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

ABSTRACT: 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.

SUBMITTER: Rodriguez-Gallardo S

PROVIDER: S-EPMC8486111 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In the last years, microdosimetric numerical models of cells including intracellular compartments have been proposed, aiming to investigate the poration induced by the application of nanosecond pulsed electric fields (nsPEFs). A limitation of such models was the extremely approximate cell and organelle shapes, leading to an incorrect estimation of the electric field or transmembrane potential distribution in the studied domain. In order to obtain a reliable model of in vitro experiments and a one-to-one comparison between experimental and simulated results, here, a realistic model of 12 human mesenchymal stem cells was built starting from their optical microscopy images where different cell compartments were highlighted. The microdosimetric analysis of the cells group was quantified in terms of electric field and transmembrane potentials (TMPs) induced by an externally applied 10-ns trapezoidal pulse with rise and fall times of 2 ns, with amplitudes ranging from 2 to 30 MV/m. The obtained results showed that the plasma and endoplasmic reticulum (ER) membrane of each cell respond in a different way to the same electric field amplitude, depending on differences in shape, size, and position of the single cell with respect to the applied electric field direction. Therefore, also the threshold for an efficient electroporation is highly different from cell to cell. This difference was quantitatively estimated through the cumulative distribution function of the pore density for the plasma and ER membrane of each cell, representing the probability that a certain percentage of membrane has reached a specific value of pore density. By comparing the dose-response curves resulted from the simulations and those from the experimental study of De Menorval et al. (2016), we found a very good matching of results for plasma and ER membrane when 2% of the porated area is considered sufficient for permeabilizing the membrane. This result is worth of noting as it highlights the possibility to effectively predict the behavior of a cell (or of a population of cells) exposed to nsPEFs. Therefore, the microdosimetric realistic model described here could represent a valid tool in setting up more efficient and controlled electroporation protocols.

Dataset Information

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

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