Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane-associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high-stoichiometric presence of actin-binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F-actin causes further enrichment of the actin-binding and actin-related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin-binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.

Mutations affecting the integrity and function of cilia have been identified in various genes over the last decade accounting for a group of diseases called ciliopathies. Ciliopathies display a broad spectrum of phenotypes ranging from mild manifestations to lethal combinations of multiple severe symptoms and most of them share cystic kidneys as a common feature. Our starting point was a consanguineous pedigree with three affected fetuses showing an early embryonic phenotype with enlarged cystic kidneys, liver and pancreas and developmental heart disease. By genome-wide linkage analysis, we mapped the disease locus to chromosome 17q11 and identified a homozygous nonsense mutation in NEK8/NPHP9 that encodes a kinase involved in ciliary dynamics and cell cycle progression. Missense mutations in NEK8/NPHP9 have been identified in juvenile cystic kidney jck mice and in patients suffering from nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. This work confirmed a complete loss of NEK8 expression in the affected fetuses due to nonsense-mediated decay. In cultured fibroblasts derived from these fetuses, the expression of prominent polycystic kidney disease genes (PKD1 and PKD2) was decreased, whereas the oncogene c-MYC was upregulated, providing potential explanations for the observed renal phenotype. We furthermore linked NEK8 with NPHP3, another NPH protein known to cause a very similar phenotype in case of null mutations. Both proteins interact and activate the Hippo effector TAZ. Taken together, our study demonstrates that NEK8 is essential for organ development and that the complete loss of NEK8 perturbs multiple signalling pathways resulting in a severe early embryonic phenotype.

Podocytes are terminally differentiated cells of the kidney filtration barrier. They are subjected to physiological filtration pressure and considerable mechanical strain, which can be further increased in various kidney diseases. When injury causes cytoskeletal reorganization and morphological alterations of these cells, the filtration barrier may become compromised and allow proteins to leak into the urine (a condition called proteinuria). Using time-resolved proteomics, we showed that podocyte injury stimulated the activity of the transcriptional coactivator YAP and the expression of YAP target genes in a rat model of glomerular disease before the development of proteinuria. Although the activities of YAP and its ortholog TAZ are activated by mechanical stress in most cell types, injury reduced YAP and TAZ activity in cultured human and mouse podocyte cell lines grown on stiff substrates. Culturing these cells on soft matrix or inhibiting stress fiber formation recapitulated the damage-induced YAP up-regulation observed in vivo, indicating a mechanotransduction-dependent mechanism of YAP activation in podocytes. YAP overexpression in cultured podocytes increased the abundance of extracellular matrix-related proteins that can contribute to fibrosis. YAP activity was increased in mouse models of diabetic nephropathy, and the YAP target was highly expressed in renal biopsies from glomerular disease patients. Although overexpression of human YAP in mice induced mild proteinuria, pharmacological inhibition of the interaction between YAP and its partner TEAD in rats ameliorated glomerular disease and reduced damage-induced mechanosignaling in the glomeruli. Thus, perturbation of YAP-dependent mechanosignaling is a potential therapeutic target for treating some glomerular diseases.

The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. We present a fast and straightforward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD-dependent transcription. Among these newly identified candidates are Rassf8, thymopoetin, and the transcription factors CCAAT/enhancer-binding protein (C/EBP)β/δ and core-binding factor subunit β (Cbfb). In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.

The PHB-domain protein podocin maintains the renal filtration barrier and its mutation is an important cause of hereditary nephrotic syndrome. Podocin and its Caenorhabditis elegans orthologue MEC-2 have emerged as key components of mechanosensitive membrane protein signalling complexes. Whereas podocin resides at a specialized cell junction at the podocyte slit diaphragm, MEC-2 is found in neurons required for touch sensitivity. Here, we show that the ubiquitin ligase Ubr4 is a key component of the podocin interactome purified both from cultured podocytes and native glomeruli. It colocalizes with podocin and regulates its stability. In C. elegans, this process is conserved. Here, Ubr4 is responsible for the degradation of mislocalized MEC-2 multimers. Ubiquitylomic analysis of mouse glomeruli revealed that podocin is ubiquitylated at two lysine residues. These sites were Ubr4-dependent and were conserved across species. Molecular dynamics simulations revealed that ubiquitylation of one site, K301, do not only target podocin/MEC-2 for proteasomal degradation, but may also affect stability and disassembly of the multimeric complex. We suggest that Ubr4 is a key regulator of podocyte foot process proteostasis.

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulusexpressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.

Cilia have recently emerged as sensory organelles present in many mammalian tissues involved in a wide variety of cellular processes such as fluid flow, cell movement as well as multiple signaling pathways. Over the last years an increasing number of human diseases have been linked to defects in cilia with autosomal‐dominant polycystic kidney disease (ADPKD) perhaps being the most prominent example. However, very little is known about the protein composition as well as the time‐resolved dynamic processes within this important organelle.To characterize the ciliary membrane‐associated proteome, we used the genetically‐engineered enzyme ascorbate peroxidase (APEX) and targeted it to the ciliary membrane (cmAPEX). With STED microscopy, we could confirm the subciliary localization of the tag specifically at the membrane of the cilium. Upon sequential addition of biotin phenol and H2O2 to the cells, APEX generates free biotin radicals that label proteins within the closest proximity, in particular at the ciliary membrane. Subsequently, cells are lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry.We identified numerous known ciliary proteins, in addition to novel ciliary membrane proteins and several actin modifying proteins. Inhibiting these actin‐modulating proteins regulated ciliary length, reaffirming the importance of actin dynamics in ciliogenesis. The results also identified ciliary proteins that are differentially regulated upon inhibition of actin polymerization and functional assays characterized their effect on ciliogenesis.cmAPEX is a versatile tool to study perturbations in this small and dynamic organelle. This approach provides quantitative analysis of the proteomic composition of the cilium and at the same time eliminates the need to isolate ciliary factions. We believe that this work will provide further insights into the cilium biology and suggest potential therapeutic targets for cilia‐associated genetic disorders.Support or Funding InformationDFG, CECAD Graduate School of Ageing.

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