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

• DOI: 10.1371/journal.pone.0258111
• Publication Date: 2021-10-01T18:53:16Z
• Authors: Sofia Rodriguez-G...; Kazuo Kurokawa; Susana Sabido-Bozo; Alejandro Cortes-...; Ana Maria Perez-L...; Auxiliadora Aguil...; Sergio Lopez; Miho Waga; Akihiko Nakano; Manuel Muñiz
• Keywords: Live cell imaging; COPII; Organelle

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 cell biology. We have further investigated yeast two distinct types cargo into different endoplasmic reticulum (ER) exit sites (ERES) for differential ER export Golgi apparatus. used an optimized protocol that combines live dual-cargo system with 3D simultaneous multi-color high-resolution microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which based on reversible retention de novo co-expressed cargos by blocking COPII function upon incubation thermo-sensitive allele sec31-1 at restrictive temperature (37°C). restored shifting down permissive (24°C) progressive incorporation fluorescently labelled ERES can be then simultaneously captured high spatial resolution SCLIM microscopy. By using shown newly synthesized glycosylphosphatidylinositol (GPI)-anchored having very long chain ceramide lipid moiety clustered specialized those transmembrane proteins. Furthermore, showed length present membrane critical sorting selectivity. Therefore, thanks presented method could obtain first direct vivo evidence length-based protein selective ERES.