Functional Study of Mammalian Neph Proteins in Drosophila melanogaster

Martin Helmstädter,Kevin Lüthy,Deepak Nihalani,Stefan A Rensing,Karl-Friedrich Fischbach,Ashish Ashish,Matias Simons,Markus Gödel,Tobias B Huber,Susan Broughton

doi:10.1371/journal.pone.0040300

Abstract

Neph molecules are highly conserved immunoglobulin superfamily proteins (IgSF) which are essential for multiple morphogenetic processes, including glomerular development in mammals and neuronal as well as nephrocyte development in D. melanogaster. While D. melanogaster expresses two Neph-like proteins (Kirre and IrreC/Rst), three Neph proteins (Neph1–3) are expressed in the mammalian system. However, although these molecules are highly abundant, their molecular functions are still poorly understood. Here we report on a fly system in which we overexpress and replace endogenous Neph homologs with mammalian Neph1–3 proteins to identify functional Neph protein networks required for neuronal and nephrocyte development. Misexpression of Neph1, but neither Neph2 nor Neph3, phenocopies the overexpression of endogenous Neph molecules suggesting a functional diversity of mammalian Neph family proteins. Moreover, structure-function analysis identified a conserved and specific Neph1 protein motif that appears to be required for the functional replacement of Kirre. Hereby, we establish D. melanogaster as a genetic system to specifically model molecular Neph1 functions in vivo and identify a conserved amino acid motif linking Neph1 to Drosophila Kirre function.

Highlights

The immunoglobulin superfamily proteins (IgSF) proteins of Nephrin and Neph families have been conserved throughout Metazoan evolution
An interesting example of such a highly specialized cell-cell contact is the slit diaphragm at the kidney filtration barrier, which consists of cis and trans- interacting Nephrin and Neph1 molecules (Fig. 1C e-h)
Recent investigations revealed that Sns and Kirre form a filtration slit in Garland cell nephrocytes (GCNs) of Drosophila (Fig. 1C a-d) which is very similar to the mammalian slit diaphragm of podocytes [15,22,23]

Summary

Introduction

The IgSF proteins of Nephrin and Neph families ( called IRM proteins [1]) have been conserved throughout Metazoan evolution. The extracellular domains of Nephrin and Neph proteins bind to each other in cis- and/or trans- interactions [4]. In C. elegans, synapse development and synaptic target recognition employ members of the Nephrin-Neph protein family. An interesting example of such a highly specialized cell-cell contact is the slit diaphragm at the kidney filtration barrier, which consists of cis and trans- interacting Nephrin and Neph molecules (Fig. 1C e-h). The Nephrin-Neph protein complex has been linked to several signaling processes at the slit diaphragm, like actin regulation, polarity signaling and cell survival [20,21]. Recent investigations revealed that Sns and Kirre form a filtration slit in Garland cell nephrocytes (GCNs) of Drosophila (Fig. 1C a-d) which is very similar to the mammalian slit diaphragm of podocytes [15,22,23]. Sns and Kirre are involved in the slit diaphragm formation of GCNs, and mediate the fusion process of GCNs that results in binuclear GCNs [15]

Methods

Results

Conclusion