Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation.

<h4>Background</h4>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 nuc...

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Autores principales:	Florian Riese, Sonja Grinschgl, Manuel T Gersbacher, Natalie Russi, Christoph Hock, Roger M Nitsch, Uwe Konietzko
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5407866b71174eafabc1cd7e9e47c6dd
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spelling	oai:doaj.org-article:5407866b71174eafabc1cd7e9e47c6dd2021-11-18T08:53:52ZVisualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation.1932-620310.1371/journal.pone.0076094https://doaj.org/article/5407866b71174eafabc1cd7e9e47c6dd2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24086696/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Objective</h4>To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells.<h4>Methods</h4>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).<h4>Results</h4>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.<h4>Conclusion</h4>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.Florian RieseSonja GrinschglManuel T GersbacherNatalie RussiChristoph HockRoger M NitschUwe KonietzkoPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 9, p e76094 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Florian Riese Sonja Grinschgl Manuel T Gersbacher Natalie Russi Christoph Hock Roger M Nitsch Uwe Konietzko Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation.
description	<h4>Background</h4>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.<h4>Objective</h4>To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells.<h4>Methods</h4>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).<h4>Results</h4>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.<h4>Conclusion</h4>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	article
author	Florian Riese Sonja Grinschgl Manuel T Gersbacher Natalie Russi Christoph Hock Roger M Nitsch Uwe Konietzko
author_facet	Florian Riese Sonja Grinschgl Manuel T Gersbacher Natalie Russi Christoph Hock Roger M Nitsch Uwe Konietzko
author_sort	Florian Riese
title	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_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_sort	visualization and quantification of app intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/5407866b71174eafabc1cd7e9e47c6dd
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_version_	1718421200650108928

Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation.

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