Phase Separation of MAGI2-Mediated Complex Underlies Formation of Slit Diaphragm Complex in Glomerular Filtration Barrier

2021 ◽  
Vol 32 (8) ◽  
pp. 1946-1960
Author(s):  
Haijiao Zhang ◽  
Lin Lin ◽  
Jianping Liu ◽  
Lifeng Pan ◽  
Zhijie Lin ◽  
...  
Keyword(s):  
Phase Separation ◽  
Insertion Site ◽  
Adaptor Proteins ◽  
Slit Diaphragm ◽  
Protein Loss ◽  
Glomerular Diseases ◽  
Content Type ◽  
Filtration Barrier ◽  
Core Components ◽  
Protein Rich

BackgroundSlit diaphragm is a specialized adhesion junction between the opposing podocytes, establishing the final filtration barrier to urinary protein loss. At the cytoplasmic insertion site of each slit diaphragm there is an electron-dense and protein-rich cellular compartment that is essential for slit diaphragm integrity and signal transduction. Mutations in genes that encode components of this membrane-less compartment have been associated with glomerular diseases. However, the molecular mechanism governing formation of compartmentalized slit diaphragm assembly remains elusive.MethodsWe systematically investigated the interactions between key components at slit diaphragm, such as MAGI2, Dendrin, and CD2AP, through a combination of biochemical, biophysical, and cell biologic approaches.ResultsWe demonstrated that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the highly dense slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively recruit Nephrin. A nephrotic syndrome–associated mutation of MAGI2 interfered with formation of the slit diaphragm condensates, thus leading to impaired enrichment of Nephrin.ConclusionsKey components at slit diaphragm (e.g., MAGI2 and its complex) can spontaneously undergo phase separation. The reconstituted slit diaphragm condensates can be enriched in adhesion molecules and cytoskeletal adaptor proteins. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of core components of the slit diaphragm in podocytes.

scholarly journals Early Glomerular Filtration Defect and Severe Renal Disease in Podocin-Deficient Mice

2004 ◽  
Vol 24 (2) ◽  
pp. 550-560 ◽  
Author(s):  
Séverine Roselli ◽  
Laurence Heidet ◽  
Mireille Sich ◽  
Anna Henger ◽  
Matthias Kretzler ◽  
...  
Keyword(s):  
Renal Disease ◽  
Glomerular Filtration ◽  
Molecular Mechanisms ◽  
Slit Diaphragm ◽  
Vascular Lesions ◽  
Suitable Model ◽  
Glomerular Diseases ◽  
Filtration Barrier ◽  
Severe Renal Disease

ABSTRACT Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2 −/−) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2 −/− mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2 −/− kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.

WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment

2005 ◽  
Vol 289 (2) ◽  
pp. F431-F441 ◽  
Author(s):  
Maribel Rico ◽  
Amitava Mukherjee ◽  
Martha Konieczkowski ◽  
Leslie A. Bruggeman ◽  
R. Tyler Miller ◽  
...  
Keyword(s):  
Adherens Junctions ◽  
Slit Diaphragm ◽  
Cell Junctions ◽  
Puromycin Aminonucleoside ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Interacting Protein ◽  
Filtration Barrier ◽  
Cell Cell

Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 μg/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PAN-treated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype.

mTOR regulates expression of slit diaphragm proteins and cytoskeleton structure in podocytes

2009 ◽  
Vol 296 (2) ◽  
pp. F418-F426 ◽  
Author(s):  
Beate Vollenbröker ◽  
Britta George ◽  
Maria Wolfgart ◽  
Moin A. Saleem ◽  
Hermann Pavenstädt ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Mtor Inhibitor ◽  
Adaptor Protein ◽  
Receptor Potential ◽  
Mtor Inhibitors ◽  
Slit Diaphragm ◽  
Glomerular Diseases ◽  
Rapamycin Treatment ◽  
Podocyte Damage ◽  
Filtration Barrier

The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors can cause proteinuria, especially in kidney and heart transplanted patients. Podocytes play a major role in establishing the selective permeability of the blood-urine filtration barrier. Damage of these cells leads to proteinuria, a hallmark of most glomerular diseases. Interestingly, podocyte damage and focal segmental glomerulosclerosis can occur after treatment with an mTOR inhibitor in some transplant patients. To investigate the mechanisms of mTOR inhibitor-induced podocyte damage, we analyzed the effect of rapamycin on mTOR signaling and cellular function in human podocytes. We found that prolonged rapamycin treatment reduced the expression of total mTOR, which correlates with diminished levels of mTOR phosphorylation at Ser2448 and Ser2481. In addition, treatment with rapamycin reduced rictor expression and mTORC2 formation, resulting in a reduced phosphorylation of protein kinase B at Ser473. The expression level of the slit-diaphragm proteins nephrin and transient receptor potential cation channel 6 as well as the cytoskeletal adaptor protein Nck significantly decreased. Moreover, rapamycin reduced cell adhesion and cell motility, which was accompanied by an enhanced formation of dot-like actin-rich structures. Our data provide new molecular insights explaining which pathways and molecules are affected in podocytes by an imbalanced mTOR function because of rapamycin treatment.

scholarly journals Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury

2015 ◽  
Vol 36 (4) ◽  
pp. 596-614 ◽  
Author(s):  
Rakesh Verma ◽  
Madhusudan Venkatareddy ◽  
Anne Kalinowski ◽  
Sanjeevkumar R. Patel ◽  
David J. Salant ◽  
...  
Keyword(s):  
Cell Culture ◽  
Tyrosine Phosphorylation ◽  
Sh2 Domain ◽  
Slit Diaphragm ◽  
Dependent Manner ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Filtration Barrier ◽  
Signaling Events ◽  
Foot Process

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injuryin vivousing protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury.

scholarly journals Structural Basis of Highly Specific Interaction between Nephrin and MAGI1 in Slit Diaphragm Assembly and Signaling

2018 ◽  
Vol 29 (9) ◽  
pp. 2362-2371 ◽  
Author(s):  
Zhuangfeng Weng ◽  
Yuan Shang ◽  
Zeyang Ji ◽  
Fei Ye ◽  
Lin Lin ◽  
...  
Keyword(s):  
Complex Structure ◽  
Pdz Domain ◽  
Specific Interaction ◽  
Slit Diaphragm ◽  
Structural Basis ◽  
Binding Motif ◽  
Inhibitory Peptide ◽  
Glomerular Diseases ◽  
Filtration Barrier

BackgroundThe slit diaphragm is a specialized adhesion junction between opposing podocytes, establishing the final filtration barrier that prevents passage of proteins from the capillary lumen into the urinary space. Nephrin, the key structural and signaling adhesion molecule expressed in the slit diaphragm, contains an evolutionally conserved, atypical PDZ-binding motif (PBM) reported to bind to a variety of proteins in the slit diaphragm. Several mutations in NPHS1 (the gene encoding nephrin) that result in nephrin lacking an intact PBM are associated with glomerular diseases. However, the molecular basis of nephrin-PBM–mediated protein complexes is still unclear.MethodsUsing a combination of biochemic, biophysic, and cell biologic approaches, we systematically investigated the interactions between nephrin-PBM and PDZ domain–containing proteins in the slit diaphragm.ResultsWe found that nephrin-PBM specifically binds to one member of the membrane-associated guanylate kinase family of scaffolding proteins, MAGI1, but not to another, MAGI2. The complex structure of MAGI1-PDZ3/nephrin-PBM reveals that the Gly at the −3 position of nephrin-PBM is the determining feature for MAGI1-PDZ3 recognition, which sharply contrasts with the typical PDZ/PBM binding mode. A single gain-of-function mutation within MAGI2 enabled nephrin-PBM binding. In addition, using our structural analysis, we developed a highly efficient inhibitory peptide capable of specifically blocking the nephrin/MAGI1 interaction.ConclusionsMAGI1 interacts with nephrin-PBM with exquisite specificity. A newly developed, potent inhibitory peptide that blocks this interaction may be useful for future functional investigations in vivo. Our findings also provide possible explanations for the diseases caused by NPHS1 mutations.

scholarly journals Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yi Zhang ◽  
Kyle Brown ◽  
Yucong Yu ◽  
Ziad Ibrahim ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Phase Separation ◽  
Cell Nucleus ◽  
Active Site ◽  
Histone Acetyltransferase ◽  
Histone H3 ◽  
Catalytic Core ◽  
Cellular Processes ◽  
Storage Pool ◽  
Core Components ◽  
Acetyltransferase P300

AbstractThe transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

scholarly journals Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress

2016 ◽  
Vol 311 (6) ◽  
pp. F1308-F1317 ◽  
Author(s):  
Leopoldo Raij ◽  
Runxia Tian ◽  
Jenny S. Wong ◽  
John C. He ◽  
Kirk N. Campbell
Keyword(s):  
Oxidative Stress ◽  
Glomerular Filtration ◽  
Biologically Active ◽  
Current Evidence ◽  
Glomerular Sclerosis ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Filtration Barrier ◽  
Focal Segmental Glomerular Sclerosis

Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS), and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of plasminogen (Plg) is activated to the biologically active serine protease plasmin by urokinase-type plasminogen activator (uPA). In vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here, we show that Plg is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap −/− model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA, and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2−) that promotes endothelin-1 synthesis. Plg via O2− also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together, our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a “second hit” in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention.

In situ microscopic studies on the structures and phase behaviors of SF/PEG films using solid-state NMR and Raman imaging

2016 ◽  
Vol 18 (24) ◽  
pp. 16353-16360 ◽  
Author(s):  
Congheng Chen ◽  
Ting Yao ◽  
Sidong Tu ◽  
Weijie Xu ◽  
Yi Han ◽  
...  
Keyword(s):  
Phase Separation ◽  
Solid State Nmr ◽  
Random Coil ◽  
Rich Domain ◽  
Microscopic Studies ◽  
Helix Conformation ◽  
Phase Behaviors ◽  
Protein Rich ◽  
Β Sheet

SF was incompatible with PEG in some extent, and the phase separation took place in their blend film. The conformation of SF in the interface between SF and PEG was changed to the β-sheet, while that in the protein-rich domain remained in the random coil and/or helix conformation.

Preservation for diverse users: digital preservation and the “Designated Community” at the Ontario Jewish Archives

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Nathan Moles
Keyword(s):  
Digital Preservation ◽  
Reference Points ◽  
Digital Information ◽  
User Group ◽  
Content Type ◽  
Open Archival Information System ◽  
Novel Approach ◽  
Core Components ◽  
Unique Approach

PurposeConventional approaches to digital preservation posit that archives should define a Designated Community, or future user group, for whom they preserve digital information. Archivists can then use their knowledge of these users as a reference to help them deliver digital information that is intelligible and usable. However, this approach is challenging for archives with mandates to serve wide and diverse audiences; these archives risk undermining their efforts by focusing on the interests of a narrow user group.Design/methodology/approachA unique approach to this challenge was developed in the context of a project to build a digital preservation program at the Ontario Jewish Archives (OJA). It draws from previous research on this topic and is based on a combination of practical and theoretical considerations.FindingsThe approach described here replaces the reference of a Designated Community with three core components: a re-articulation of the Open Archival Information System (OAIS) mandatory responsibilities; the identification of three distinct tiers of access for digital records; and the implementation of an access portal that allows digital records to be accessed and rendered online. Together with supplemental shifts in reference points, they provide an alternative to the concept of a Designated Community in the determination of preservation requirements, the identification of significant properties, the creation of Representation Information and in the evaluation of success.Originality/valueThis article contributes a novel approach to the ongoing conversation about the Designated Community in digital preservation, its application and its limitations in an archival context.

Post-war tourism as an urban reconstruction strategy case study: Khorramshahr

2018 ◽  
Vol 4 (1) ◽  
pp. 81-97 ◽  
Author(s):  
Seyed Mehdi Mirisaee ◽  
Yahaya Ahmad
Keyword(s):  
Mixed Method ◽  
Urban Areas ◽  
Urban Policy ◽  
Urban Tourism ◽  
Probability Method ◽  
Content Type ◽  
Qualitative And Quantitative ◽  
The Core ◽  
Core Components ◽  
Post War

Purpose Tourism development has been perceived as a promoter of city restoration and can also affect the post-war city reconstruction. Questions on how to reconstruct ruined buildings and urban areas through a post-war tourism-oriented approach based on the expectations of residents and tourists profound answers. The purpose of this paper is to adopt the sequential mixed method (qualitative and quantitative) with purposive sampling which is a non-probability method to investigate tourism-oriented approaches in the reconstruction of buildings and landmarks as the core components of urban tourism. Design/methodology/approach The study adopted the sequential mixed method (qualitative and quantitative) to investigate tourism-oriented approaches in the reconstruction of buildings and landmarks as the core components of urban tourism. Findings The findings of the study point that the preferred strategy for the reconstruction of damaged symbolic building is the preservation of the war effects in regard maintaining the buildings’ history to be considered by urban policy makers, urban designers, and authorities. Research limitations/implications The constraint was associated with the time-consuming nature of this type of research. Original documents of the research context and all the interview data were in the Persian language, making the translating process a time-consuming matter. Furthermore, data collection in the area located near the Iran-Iraq border (500 meters) presented a number of security caveats as limitations. Originality/value The research found a majority of tourists and the residents preferred tourism zone where the combination of post-war and natural attraction across riverside area. In other word, most considerable post-war attractions are those that combined with the appeal of the other tourism potentials like eco-leisure tourism. The preferred strategy for the reconstruction of damaged building reconstruction as post-war tourism attractions is the preservation of the war effects in regard maintaining the buildings history rather than reconstruction as the most likely to pre-war conditions with less attention paid to the war effects.

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