Claire E Martin1,2, Noah J Phippen1, Ava Keyvani Chahi1,3, Manali Tilak1, Sara L Banerjee4,5, Peihua Lu1, Laura A New1, Casey R Williamson1, Mathew J Platt6, Jeremy A Simpson6, Mira Krendel7, Nicolas Bisson4,5,8, Anne-Claude Gingras2,9, Nina Jones10. 1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada. 2. Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada. 3. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada. 4. Division of Oncology, Centre de Recherche du Centre Hospitalier Universitaire de Quebec-Laval University, Quebec City, Quebec, Canada. 5. Centre de Recherche sur le Cancer de l'Université Laval, Quebec City, Quebec, Canada. 6. Department of Human Health and Nutritional Science, University of Guelph, Guelph, Ontario, Canada. 7. Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York. 8. PROTEO-Quebec Network for Research on Protein Function, Engineering, and Applications Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University, Quebec City, Quebec, Canada. 9. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. 10. Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada jonesmcb@uoguelph.ca.
Abstract
BACKGROUND: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers 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 adaptors in podocytes remain to be fully elucidated. METHODS: We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified 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 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 at focal adhesions via α actinin-4. Furthermore, loss of 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 reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.
BACKGROUND: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers 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 adaptors in podocytes remain to be fully elucidated. METHODS: We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified 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 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 at focal adhesions via α actinin-4. Furthermore, loss of 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 reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.
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