Linking Basement Membrane and Slit Diaphragm in Drosophila Nephrocytes.

BACKGROUND: The glomerular basement membrane and the slit diaphragm are essential parts of the filtration barrier. How these layers collaborate remains unclear. The podocyte-like nephrocytes in Drosophila harbor both a slit diaphragm and a basement membrane, serving as a model to address this critical question. METHODS: Basement membrane components and matrix receptors were silenced using RNAi in nephrocytes. Slit diaphragms were analyzed by immunofluorescence followed by automated quantification. Tracer endocytosis was applied for functional readouts. RESULTS: Immunofluorescence indicated a significant reduction of slit diaphragm density upon loss of laminin and collagen IV components. This was accompanied by reduced expression of fly nephrin and shallower membrane invaginations. Tracer studies revealed that the basement membrane defines properties of the nephrocyte filtration barrier. Acute enzymatic disruption of the basement membrane via collagenase rapidly caused slit diaphragm mislocalization and disintegration, which was independent of cell death. Loss of matrix-interacting receptors, particularly integrins mys and mew, phenocopied basement membrane disruption. Integrins and nephrin co-localized at the slit diaphragm in nephrocytes in a mutually dependent manner, interacting genetically. Human integrin α3 interacted physically with nephrin. CONCLUSIONS: The glomerular basement membrane model in Drosophila nephrocytes reveals that matrix receptor-mediated cues ensure correct positioning of the slit diaphragm and the overall filtration barrier architecture.

CyVerse: Cyberinfrastructure for open science.

CyVerse, the largest publicly-funded open-source research cyberinfrastructure for life sciences, has played a crucial role in advancing data-driven research since the 2010s. As the technology landscape evolved with the emergence of cloud computing platforms, machine learning and artificial intelligence (AI) applications, CyVerse has enabled access by providing interfaces, Software as a Service (SaaS), and cloud-native Infrastructure as Code (IaC) to leverage new technologies. CyVerse services enable researchers to integrate institutional and private computational resources, custom software, perform analyses, and publish data in accordance with open science principles. Over the past 13 years, CyVerse has registered more than 124,000 verified accounts from 160 countries and was used for over 1,600 peer-reviewed publications. Since 2011, 45,000 students and researchers have been trained to use CyVerse. The platform has been replicated and deployed in three countries outside the US, with additional private deployments on commercial clouds for US government agencies and multinational corporations. In this manuscript, we present a strategic blueprint for creating and managing SaaS cyberinfrastructure and IaC as free and open-source software.

Nephrotic Syndrome Gene TBC1D8B Is Required for Endosomal Maturation and Nephrin Endocytosis in Drosophila.

BACKGROUND: Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. METHODS: We generated a stable deletion in Tbc1d8b and used microhomology-mediated end-joining for genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulation of endocytic regulators and transgenesis of murine Tbc1d8b provided a comprehensive functional analysis of Tbc1d8b. RESULTS: A null allele of Drosophila TBC1D8B exhibited a nephrocyte-restricted phenotype of nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from the Tbc1d8b locus bearing the edited tag predominantly localized to mature early and late endosomes. Tbc1d8b was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of Tbc1d8b. Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel TBC1D8B variants within a cohort of 363 patients with FSGS and validated a functional effect of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. CONCLUSIONS: Variants in TBC1D8B are not infrequent among patients with FSGS. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.

Selective endocytosis controls slit diaphragm maintenance and dynamics in Drosophila nephrocytes.

The kidneys generate about 180 l of primary urine per day by filtration of plasma. An essential part of the filtration barrier is the slit diaphragm, a multiprotein complex containing nephrin as major component. Filter dysfunction typically manifests with proteinuria and mutations in endocytosis regulating genes were discovered as causes of proteinuria. However, it is unclear how endocytosis regulates the slit diaphragm and how the filtration barrier is maintained without either protein leakage or filter clogging. Here, we study nephrin dynamics in podocyte-like nephrocytes of Drosophila and show that selective endocytosis either by dynamin- or flotillin-mediated pathways regulates a stable yet highly dynamic architecture. Short-term manipulation of endocytic functions indicates that dynamin-mediated endocytosis of ectopic nephrin restricts slit diaphragm formation spatially while flotillin-mediated turnover of nephrin within the slit diaphragm is needed to maintain filter permeability by shedding of molecules bound to nephrin in endosomes. Since slit diaphragms cannot be studied in vitro and are poorly accessible in mouse models, this is the first analysis of their dynamics within the slit diaphragm multiprotein complex. Identification of the mechanisms of slit diaphragm maintenance will help to develop novel therapies for proteinuric renal diseases that are frequently limited to symptomatic treatment.

Inhibition of endoplasmic reticulum stress signaling rescues cytotoxicity of human apolipoprotein-L1 risk variants in Drosophila.

Risk variants of the apolipoprotein-L1 (APOL1) gene are associated with severe kidney disease, putting homozygous carriers at risk. Since APOL1 lacks orthologs in all major model organisms, a wide range of mechanisms frequently in conflict have been described for APOL1-associated nephropathies. The genetic toolkit in Drosophila allows unique in vivo insights into disrupted cellular homeostasis. To perform a mechanistic analysis, we expressed human APOL1 control and gain-of-function kidney risk variants in the podocyte-like garland cells of Drosophila nephrocytes and a wing precursor tissue. Expression of APOL1 risk variants was found to elevate endocytic function of garland cell nephrocytes that simultaneously showed early signs of cell death. Wild-type APOL1 had a significantly milder effect, while a control transgene with deletion of the short BH3 domain showed no overt phenotype. Nephrocyte endo-lysosomal function and slit diaphragm architecture remained unaffected by APOL1 risk variants, but endoplasmic reticulum (ER) swelling, chaperone induction, and expression of the reporter Xbp1-EGFP suggested an ER stress response. Pharmacological inhibition of ER stress diminished APOL1-mediated cell death and direct ER stress induction enhanced nephrocyte endocytic function similar to expression of APOL1 risk variants. We confirmed APOL1-dependent ER stress in the Drosophila wing precursor where silencing the IRE1-dependent branch of ER stress signaling by inhibition with Xbp1-RNAi abrogated cell death, representing the first rescue of APOL1-associated cytotoxicity in vivo. Thus, we uncovered ER stress as an essential consequence of APOL1 risk variant expression in vivo in Drosophila, suggesting a central role of this pathway in the pathogenesis of APOL1-associated nephropathies.

A local platform for user-friendly FAIR data management and reproducible analytics.

Collaborative research is common practice in modern life sciences. For most projects several researchers from multiple universities collaborate on a specific topic. Frequently, these research projects produce a wealth of data that requires central and secure storage, which should also allow for easy sharing among project participants. Only under best circumstances, this comes with minimal technical overhead for the researchers. Moreover, the need for data to be analyzed in a reproducible way often poses a challenge for researchers without a data science background and thus represents an overly time-consuming process. Here, we report on the integration of CyVerse Austria (CAT), a new cyberinfrastructure for a local community of life science researchers, and provide two examples how it can be used to facilitate FAIR data management and reproducible analytics for teaching and research. In particular, we describe in detail how CAT can be used (i) as a teaching platform with a defined software environment and data management/sharing possibilities, and (ii) to build a data analysis pipeline using the Docker technology tailored to the needs and interests of the researcher.