ABSTRACT
The subcellular distribution of the polarity protein Yurt (Yrt) is subjected to a spatio-temporal regulation in Drosophila melanogaster embryonic epithelia. After cellularization, Yrt binds to the lateral membrane of ectodermal cells and maintains this localization throughout embryogenesis. During terminal differentiation of the epidermis, Yrt accumulates at septate junctions and is also recruited to the apical domain. Although the mechanisms through which Yrt associates with septate junctions and the apical domain have been deciphered, how Yrt binds to the lateral membrane remains as an outstanding puzzle. Here, we show that the FERM domain of Yrt is necessary and sufficient for membrane localization. Our data also establish that the FERM domain of Yrt directly binds negatively charged phospholipids. Moreover, we demonstrate that positively charged amino acid motifs embedded within the FERM domain mediates Yrt membrane association. Finally, we provide evidence suggesting that Yrt membrane association is functionally important. Overall, our study highlights the molecular basis of how Yrt associates with the lateral membrane during the developmental time window where it is required for segregation of lateral and apical domains.
Subject(s)
Cell Membrane , Cell Polarity , Drosophila Proteins , Protein Domains , Animals , Amino Acid Motifs , Cell Membrane/metabolism , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/chemistry , Hydrophobic and Hydrophilic Interactions , Membrane Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/chemistry , Phospholipids/metabolism , Protein BindingABSTRACT
Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. Here we show that Drosophila Girdin and its human ortholog (GIRDIN) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, we observed cell dissemination from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, we discovered a core mechanism contributing to epithelial cell polarization from flies to humans. Our data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination.
Subject(s)
Drosophila Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Microfilament Proteins/metabolism , Vesicular Transport Proteins/metabolism , Animals , Caco-2 Cells , Cell Differentiation/physiology , Cell Polarity/physiology , Cell Proliferation/physiology , Drosophila Proteins/genetics , Drosophila melanogaster , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Membrane Proteins/metabolism , Microfilament Proteins/genetics , Morphogenesis/physiology , Protein Interaction Maps , Protein Kinase C/metabolism , Vesicular Transport Proteins/geneticsABSTRACT
Drosophila melanogaster Yurt (Yrt) and its mammalian orthologue EPB41L5 limit apical membrane growth in polarized epithelia. EPB41L5 also supports epithelial-mesenchymal transition and metastasis. Yrt and EPB41L5 contain a four-point-one, ezrin, radixin, and moesin (FERM) domain and a FERM-adjacent (FA) domain. The former contributes to the quaternary structure of 50 human proteins, whereas the latter defines a subfamily of 14 human FERM proteins and fulfills unknown roles. In this study, we show that both Yrt and EPB41L5 oligomerize. Our data also establish that the FERM-FA unit forms an oligomeric interface and that multimerization of Yrt is crucial for its function in epithelial cell polarity regulation. Finally, we demonstrate that aPKC destabilizes the Yrt oligomer to repress its functions, thereby revealing a mechanism through which this kinase supports apical domain formation. Overall, our study highlights a conserved biochemical property of fly and human Yrt proteins, describes a novel function of the FA domain, and further characterizes the molecular mechanisms sustaining epithelial cell polarity.
Subject(s)
Cell Polarity , Drosophila Proteins/metabolism , Epithelial Cells/metabolism , Protein Multimerization , Animals , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster , Epithelial Cells/chemistry , Epithelial Cells/cytology , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein DomainsABSTRACT
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.