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Visualization and Quantification of APP Intracellular Domain-Mediated Nuclear Signaling by Bimolecular Fluorescence Complementation
BACKGROUND: The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein c...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3783399/ https://www.ncbi.nlm.nih.gov/pubmed/24086696 http://dx.doi.org/10.1371/journal.pone.0076094 |
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author | Riese, Florian Grinschgl, Sonja Gersbacher, Manuel T. Russi, Natalie Hock, Christoph Nitsch, Roger M. Konietzko, Uwe |
author_facet | Riese, Florian Grinschgl, Sonja Gersbacher, Manuel T. Russi, Natalie Hock, Christoph Nitsch, Roger M. Konietzko, Uwe |
author_sort | Riese, Florian |
collection | PubMed |
description | BACKGROUND: The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes) that function in transcriptional regulation. OBJECTIVE: To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells. METHODS: We used cotransfection of bimolecular fluorescence complementation (BiFC) fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC). RESULTS: Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer’s disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic β-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals. CONCLUSION: We established a new method for visualization and FC quantification of the interaction between AICD, Fe65 and Tip60 in the nucleus based on BiFC. It enables flow cytometric analysis of AICD nuclear signaling and is characterized by scalability and low background fluorescence. |
format | Online Article Text |
id | pubmed-3783399 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-37833992013-10-01 Visualization and Quantification of APP Intracellular Domain-Mediated Nuclear Signaling by Bimolecular Fluorescence Complementation Riese, Florian Grinschgl, Sonja Gersbacher, Manuel T. Russi, Natalie Hock, Christoph Nitsch, Roger M. Konietzko, Uwe PLoS One Research Article BACKGROUND: The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes) that function in transcriptional regulation. OBJECTIVE: To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells. METHODS: We used cotransfection of bimolecular fluorescence complementation (BiFC) fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC). RESULTS: Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer’s disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic β-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals. CONCLUSION: We established a new method for visualization and FC quantification of the interaction between AICD, Fe65 and Tip60 in the nucleus based on BiFC. It enables flow cytometric analysis of AICD nuclear signaling and is characterized by scalability and low background fluorescence. Public Library of Science 2013-09-25 /pmc/articles/PMC3783399/ /pubmed/24086696 http://dx.doi.org/10.1371/journal.pone.0076094 Text en © 2013 Riese et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Riese, Florian Grinschgl, Sonja Gersbacher, Manuel T. Russi, Natalie Hock, Christoph Nitsch, Roger M. Konietzko, Uwe Visualization and Quantification of APP Intracellular Domain-Mediated Nuclear Signaling by Bimolecular Fluorescence Complementation |
title | Visualization and Quantification of APP Intracellular Domain-Mediated Nuclear Signaling by Bimolecular Fluorescence Complementation |
title_full | Visualization and Quantification of APP Intracellular Domain-Mediated Nuclear Signaling by Bimolecular Fluorescence Complementation |
title_fullStr | Visualization and Quantification of APP Intracellular Domain-Mediated Nuclear Signaling by Bimolecular Fluorescence Complementation |
title_full_unstemmed | Visualization and Quantification of APP Intracellular Domain-Mediated Nuclear Signaling by Bimolecular Fluorescence Complementation |
title_short | Visualization and Quantification of APP Intracellular Domain-Mediated Nuclear Signaling by Bimolecular Fluorescence Complementation |
title_sort | visualization and quantification of app intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3783399/ https://www.ncbi.nlm.nih.gov/pubmed/24086696 http://dx.doi.org/10.1371/journal.pone.0076094 |
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