Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into differen...

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Autores principales:	Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortes-Gomez, Ana Maria Perez-Linero, Auxiliadora Aguilera-Romero, Sergio Lopez, Miho Waga, Akihiko Nakano, Manuel Muñiz
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/559aca166abe4d3295be01873b9fb3a5
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spelling	oai:doaj.org-article:559aca166abe4d3295be01873b9fb3a52021-12-02T20:17:25ZAssay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).1932-620310.1371/journal.pone.0258111https://doaj.org/article/559aca166abe4d3295be01873b9fb3a52021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0258111https://doaj.org/toc/1932-6203Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.Sofia Rodriguez-GallardoKazuo KurokawaSusana Sabido-BozoAlejandro Cortes-GomezAna Maria Perez-LineroAuxiliadora Aguilera-RomeroSergio LopezMiho WagaAkihiko NakanoManuel MuñizPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 10, p e0258111 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sofia Rodriguez-Gallardo Kazuo Kurokawa Susana Sabido-Bozo Alejandro Cortes-Gomez Ana Maria Perez-Linero Auxiliadora Aguilera-Romero Sergio Lopez Miho Waga Akihiko Nakano Manuel Muñiz Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).
description	Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.
format	article
author	Sofia Rodriguez-Gallardo Kazuo Kurokawa Susana Sabido-Bozo Alejandro Cortes-Gomez Ana Maria Perez-Linero Auxiliadora Aguilera-Romero Sergio Lopez Miho Waga Akihiko Nakano Manuel Muñiz
author_facet	Sofia Rodriguez-Gallardo Kazuo Kurokawa Susana Sabido-Bozo Alejandro Cortes-Gomez Ana Maria Perez-Linero Auxiliadora Aguilera-Romero Sergio Lopez Miho Waga Akihiko Nakano Manuel Muñiz
author_sort	Sofia Rodriguez-Gallardo
title	Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).
title_short	Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).
title_full	Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).
title_fullStr	Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).
title_full_unstemmed	Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).
title_sort	assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3d super-resolution confocal live imaging microscopy (sclim).
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/559aca166abe4d3295be01873b9fb3a5
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Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).

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