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In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution
The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose tempo...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724601/ https://www.ncbi.nlm.nih.gov/pubmed/30892990 http://dx.doi.org/10.1091/mbc.E18-12-0799 |
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author | Remnant, Lucy Booth, Daniel G. Vargiu, Giulia Spanos, Christos Kerr, Alastair R. W. Earnshaw, William C. |
author_facet | Remnant, Lucy Booth, Daniel G. Vargiu, Giulia Spanos, Christos Kerr, Alastair R. W. Earnshaw, William C. |
author_sort | Remnant, Lucy |
collection | PubMed |
description | The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose temporal characterization during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a “promiscuous” biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion protein of interest. As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 h during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighborhood could then be characterized by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterized within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is “frozen.” This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighborhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology. |
format | Online Article Text |
id | pubmed-6724601 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-67246012019-09-06 In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution Remnant, Lucy Booth, Daniel G. Vargiu, Giulia Spanos, Christos Kerr, Alastair R. W. Earnshaw, William C. Mol Biol Cell Articles The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose temporal characterization during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a “promiscuous” biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion protein of interest. As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 h during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighborhood could then be characterized by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterized within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is “frozen.” This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighborhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology. The American Society for Cell Biology 2019-05-15 /pmc/articles/PMC6724601/ /pubmed/30892990 http://dx.doi.org/10.1091/mbc.E18-12-0799 Text en © 2019 Remnant, Booth, et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. http://creativecommons.org/licenses/by-nc-sa/3.0 This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License. |
spellingShingle | Articles Remnant, Lucy Booth, Daniel G. Vargiu, Giulia Spanos, Christos Kerr, Alastair R. W. Earnshaw, William C. In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution |
title | In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution |
title_full | In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution |
title_fullStr | In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution |
title_full_unstemmed | In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution |
title_short | In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution |
title_sort | in vitro bioid: mapping the cenp-a microenvironment with high temporal and spatial resolution |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724601/ https://www.ncbi.nlm.nih.gov/pubmed/30892990 http://dx.doi.org/10.1091/mbc.E18-12-0799 |
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