Rodriguez-Gallardo, SofiaKurokawa, KazuoSabido-Bozo, SusanaCortes-Gomez, AlejandroPerez-Linero, Ana MariaAguilera-Romero, AuxiliadoraLopez, SergioWaga, MihoNakano, AkihikoMuñiz, Manuel2022-07-262022-07-262021-10-01Rodriguez-Gallardo S, Kurokawa K, Sabido-Bozo S, Cortes-Gomez A, Perez-Linero AM, Aguilera-Romero A, et al. Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM). PLoS One. 2021 Oct 1;16(10):e0258111http://hdl.handle.net/10668/3822Otro material suplementario: Video 1: Multi-angle 3D reconstructed movie representing sorting of Gas1-GFP (green) and Mid2-iRFP (blue) into different ERES (red) in sec31-1 cells. https://doi.org/10.1371/journal.pone.0258111.s002. Video 2: Multi-angle 3D reconstructed movie representing incorporation of Gas1-GFP (green) and Mid2-iRFP (blue) into the same ERES (red) in sec31-1 Ghlag1 cells. https://doi.org/10.1371/journal.pone.0258111.s003Understanding 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.enAtribución 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/Endoplasmic reticulumCeramideMicroscopyProteinERESMembraneRetículo endoplásmicoCeramidasMicroscopíaProteínaMembranasMedical Subject Headings::Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological TransportMedical Subject Headings::Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Endoplasmic ReticulumMedical Subject Headings::Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Golgi ApparatusMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Diagnostic Imaging::Imaging, Three-DimensionalMedical Subject Headings::Anatomy::Cells::Cellular Structures::Cell Membrane::Intracellular MembranesMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Diagnostic Imaging::Microscopy::Microscopy, ConfocalMedical Subject Headings::Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological Transport::Protein Transportresearch article34597321open access10.1371/journal.pone.02581111932-6203PMC8486111