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Juno and CD9 protein network organization in oolemma of mouse oocyte
Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metap...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450504/ https://www.ncbi.nlm.nih.gov/pubmed/37635875 http://dx.doi.org/10.3389/fcell.2023.1110681 |
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author | Frolikova, Michaela Sur, Vishma Pratap Novotny, Ivan Blazikova, Michaela Vondrakova, Jana Simonik, Ondrej Ded, Lukas Valaskova, Eliska Koptasikova, Lenka Benda, Ales Postlerova, Pavla Horvath, Ondrej Komrskova, Katerina |
author_facet | Frolikova, Michaela Sur, Vishma Pratap Novotny, Ivan Blazikova, Michaela Vondrakova, Jana Simonik, Ondrej Ded, Lukas Valaskova, Eliska Koptasikova, Lenka Benda, Ales Postlerova, Pavla Horvath, Ondrej Komrskova, Katerina |
author_sort | Frolikova, Michaela |
collection | PubMed |
description | Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson’s correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion. |
format | Online Article Text |
id | pubmed-10450504 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-104505042023-08-26 Juno and CD9 protein network organization in oolemma of mouse oocyte Frolikova, Michaela Sur, Vishma Pratap Novotny, Ivan Blazikova, Michaela Vondrakova, Jana Simonik, Ondrej Ded, Lukas Valaskova, Eliska Koptasikova, Lenka Benda, Ales Postlerova, Pavla Horvath, Ondrej Komrskova, Katerina Front Cell Dev Biol Cell and Developmental Biology Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson’s correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion. Frontiers Media S.A. 2023-08-10 /pmc/articles/PMC10450504/ /pubmed/37635875 http://dx.doi.org/10.3389/fcell.2023.1110681 Text en Copyright © 2023 Frolikova, Sur, Novotny, Blazikova, Vondrakova, Simonik, Ded, Valaskova, Koptasikova, Benda, Postlerova, Horvath and Komrskova. 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 Frolikova, Michaela Sur, Vishma Pratap Novotny, Ivan Blazikova, Michaela Vondrakova, Jana Simonik, Ondrej Ded, Lukas Valaskova, Eliska Koptasikova, Lenka Benda, Ales Postlerova, Pavla Horvath, Ondrej Komrskova, Katerina Juno and CD9 protein network organization in oolemma of mouse oocyte |
title | Juno and CD9 protein network organization in oolemma of mouse oocyte |
title_full | Juno and CD9 protein network organization in oolemma of mouse oocyte |
title_fullStr | Juno and CD9 protein network organization in oolemma of mouse oocyte |
title_full_unstemmed | Juno and CD9 protein network organization in oolemma of mouse oocyte |
title_short | Juno and CD9 protein network organization in oolemma of mouse oocyte |
title_sort | juno and cd9 protein network organization in oolemma of mouse oocyte |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450504/ https://www.ncbi.nlm.nih.gov/pubmed/37635875 http://dx.doi.org/10.3389/fcell.2023.1110681 |
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