promoting basolateral identity during polarity maintenance. Here, we report a surprising role for Scribble/Dlg in polarity initiation, placing it near the top of the network-positioning adherens junctions. Scribble and Dlg are enriched in nascent adherens junctions, are essential for adherens junction positioning and supermolecular assembly, and also play a role in basal junction assembly. We test the hypotheses for the underlying mechanisms, exploring potential effects on protein trafficking, cytoskeletal polarity or Par-1 localization/function. Our data suggest that the Scribble/Dlg module plays multiple roles in polarity initiation. Different domains of Scribble contribute to these distinct roles. Together, these data reveal novel roles for Scribble/Dlg as master scaffolds regulating assembly of distinct junctional complexes at different times and places.

Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

Establishment of apical-basal polarity, through correct targeting of polarity determinants to distinct domains of the plasma membrane, is a fundamental process for the development of functioning epithelial tubules. Here we report that galectin (Gal)-8 regulates apical-basal polarity of Madin-Darby canine kidney (MDCK) cells via apical targeting of 135-kDa glycoprotein (Gp135). Gal-8 interacts with newly synthesized Gp135 in a glycan-dependent manner. Gal-8 knockdown induces aberrant lumens at the lateral domain and mistargeting of Gp135 to this structure, thus disrupting the kidney epithelial polarity of MDCK cells, which organize lumens at the apical surface. The O-glycosylation deletion mutant of Gp135 phenocopies the effect of Gal-8 knockdown, which suggests that Gal-8 is the decoding machinery for the apical sorting signals of Gp135 residing at its O-glycosylation-rich region. Collectively, our results reveal a new role of Gal-8 in the development of luminal organs by regulating targeting of apical polarity protein Gp135.-Lim, H., Yu, C.-Y., Jou, T.-S. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment.

Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.

Membrane palmitoylated proteins regulate trafficking and processing of nectins

Nectins are cell–cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell–cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell–cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell–cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell–cell junctions and that these associations may regulate trafficking and processing of nectins.

O26. Role of cell division during neural tube formation in the zebrafish

The vertebrate neural tube is a highly polarised tissue consisting of epithelial cells organised around a central lumen with their apical surfaces facing the lumen. In zebrafish the neural tube is generated by converting a soild neural rod into a tube. During rod stage, a polarised midline becomes established and eventually a small lumen opens in a roughly ventral to dorsal progression. The lumen inflates to become the ventricle later in development. We have previously suggested the transition from rod to tube is orchestrated by an unique mirror-symmetric cell division which deposits one daughter cell on either side of the future midline, and at the same time establishes the apical nature of the midline by depositing Par3 protein at the cleavage plane. To investigate the advantages of this novel division in establishment of apical-basal polarity and lumen formation we have inhibited cell division and examined the consequences on neural tube formation. We find that cells can still polarize when division is blocked, however polarization and cellular organization at the midline is more disorganized and lumen formation is disrupted. To better understand the roles of the mirror-symmetric division, we are using time-lapse microscopy to compare cell behaviour and polarization between wild-type and division-blocked embryos and we observe novel cell behaviours that are helping us understand the mechanisms that direct cell polarization and morphogenesis of the neural tube lumen.

Cdc42 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells through PAK4 and Par6B

Cdc42 has been implicated in numerous biochemical pathways during epithelial morphogenesis, including the control of spindle orientation during mitosis, the establishment of apical-basal polarity, the formation of apical cell-cell junctions, and polarized secretion. To investigate the signaling pathways through which Cdc42 mediates these diverse effects, we have screened an siRNA library corresponding to the 36 known Cdc42 target proteins, in a human bronchial epithelial cell line. Two targets, PAK4 and Par6B, were identified as necessary for the formation of apical junctions. PAK4 is recruited to nascent cell-cell contacts in a Cdc42-dependent manner, where it is required for the maturation of primordial junctions into apical junctions. PAK4 kinase activity is essential for junction maturation, but overexpression of an activated PAK4 mutant disrupts this process. Par6B, together with its binding partner aPKC, is necessary both for junction maturation and for the retention of PAK4 at sites of cell-cell contact. This study demonstrates that controlled regulation of PAK4 is required for apical junction formation in lung epithelial cells and highlights potential cross-talk between two Cdc42 targets, PAK4 and Par6B.

Epithelial Polarity Proteins Regulate Drosophila Tracheal Tube Size in Parallel to the Luminal Matrix Pathway

Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). Here, we show that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. Our data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size.