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Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process

Introduction: The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. Howeve...

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Autores principales: Gerlach, Gary F., Imseis, Zachary H., Cooper, Shamus L., Santos, Anabella N., O’Brien, Lori L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10262054/
https://www.ncbi.nlm.nih.gov/pubmed/37325559
http://dx.doi.org/10.3389/fcell.2023.1195037
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author Gerlach, Gary F.
Imseis, Zachary H.
Cooper, Shamus L.
Santos, Anabella N.
O’Brien, Lori L.
author_facet Gerlach, Gary F.
Imseis, Zachary H.
Cooper, Shamus L.
Santos, Anabella N.
O’Brien, Lori L.
author_sort Gerlach, Gary F.
collection PubMed
description Introduction: The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remain to be elucidated. Methods: Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin–BioID fusion. Podocin–BioID localizes to the slit diaphragm, and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Results and Discussion: Gene ontology analysis of 54 proteins specifically enriched in our podocin–BioID sample revealed ‘cell junctions,’ ‘actin binding,’ and ‘cytoskeleton organization’ as top terms. Known foot process components were identified, and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed that Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and support the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.
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spelling pubmed-102620542023-06-15 Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process Gerlach, Gary F. Imseis, Zachary H. Cooper, Shamus L. Santos, Anabella N. O’Brien, Lori L. Front Cell Dev Biol Cell and Developmental Biology Introduction: The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remain to be elucidated. Methods: Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin–BioID fusion. Podocin–BioID localizes to the slit diaphragm, and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Results and Discussion: Gene ontology analysis of 54 proteins specifically enriched in our podocin–BioID sample revealed ‘cell junctions,’ ‘actin binding,’ and ‘cytoskeleton organization’ as top terms. Known foot process components were identified, and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed that Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and support the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease. Frontiers Media S.A. 2023-05-30 /pmc/articles/PMC10262054/ /pubmed/37325559 http://dx.doi.org/10.3389/fcell.2023.1195037 Text en Copyright © 2023 Gerlach, Imseis, Cooper, Santos and O’Brien. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Gerlach, Gary F.
Imseis, Zachary H.
Cooper, Shamus L.
Santos, Anabella N.
O’Brien, Lori L.
Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process
title Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process
title_full Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process
title_fullStr Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process
title_full_unstemmed Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process
title_short Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process
title_sort mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10262054/
https://www.ncbi.nlm.nih.gov/pubmed/37325559
http://dx.doi.org/10.3389/fcell.2023.1195037
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