Fluorescence intensity of the wild type was set to 100%

Fluorescence intensity of the wild type was set to 100%. Fluorescence and DIC images for the publication were taken with a Zeiss AxioImager Z1 microscope. == Cell culture and transfection == For co-immunoprecipitations, HEK 293T cells (received from American Type Culture Collection, ATCC, Manassas, VA) were grown in DMEM with 10% FBS. not SYG-2 binding indicating a critical role of SYG-1 intracellular signalling for morphogenetic events and pointing toward the dynamic and stochastic nature of extra- and intracellular nephrin-Neph interactions to generate reproducible patterns of synaptic connectivity. == Introduction == A striking property of the nervous system is the precision of its uncountable numbers of synaptic connections, which are organized into specific neural circuits. Despite the diversity of mechanisms and molecules that can give rise to synaptic specificity, some important themes are beginning to emerge: Synaptic specification can operate both at the level of partner choice and at the level of synapse formation onto a specific subcellular compartment and there is now strong evidence that immunoglobulin superfamily proteins serve as molecular tags for both processes[1]. The IgSF proteins nephrin and Neph are conserved through evolution. All Neph proteins share four to five extracellular immunoglobulin-like domains and a short cytoplasmic tail that contains a conserved PDZ binding motif at the very carboxy terminus[2]. The PDZ binding motif serves as a scaffold for protein complex binding to facilitate intracellular signaling events[3]. The extracellular domains of nephrin and Neph proteins bind to each other incis- and/ortrans- interactions[4]. Two Neph1 (Roughest, Kirre) and two nephrin homologs (Hibris, Sticks-and-stones) are involved in pupal eye development and axonal guidance inDrosophila[5],[6],[7]. InC. elegans,synapse development and synaptic target recognition also involves members of the nephrin-Neph protein family. 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)[8],[9]. The HSNL controls egg-laying Glycine behaviour by forming stereotypicen passantsynapses on vulva muscle cells and ventral cord (VC) motor neurons. The recognition between HSNL and its targets and the precise positioning of synapses is initiated by adjacent vulva epithelial guidepost cells that express SYG-2/nephrin. SYG-2 interacts with SYG-1/Neph1 that is expressed in the HSNL, and thus recruits SYG-1 to the location along the HSNL axon where presynaptic sites are formed[8],[9]. In an exciting analogy to the nervous system nephrin and Neph molecules Glycine seem also to instruct the formation of the slit diaphragm. The slit diaphragm is a highly specialized cell-cell contact bridging the secondary foot Rabbit Polyclonal to KCNK1 processes of podocytes to form the most outer part of the filtrations barrier[10]. The importance of nephrin and Neph1 for the development and function of glomerular podocytes and the slit diaphragm is exemplified by the defects that have been discovered in mice and humans deficient in these molecules[11],[12],[13],[14]. Given the central role of podocytes in glomerular pathology, a lot of effort has been made to uncover the mechanisms of podocyte development, maintenance and signaling pathways. However, even though mouse models have been a useful tool in investigating podocyte functions, the mammalian glomerulus is a poorly accessible structure making it difficult to performin vivoexperiments. In this respect, genetically tractable model organisms such asC. elegans, D. melanogasterand zebrafish can overcome the difficult accessibility of slit diaphragm molecules and allow efficient functional analysis. We have previously utilizedC. elegansto exemplify the functional complementarities betweenC. elegansSYG-1 and all three mammalian Neph proteins in regulating synaptic connectivity[15]. The asymmetric distribution of the nephrin/Neph orthologs on different cell types provides a perfect setting for further mechanistic studies of nephrin-Neph protein interactions and domain function. Here, we now show that expression of mammalian nephrin can fully compensate loss Glycine ofsyg-2function and restore synapse formation inC. elegans. Furthermore, we investigated the ability of SYG-1 and SYG-2, respectively, to engage homophilic interactionsin transbetween the HSN and vulva epithelial cells. Strikingly, a chimeric construct with the extracellular domain of SYG-2 and the intracellular domain of SYG-1 failed to replace full length SYG-1 in HSN, whereas full length SYG-1 was at least partially able to substitute for SYG-2 in epithelial cells. == Results == == Expression of human nephrin can rescue defective synaptogenesis insyg-2mutants == We have previously reported that the Neph/nephrin family proteins can form cell-cell adhesion modules across species[15](Fig. 1A, B). All three mammalian Neph proteins were able.