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Analysis of podocyte adhesome proteomes

ABSTRACT: To elucidate potential mechanisms how EPB41L5 modulates integrin adhesion complex maturation, we performed quantitative SILAC-based integrin adhesome proteomics. For MS analysis of the podocyte adhesome, EPB41L5 or Luciferase (as negative control) was transient expressed in SILAC labeled podocytes by nucleofection (Amaxa Nucleofector, Lonza, Switzerland). Podocytes were seeded on cell culture dishes for 24 hours. Isolation of integrin adhesion complexes from podocytes was performed prior LC-MSMS analysis.In a second set of experiments adhesion complexes of collagen IV and fibronectin coated dishes were purified and analysed by LC-MSMS.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Podocyte (sensu Diptera)

SUBMITTER: Joern Dengjel

LAB HEAD: Joern Dengjel

PROVIDER: PXD023821 | Pride | 2021-08-05

REPOSITORIES: pride

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			Action	DRS
	20140416_VKNT2_A_01.RAW	Raw
	20140416_VKNT2_A_02.RAW	Raw
	20140416_VKNT2_A_03.RAW	Raw
	20140416_VKNT2_A_04.RAW	Raw
	20140416_VKNT2_A_05.RAW	Raw

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Publications

EPB41L5 controls podocyte extracellular matrix assembly by adhesome-dependent force transmission.

Maier Jasmin I JI Rogg Manuel M Helmstädter Martin M Sammarco Alena A Schilling Oliver O Sabass Benedikt B Miner Jeffrey H JH Dengjel Jörn J Walz Gerd G Werner Martin M Huber Tobias B TB Schell Christoph C

Cell reports 20210301 12

The integrity of the kidney filtration barrier essentially relies on the balanced interplay of podocytes and the glomerular basement membrane (GBM). Here, we show by analysis of in vitro and in vivo models that a loss of the podocyte-specific FERM-domain protein EPB41L5 results in impaired extracellular matrix (ECM) assembly. By using quantitative proteomics analysis of the secretome and matrisome, we demonstrate a shift in ECM composition characterized by diminished deposition of core GBM compo ...[more]

PMID: 33761352

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EPB41L5 controls podocyte extracellular matrix assembly by promoting integrin adhesome reinforcement - CCM dataset

Project description:The integrity of the kidney filtration barrier essentially relies on the balanced interplay of podocytes, endothelial cells and the glomerular basement membrane. Dysfunction in any of these components results in proteinuria and can progress to chronic kidney disease. While the relevance of cell-matrix interactions for podocytes is well established, it is only incompletely understood whether and how adhesion receptors reciprocally influence the basement membrane and maintain glomerular function. Here, we show by analysis of in vitro and in vivo models that a loss of the podocyte specific FERM-domain protein EPB41L5 results in impaired extracellular matrix integrity and stability. Employing quantitative proteomics analysis of the secretome, matrisome and integrin adhesome revealed that EPB41L5 promotes mechanosensitive properties of podocyte integrin complexes by recruitment of PDLIM5/ACTN4. Consecutively, EPB41L5 knockout podocytes showed insufficient reinforcement of integrin adhesion sites, which translated into impaired ECM assembly. These observations build the framework for a model where EPB41L5 as a cell type specific regulator of the podocyte adhesome controls a localized adaptive module in order to prevent podocyte detachment and ensures GBM integrity.

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EPB41L5 controls podocyte extracellular matrix assembly by promoting integrin adhesome reinforcement - CDM dataset

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The melanoma cell line MDA-MB-435 adhesome in long-term culture and its comparison to the adhesome upon integrin αV knockdown

Project description:Using mass spectrometry–based proteomics, we analysed the components of integrin adhesion complexes of MDA-MB-435S cells and two MDA-MB-435S-derived integrin αV-specific shRNA-expressing cell clones. Among integrins, we detected αV and β5 integrin subunits in the MDA-MB-435S adhesome, thus showing that in long term culture these cells use preferentially integrin αVβ5 for adhesion. Our data represents the first published adhesome of αVβ5 integrin heterodimer.

2020-02-17 | PXD016837 | Pride

Characterisation of a nucleo-adhesome

Project description:In addition to central functions in cell adhesion signalling, integrin-associated proteins have wider roles at sites distal to adhesion receptors. In experimentally defined adhesomes, we noticed that there is clear enrichment of proteins that localise to the nucleus, and conversely, we now report that nuclear proteomes contain a class of adhesome components that localise to the nucleus. We here defined a nucleo-adhesome, providing experimental evidence for a remarkable scale of nuclear localisation of adhesion proteins, establishing a framework for interrogating nuclear adhesion protein functions.

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Characterisation of the adhesome of patient-derived cutaneous squamous carcinoma cells

Project description:Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes (IACs), play key roles in cancer progression and metastasis. We report here systems-level changes in the adhesome during progression of a patient-derived cutaneous squamous cell carcinoma (cSCC). We found that the actin regulatory protein Mena is enriched in IACs in metastatic cSCC cells and is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2.

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Expression data from cultured WT and NCK1/2 knockout mouse podocyte cells

Project description:Background: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins to trigger actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 in podocytes remain to be fully elucidated. Methods: We generated podocytes deficient in Nck1 and Nck2, and used transcriptomic approaches to profile expression differences. We further applied proteomic techniques to identify specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2 along with podocyte injury models to comprehensively verify our findings. Results: Compound loss of Nck1/2 resulted in altered expression of genes involved in actin binding, cell adhesion and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2, and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling and integrin activation at focal adhesions via α-actinin-4. Furthermore, we found that loss of either Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2 deficient mice, consistent with enhanced susceptibility to podocyte injury. Conclusion: These findings highlight distinct yet complementary roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition and sustaining filtration barrier integrity.

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The Itgb3 Adhesome - Integrin β3 is seen as a key anti-angiogenic target for cancer treatment due to its expression on neovasculature

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Definition of the fibroblast adhesome using multiplexed proximity biotinylation

Project description:Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry and topological organisation of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs on a global level.

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Podocyte injury in Fabry nephropathy

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