Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier

Background Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS).Methods To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2(podKO) knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants.Results Crb2(podKO) mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response.Conclusions Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.Ophthalmic researc

Development of Oculimacula yallundae and O-acuformis (eyespot) on leaf sheaths of winter wheat in the UK in relation to thermal time

In a controlled environment (15/10 degrees C) (day/night) container experiment on winter wheat (cv. Avalon), eyespot incidence (percentage of plants affected) and number of leaf sheaths penetrated after 6 weeks increased with inoculum concentration (10(2)-10(6) conidia mL(-1)) of Oculimacula yallundae (OY) or Oculimacula acuformis (OA), but there was no difference between the two species. In an outdoor container experiment, seedlings inoculated with OY 2 weeks after sowing had a greater incidence of eyespot than those inoculated with OA, when assessed 7 weeks after inoculation. Seedlings inoculated with OA at 10 or 20 weeks after sowing developed more severe eyespot by maturity than those inoculated with OY. In an experiment at 15/10 degrees C with seedlings inoculated with OY + OA 2 weeks after sowing, more leaf sheaths were penetrated by OY (3.0 per plant) than OA (2.3 per plant) 6 weeks after inoculation. Field experiments with winter wheat consistently showed leaf sheath production, leaf sheath death, and number of leaf sheaths infected or penetrated by OA or OY were related linearly to thermal time (degrees C days) after sowing. Depending on cultivar, season and sample, a new leaf sheath was produced in 116-216 degrees C days; a leaf sheath died in 221-350 degrees C days; and infection of a new leaf sheath occurred in 129-389 degrees C days. The mean number of living leaf sheaths infected differed between samples, cultivars and seasons for both OY and OA. Regression analysis of the 1985/86 data suggested that OY progressed more rapidly than OA through the leaf sheaths, and that both the pathogens progressed more rapidly than the rate of leaf sheath death, but more slowly than the rate at which leaf sheaths were produced. It also suggested that OA progressed more slowly than the rate at which leaf sheaths died in 1987/88, but OY did not.Peer reviewe