Abstract. Previous reports indicate that endothelial fenestrae in vitro can form by fusion of caveolae or caveolae-like vesicles. The principal aim of this study was to determine whether formation of glomerular endothelial cell fenestrae in vivo similarly involves caveolae and caveolin-1. Whereas caveolin-1 immunofluorescence was found around the circumference of human and mouse glomerular capillary loops, it co-localized only partially with the endothelium-specific lectin Ulex Europaeus I in human glomeruli, leaving portions of the endothelium devoid of caveolin-1. Immunogold electron microscopy, used to definitively localize caveolin-1 in glomeruli, showed that caveolin-1 was completely excluded from the fenestrated portion of the endothelium. Moreover, in caveolin-1-deficient mice, which cannot form caveolae, the ultrastructure of glomerular endothelial fenestrae appeared entirely normal. Interestingly, strong caveolin-1 immunogold labeling was observed in podocytes, where some caveolin-1 localized to filtration slits. Caveolin-1 co-immunoprecipitated with the podocyte slit diaphragm proteins nephrin and CD2AP, and dual immunofluorescence confirmed co-localization of caveolin-1 and nephrin. Nevertheless, in caveolin-1-deficient mice, podocyte ultrastructure appeared normal, and the podocyte proteins synaptopodin, nephrin, and podocin were expressed normally. In addition, blood urea nitrogen concentrations and urinary protein excretion in these mice were similar to those in wild-type mice. Thus, unlike caveolae formation, glomerular endothelial cell fenestrae formation in vivo does not require caveolin-1, ruling out the previous hypothesis that endothelial fenestrae represent fused caveolae, at least for glomerular endothelial cells. Localization of caveolin-1 to podocytes and their filtration slits is consistent with the view that the filtration slit plasma membrane represents a type of lipid raft microdomain.

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...

A 53-year-old man was referred for progressive leucocytosis. The patient showed a complete blood count (CBC) of red blood cells (RBCs) 4´14