Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM)
| Autor: | Miho Waga, Akihiko Nakano, Auxiliadora Aguilera-Romero, Alejandro Cortes-Gomez, Sergio López, Sofia Rodriguez-Gallardo, Manuel Muñiz, Kazuo Kurokawa, Susana Sabido-Bozo, Ana Maria Perez-Linero |
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| Přispěvatelé: | [Rodriguez-Gallardo,S, Sabido-Bozo,S, Cortes-Gomez,A, Perez-Linero,AM, Aguilera-Romero,A, Lopez,S, Muñiz,M] Department of Cell Biology, University of Seville and Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain. [Kurokawa,K, Waga,M, Nakano,A] Live Cell Super-Resolution Imaging Research Team, RIKEN Center for Advanced Photonics, Saitama, Japan., MM, BFU2017-89700-P, FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación, https://www.ciencia.gob.es/portal/site/MICINN/aei AN and KK, JP25221103, JP17H06420 and JP18H05275, Japan Society for the Promotion of Science (JSPS), https://www.jsps.go.jp/english/index.html, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Japan Society for the Promotion of Science, Universidad de Sevilla. Departamento de Biología Celular |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Cell Membranes Golgi Apparatus Endoplasmic Reticulum Biochemistry Anatomy::Cells::Cellular Structures::Cell Membrane::Intracellular Membranes [Medical Subject Headings] Ceramide Fluorescence Microscopy Lab Protocol Cargo Proteins Microscopía Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Endoplasmic Reticulum [Medical Subject Headings] ERES COPII Microscopy Multidisciplinary Secretory Pathway Microscopy Confocal Analytical Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Diagnostic Imaging::Imaging Three-Dimensional [Medical Subject Headings] Chemistry Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological Transport::Protein Transport [Medical Subject Headings] Membrane Eukaryota Light Microscopy ER retention Lipids Cell biology Protein Transport Cell Processes symbols Medicine Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological Transport [Medical Subject Headings] Cellular Structures and Organelles Analytical Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Diagnostic Imaging::Microscopy::Microscopy Confocal [Medical Subject Headings] Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Golgi Apparatus [Medical Subject Headings] Imaging Techniques Science Research and Analysis Methods Retículo endoplásmico symbols.namesake Imaging Three-Dimensional Live cell imaging Fluorescence Imaging Secretory pathway Endoplasmic reticulum Protein Organisms Fungi Biology and Life Sciences Proteins Membrane Proteins Biological Transport Cell Biology Intracellular Membranes Golgi apparatus Yeast Membranas Ceramidas Secretory protein Proteína |
| Zdroj: | PLoS ONE PLoS ONE, Vol 16, Iss 10, p e0258111 (2021) idUS. Depósito de Investigación de la Universidad de Sevilla instname Digital.CSIC. Repositorio Institucional del CSIC |
| Popis: | 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. MM, BFU2017-89700-P, FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación, https://www.ciencia.gob.es/portal/site/MICINN/aei AN and KK, JP25221103, JP17H06420 and JP18H05275, Japan Society for the Promotion of Science (JSPS), https://www.jsps.go.jp/english/index.html |
| Databáze: | OpenAIRE |
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