Martin Höhne

Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG/ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein–cholesterol supercomplexes in the plasma membrane.

The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth

Cilia are specialized organelles that play an important role in several biological processes, including mechanosensation, photoperception, and osmosignaling. Mutations in proteins localized to cilia have been implicated in a growing number of human diseases. In this study, we demonstrate that the von Hippel-Lindau (VHL) protein (pVHL) is a ciliary protein that controls ciliogenesis in kidney cells. Knockdown of pVHL impeded the formation of cilia in mouse inner medullary collecting duct 3 kidney cells, whereas the expression of pVHL in VHL-negative renal cancer cells rescued the ciliogenesis defect. Using green fluorescent protein–tagged end-binding protein 1 to label microtubule plus ends, we found that pVHL does not affect the microtubule growth rate but is needed to orient the growth of microtubules toward the cell periphery, a prerequisite for the formation of cilia. Furthermore, pVHL interacts with the Par3–Par6–atypical PKC complex, suggesting a mechanism for linking polarity pathways to microtubule capture and ciliogenesis.

Neuronal Expression and Interaction with the Synaptic Protein CASK Suggest a Role for Neph1 and Neph2 in Synaptogenesis

Formation, differentiation, and plasticity of synapses require interactions between pre‐ and postsynaptic partners. Recently, it was shown that the transmembrane immunoglobulin superfamily protein SYG‐1 is required for providing synaptic specificity in C. elegans. However, it is unclear whether the mammalian orthologs of SYG‐1 are also involved in local cell interactions to determine specificity during synapse formation. We used in situ hybridization, immunohistochemistry, and immunogold electron microscopy to study the temporal and spatial expression of Neph1 and Neph2 in the developing and adult mouse brain. Both proteins show similar patterns with neuronal expression starting around embryonic days 12 and 11, respectively. Expression is strongest in areas of high migratory activity. In the adult brain, Neph1 and Neph2 are predominantly seen in the olfactory nerve layer and the glomerular layer of the olfactory bulb, in the hippocampus, and in Purkinje cells of the cerebellum. At the ultrastructural level, Neph1 and Neph2 are detectable within the dendritic shafts of pyramidal neurons. To a lesser extent, there is also synaptic localization of Neph1 within the stratum pyramidale of the hippocampal CA1 and CA3 region on both pre‐ and postsynaptic sites. Here it colocalizes with the synaptic scaffolder calmodulin‐associated serin/threonin kinase (CASK), and both Neph1 and Neph2 interact with the PDZ domain of CASK via their cytoplasmic tail. Our results show that Neph proteins are expressed in the developing nervous system of mammals and suggest that these proteins may have a conserved function in synapse formation or neurogenesis. J. Comp. Neurol. 498:466–475, 2006.

Light microscopic visualization of podocyte ultrastructure demonstrates oscillating glomerular contractions.

Podocytes, the visceral epithelial cells of the kidney glomerulus, elaborate primary and interdigitating secondary extensions to enwrap the glomerular capillaries. A hallmark of podocyte injury is the loss of unique ultrastructure and simplification of the cell shape, called foot process effacement, which is a classic feature of proteinuric kidney disease. Although several key pathways have been identified that control cytoskeletal regulation, actin dynamics, and polarity signaling, studies into the dynamic regulation of the podocyte structure have been hampered by the fact that ultrastructural analyses require electron microscopic imaging of fixed tissue. We developed a new technique that allows for visualization of podocyte foot processes using confocal laser scanning microscopy. The combination of inducible and mosaic expression of membrane-tagged fluorescent proteins in a small subset of podocytes enabled us to acquire light microscopic images of podocyte foot processes in unprecedented detail, even in living podocytes of freshly isolated glomeruli. Moreover, this technique visualized oscillatory glomerular contractions and confirmed the morphometric evaluations obtained in static electron microscopic images of podocyte processes. These data suggest that the new technique will provide an extremely powerful tool for studying the dynamics of podocyte ultrastructure.

Comparative analysis of Neph gene expression in mouse and chicken development

Neph proteins are evolutionarily conserved members of the immunoglobulin superfamily of adhesion proteins and regulate morphogenesis and patterning of different tissues. They share a common protein structure consisting of extracellular immunoglobulin-like domains, a transmembrane region, and a carboxyl terminal cytoplasmic tail required for signaling. Neph orthologs have been widely characterized in invertebrates where they mediate such diverse processes as neural development, synaptogenesis, or myoblast fusion. Vertebrate Neph proteins have been described first at the glomerular filtration barrier of the kidney. Recently, there has been accumulating evidence suggesting a function of Neph proteins also outside the kidney. Here we demonstrate that Neph1, Neph2, and Neph3 are expressed differentially in various tissues during ontogenesis in mouse and chicken. Neph1 and Neph2 were found to be amply expressed in the central nervous system while Neph3 expression remained localized to the cerebellum anlage and the spinal cord. Outside the nervous system, Neph mRNAs were also differentially expressed in branchial arches, somites, heart, lung bud, and apical ectodermal ridge. Our findings support the concept that vertebrate Neph proteins, similarly to their Drosophila and C. elegans orthologs, provide guidance cues for cell recognition and tissue patterning in various organs which may open interesting perspectives for future research on Neph1-3 controlled morphogenesis.

Nephrocystin-4 regulates Pyk2-induced tyrosine phosphorylation of nephrocystin-1 to control targeting to monocilia.

Nephronophthisis is the most common genetic cause of end-stage renal failure during childhood and adolescence. Genetic studies have identified disease-causing mutations in at least 11 different genes (NPHP1–11), but the function of the corresponding nephrocystin proteins remains poorly understood. The two evolutionarily conserved proteins nephrocystin-1 (NPHP1) and nephrocystin-4 (NPHP4) interact and localize to cilia in kidney, retina, and brain characterizing nephronophthisis and associated pathologies as result of a ciliopathy. Here we show that NPHP4, but not truncating patient mutations, negatively regulates tyrosine phosphorylation of NPHP1. NPHP4 counteracts Pyk2-mediated phosphorylation of three defined tyrosine residues of NPHP1 thereby controlling binding of NPHP1 to the trans-Golgi sorting protein PACS-1. Knockdown of NPHP4 resulted in an accumulation of NPHP1 in trans-Golgi vesicles of ciliated retinal epithelial cells. These data strongly suggest that NPHP4 acts upstream of NPHP1 in a common pathway and support the concept of a role for nephrocystin proteins in intracellular vesicular transport.

Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure

Mitochondrial dysfunction and alterations in energy metabolism have been implicated in a variety of human diseases. Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin‐2 (PHB2) at the mitochondrial inner membrane. Here, we provide a link between PHB2 deficiency and hyperactive insulin/IGF‐1 signaling. Deletion of PHB2 in podocytes of mice, terminally differentiated cells at the kidney filtration barrier, caused progressive proteinuria, kidney failure, and death of the animals and resulted in hyperphosphorylation of S6 ribosomal protein (S6RP), a known mediator of the mTOR signaling pathway. Inhibition of the insulin/IGF‐1 signaling system through genetic deletion of the insulin receptor alone or in combination with the IGF‐1 receptor or treatment with rapamycin prevented hyperphosphorylation of S6RP without affecting the mitochondrial structural defect, alleviated renal disease, and delayed the onset of kidney failure in PHB2‐deficient animals. Evidently, perturbation of insulin/IGF‐1 receptor signaling contributes to tissue damage in mitochondrial disease, which may allow therapeutic intervention against a wide spectrum of diseases.

The ubiquitin ligase Ubr4 controls stability of podocin/MEC-2 supercomplexes.

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.

Identification of regulatory modules mediating specific expression of the roughest gene in Drosophila melanogaster

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily with pleiotropic functions during the development of Drosophila melanogaster. It has been shown to be involved in cell sorting before apoptosis in the developing compound eye, in fusion processes of embryonic muscle development and in axonal pathfinding. In accordance with its multiple functions, the rst gene shows a dynamic expression pattern throughout the development of Drosophila. In order to understand the transcriptional regulation of rst expression we have identified rst cis regulatory sequences in an enhancer detection screen. By dissection of the identified rst cis regulatory sequences we identified several distinct rst regulatory modules. Among others these include elements for expression in interommatidial cells of the pupal eye disc at a time when apoptotic decisions are made in these cells and elements for expression in the embryonic mesoderm. The expression of rst in the embryonic mesoderm is regulated by at least two separate modules.

Three-layered proteomic characterization of a novel ACTN4 mutation unravels its pathogenic potential in FSGS

Genetic diseases constitute the most important cause for end-stage renal disease in children and adolescents. Mutations in the ACTN4 gene, encoding the actin-binding protein α-actinin-4, are a rare cause of autosomal dominant familial focal segmental glomerulosclerosis (FSGS). Here, we report the identification of a novel, disease-causing ACTN4 mutation (p.G195D, de novo) in a sporadic case of childhood FSGS using next generation sequencing. Proteome analysis by quantitative mass spectrometry (MS) of patient-derived urinary epithelial cells indicated that ACTN4 levels were significantly decreased when compared with healthy controls. By resolving the peptide bearing the mutated residue, we could proof that the mutant protein is less abundant when compared with the wild-type protein. Further analyses revealed that the decreased stability of p.G195D is associated with increased ubiquitylation in the vicinity of the mutation site. We next defined the ACTN4 interactome, which was predominantly composed of cytoskeletal modulators and LIM domain-containing proteins. Interestingly, this entire group of proteins, including several highly specific ACTN4 interactors, was globally decreased in the patient-derived cells. Taken together, these data suggest a mechanistic link between ACTN4 instability and proteome perturbations of the ACTN4 interactome. Our findings advance the understanding of dominant effects exerted by ACTN4 mutations in FSGS. This study illustrates the potential of genomics and complementary, high-resolution proteomics analyses to study the pathogenicity of rare gene variants.