All Nephrin and Neph proteins share extracellular immunoglobulin-like domains and a short cytoplasmic tail that contains multiple signaling motifs[2],[3]. The IgSF proteins of Nephrin and Neph families (also called IRM proteins[1]) have been conserved throughout Metazoan evolution. All Nephrin and Neph proteins share extracellular immunoglobulin-like domains and a short cytoplasmic tail that contains multiple Cilastatin sodium signaling motifs[2],[3]. The extracellular domains of Nephrin and Neph proteins bind to each other incis- and/ortrans- interactions[4]. Two Neph homologs (IrreC/Rst, Kirre) and two Nephrin homologs (Hbs, Sns) Timp1 are involved in pupal eye development, muscle fusion and axonal guidance inDrosophila[5][9]. InC. elegans,synapse development and synaptic target recognition also employ members of the Nephrin-Neph protein family. In this system the Nephrin homolog SYG-2 and the Neph1 homolog SYG-1 mediate precise recognition of appropriate partners and trigger synapse formation of the hermaphrodite specific motor neuron (HSNL)[10],[11]. Interestingly, all mammalian Neph molecules (Neph13) have been shown to be able to functionally replace endogenous SYG-1 indicating a potential redundant function of Neph proteins[12],[13]. Despite the diversity of signaling mechanisms and expression patterns of IRM proteins throughout different species, some important themes are beginning to emerge: a striking house of IRM proteins is the formation of variable, homo- and heterophilic conversation modules incisandtransconformation that precisely guideline cellular connections[4],[12][15]. An interesting example of such a highly specialized cell-cell contact is the slit diaphragm at the kidney filtration barrier, which consists ofcisandtrans-interacting Nephrin and Neph1 molecules (Fig. 1Ce-h). Mutations in Nephrin lead to congenital nephrotic syndrome which is usually characterized by a disruption of the kidney filtration barrier, kidney failure and severe protein loss into the urine[16]. In addition, mice lacking Neph1 are proteinuric and reveal effacement of podocyte foot processes[17]. Nephrin and Neph1 molecules have been demonstrated to form acis-andtrans-interacting complex[4],[18],[19]. Moreover, the Nephrin-Neph1 protein complex has been linked to several signaling processes at the slit diaphragm, like actin regulation, polarity signaling and cell survival[20],[21]. Strikingly, recent investigations revealed that Sns and Kirre form a filtration slit in Garland cell nephrocytes (GCNs) ofDrosophila(Fig. 1Ca-d) which is very similar to the mammalian slit diaphragm of podocytes[15],[22],[23]. As the experimental accessibility of the mammalian slit diaphragms is very limited, this obtaining has important implications. Therefore, theDrosophilaGCN appears to be an ideal system to study Nephrin-Neph protein functions in a genetically easy tractable system[24]. Interestingly, Sns and Kirre are involved not only in the slit diaphragm formation of GCNs, but also mediate the fusion process of GCNs that results in binuclear GCNs[15]. == Physique 1. The irre cell recognition module (IRM) is usually conserved across species. == A.Similarity network of Neph13, Kirre and IrreC/Rst generated with T-Coffee multiple sequence alignment algorithm. Values represent full length protein similarity in percent.B.Protein tree of the Neph Cilastatin sodium (blue) and Nephrin (red) protein families as present inMus musculus,Danio rerio,Caenorhabditis elegans, Anopheles gambiaeand severalDrosophilaspecies(Drosophila mojavensis(DROMO),Drosophila virilis(DROVI),Drosophila grimshawi(DROGR),Drosophila willistoni(DROWI),Drosophila pseudoobscura(DROPS),Drosophila persimilis(DROPE),Drosophila ananassae(DROAN),Drosophila melanogaster(DROME),Drosophila sechellia(DROSE),Drosophila yakuba(DROYA). Arrowheads mark the proteins investigated in this study.C.Structural comparison ofM. musculuskidney glomerulus (e), podocytes (f) and slit diaphragm (g) with GCNs ofD. melanogaster(a). The rough surface of the GCN underneath the basement membrane (b) is formed by the nephrocyte diaphragm (c). In contrast to the mammalian slit diaphragm which is formed between neighboring podocytes (h), theDrosophilanephrocyte diaphragm is formed within Cilastatin sodium one GCN (d). We employedD. melanogaster as a model system to investigate the evolutionary conservation of the Nephrin-Neph proteins and to determine differences between the mammalian Neph proteins 13 in their ability to phenocopy and rescue Kirre and IrreC/Rst phenotypes. Our results demonstrate that Neph1 is the only mammalian Neph protein that can mimic the phenotypes of overexpressed Kirre or misexpressed IrreC/Rst in GCNs. Furthermore, we illustrate that only misexpressed Neph1, which partially colocalizes with Sns in GCNs, is able to rescue a GCN.