Differentially Spliced Isoforms of FAT1 Are Asymmetrically Distributed within Migrating Cells

Abstract

Cadherin FAT1 is localized along the leading edge of mammalian cells and is necessary for polarization and directed migration. It is essential for maintenance of the complex cytoarchitecture of the glomerular filtration barrier within the kidney. In this study, three novel splice isoforms of FAT1 with important functional differences in comparison with wild-type FAT1, FAT1(WT), were identified. The novel variants contained additional short peptide sequences at a specific site of the cytoplasmic domain (؉12 or ؉32 or ؉8 amino acids, the latter resulting in a premature stop codon). FAT1(؉12) was expressed in all peripheral tissues together with FAT1(WT), whereas FAT1(؉32) and -(؉8TR) were brain-specific. At the subcellular level, exclusively FAT1(WT) was localized along the cellular leading edge, whereas spliced FAT1 isoforms were confined to intercellular junctions. A shift of FAT1(WT) expression toward a predominance of FAT1(؉12) was observed in migratory versus quiescent cells. A similar shift was observed in vivo when glomeruli from healthy individuals were compared with those from patients affected by glomerulonephritis. At the molecular level, the differential subcellular localization of FAT1 isoforms was mediated by a novel region harboring a phosphotyrosine-bindinglike motif (DN_XYH), which was disrupted by the peptide inserts in the alternative splice variants. Overexpression of FAT1(WT) or specific knockdown of spliced FAT1 isoforms resulted in formation of cellular protrusions or increased wound healing, respectively. In summary, FAT1(WT) is the only FAT1 isoform located along the cellular leading edge. Only FAT1(WT) is up-regulated in migration, induces cellular process formation when overexpressed, and is necessary for efficient wound healing.FAT cadherins are large ϳ500-kDa transmembrane proteins with an extracellular domain containing 34 cadherin motifs and a cytoplasmic domain that harbors several protein interaction motifs (1). So far, Ena/VASP, Homer 3, and ␤-catenin have been reported to interact with the FAT1 cytoplasmic domain (2-5). In Drosophila, the orthologs of mammalian FAT1, dfat (ft) and dfat-like (ftl), have been implicated in the establishment of planar cell polarity and formation of tracheal epithelia, respectively (6, 7). We and others have shown that mammalian FAT1 is localized along the leading edge, filopodial protrusions, and intercellular junctions (2, 4). As demonstrated by knockdown experiments, mammalian FAT1 is essential for cellular polarization and directed cell migration (2, 4, 5). FAT1 regulates actin dynamics at least in part via a direct interaction with Ena/VASP proteins (2). Consistently, genetic inactivation of FAT1 in mice leads to loss of podocyte foot processes in the kidney, which are highly dynamic actin-based structures forming a specialized intercellular junction, termed slit diaphragm (8 -11). In this study, several novel splice isoforms of FAT1 were identified and characterized. EXPERIMENTAL PROCEDURESPlasmid Constructs-FAT1(WT)mito encoded the fusion prot...

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