Background: Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis. Methods: Glomeruli isolated from Podocin-Cre Rosa-DTR flox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification. Results: Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed. Conclusions: Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.
Background: Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis. Methods: Glomeruli isolated from Podocin-Cre Rosa-DTR flox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification. Results: Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed. Conclusions: Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.
Authors: Cristina Has; Giuseppina Spartà; Dimitra Kiritsi; Lisa Weibel; Alexander Moeller; Virginia Vega-Warner; Aoife Waters; Yinghong He; Yair Anikster; Philipp Esser; Beate K Straub; Ingrid Hausser; Detlef Bockenhauer; Benjamin Dekel; Friedhelm Hildebrandt; Leena Bruckner-Tuderman; Guido F Laube Journal: N Engl J Med Date: 2012-04-19 Impact factor: 91.245
Authors: Xuefei Tian; Jin Ju Kim; Susan M Monkley; Nanami Gotoh; Ramiro Nandez; Keita Soda; Kazunori Inoue; Daniel M Balkin; Hossam Hassan; Sung Hyun Son; Yashang Lee; Gilbert Moeckel; David A Calderwood; Lawrence B Holzman; David R Critchley; Roy Zent; Jochen Reiser; Shuta Ishibe Journal: J Clin Invest Date: 2014-02-17 Impact factor: 14.808
Authors: Sharon E Miller; Daniela A Sahlender; Stephen C Graham; Stefan Höning; Margaret S Robinson; Andrew A Peden; David J Owen Journal: Cell Date: 2011-11-23 Impact factor: 41.582
Authors: Jennifer Hirst; James R Edgar; Georg H H Borner; Sam Li; Daniela A Sahlender; Robin Antrobus; Margaret S Robinson Journal: Mol Biol Cell Date: 2015-07-15 Impact factor: 4.138