Three-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy

ABSTRACT The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM)...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Winnie Kerstens, Anneke Kremer, Michelle Holtappels, Peter Borghgraef, Saskia Lippens, Patrick Van Dijck
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	FIB-SEM electron microscopy Saccharomyces cerevisiae yeast APEX2 DAB Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/03364b78ae7b4984a449b07f752521ff
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:03364b78ae7b4984a449b07f752521ff
record_format	dspace
spelling	oai:doaj.org-article:03364b78ae7b4984a449b07f752521ff2021-11-15T15:27:53ZThree-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy10.1128/mSphere.00981-192379-5042https://doaj.org/article/03364b78ae7b4984a449b07f752521ff2020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00981-19https://doaj.org/toc/2379-5042ABSTRACT The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3′-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins. IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae. As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.Winnie KerstensAnneke KremerMichelle HoltappelsPeter BorghgraefSaskia LippensPatrick Van DijckAmerican Society for MicrobiologyarticleFIB-SEMelectron microscopySaccharomyces cerevisiaeyeastAPEX2DABMicrobiologyQR1-502ENmSphere, Vol 5, Iss 1 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	FIB-SEM electron microscopy Saccharomyces cerevisiae yeast APEX2 DAB Microbiology QR1-502
spellingShingle	FIB-SEM electron microscopy Saccharomyces cerevisiae yeast APEX2 DAB Microbiology QR1-502 Winnie Kerstens Anneke Kremer Michelle Holtappels Peter Borghgraef Saskia Lippens Patrick Van Dijck Three-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy
description	ABSTRACT The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3′-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins. IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae. As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.
format	article
author	Winnie Kerstens Anneke Kremer Michelle Holtappels Peter Borghgraef Saskia Lippens Patrick Van Dijck
author_facet	Winnie Kerstens Anneke Kremer Michelle Holtappels Peter Borghgraef Saskia Lippens Patrick Van Dijck
author_sort	Winnie Kerstens
title	Three-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy
title_short	Three-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy
title_full	Three-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy
title_fullStr	Three-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy
title_full_unstemmed	Three-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy
title_sort	three-dimensional visualization of apex2-tagged erg11 in <named-content content-type="genus-species">saccharomyces cerevisiae</named-content> using focused ion beam scanning electron microscopy
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/03364b78ae7b4984a449b07f752521ff
work_keys_str_mv	AT winniekerstens threedimensionalvisualizationofapex2taggederg11innamedcontentcontenttypegenusspeciessaccharomycescerevisiaenamedcontentusingfocusedionbeamscanningelectronmicroscopy AT annekekremer threedimensionalvisualizationofapex2taggederg11innamedcontentcontenttypegenusspeciessaccharomycescerevisiaenamedcontentusingfocusedionbeamscanningelectronmicroscopy AT michelleholtappels threedimensionalvisualizationofapex2taggederg11innamedcontentcontenttypegenusspeciessaccharomycescerevisiaenamedcontentusingfocusedionbeamscanningelectronmicroscopy AT peterborghgraef threedimensionalvisualizationofapex2taggederg11innamedcontentcontenttypegenusspeciessaccharomycescerevisiaenamedcontentusingfocusedionbeamscanningelectronmicroscopy AT saskialippens threedimensionalvisualizationofapex2taggederg11innamedcontentcontenttypegenusspeciessaccharomycescerevisiaenamedcontentusingfocusedionbeamscanningelectronmicroscopy AT patrickvandijck threedimensionalvisualizationofapex2taggederg11innamedcontentcontenttypegenusspeciessaccharomycescerevisiaenamedcontentusingfocusedionbeamscanningelectronmicroscopy
_version_	1718427927296606208

Three-Dimensional Visualization of APEX2-Tagged Erg11 in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content> Using Focused Ion Beam Scanning Electron Microscopy

Ejemplares similares