TITLE: Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by super-resolution confocal live imaging microscopy (SCLIM)
AUTHORS: 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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>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 SCLIM (Super-resolution Confocal Live Imaging Microscopy). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking the COPII function upon incubation of the thermo-sensitive COPII allele </span><span style = "font-style:italic;">sec31-1</span><span> at restrictive temperature (37ºC). ER export is restored by shifting down to permissive temperature (24ºC) and the progressive incorporation of the two different types of cargos into the fluorescently labeled 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 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.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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[STEPS]
?. Pick up sec31-1 temperature-sensitive mutant yeast cells expressing two galactose-inducible secretory cargos (i.e. the GPI-AP cargo Gas1-GFP and the transmembrane plasma membrane protein Mid2-iRFP) and the constitutive COPII outer coat protein Sec13-mCherry as ERES marker from a frozen stock using a sterile toothpick, streak yeast cells on a SD agar plate with appropriate amino acid supplements and incubate them at 24 °C for 2-3 days.
?. [Yeast growth and culture]
Inoculate yeast cells into 3 ml YPD medium in a 5 ml sterile tube and growth them for 6-8 h to 0,5 x 107 cells/ml at 24°C with shaking at 500 rpm.
?. [Yeast growth and culture]
Pick up sec31-1 temperature-sensitive mutant yeast cells expressing two galactose-inducible secretory cargos (i.e. the GPI-AP cargo Gas1-GFP and the transmembrane plasma membrane protein Mid2-iRFP) and the constitutive COPII outer coat protein Sec13-mCherry as ERES marker from a frozen stock using a sterile toothpick, streak yeast cells on a SD agar plate with appropriate amino acid supplements and incubate them at 24 °C for 2-3 days.
?. [Yeast growth and culture]
Pellet yeast cells by centrifugation at 3000 x g for 5 min, discard the supernatant and resuspend in 4 ml of YPR medium.
?. [Yeast growth and culture]
Pellet yeast cells again by centrifugation at 3000 x g for 5 min, discard the supernatant, resuspend in 3 ml of YPR and growth them to 0,4 x 107 cells/ml overnight at 24°C with shaking at 500 rpm.
?. [Yeast sample preparation]
During the 30 min incubation at 37°C in SG (see step 8), prepare a fresh Concanavalin A-coated well chamber slide to immobilize the yeast cells for microscopic observation.
?. [Yeast sample preparation]
After incubation for 30 min at 37°C in SG (see step 8), apply about 100 µl of yeast liquid culture into a well of the Concanavalin A-coated chamber slide.
?. [Yeast sample preparation]
Wait for 2 min for immobilization of yeast cells on the Concanavalin A-coated chamber slide.
?. [Yeast sample preparation]
Place the Concanavalin A-coated chamber slide with immobilized yeast cells on the microscope stage, which has been already pre-warmed at 24°C by an automatic thermo-control system.
?. [Yeast sample preparation]
Image yeast cells after 20 min of shift down to 24ºC (permissive temperature) to simultaneously visualize the entry of the two cargo proteins into the ERES.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Set up of SCLIM before observation.Due to several factors, such as chromatic aberrations of lenses in the whole microscopic and spectroscopic system, the 3 different fluorescence signals coming from the same position (XYZ) may be projected to different positions (XYZ) of 3 cameras. To conduct simultaneous 3-color 3D observation, it is necessary first to check and correct the XYZ misalignments of these fluorescence signals by using pinhole images of 3 cameras.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Observe the yeast cells expressing cargos (Galactose-inducible Gas1-GFP and Mid2-iRFP) and ERES marker (Sec13-mCherry) by SCLIM. In these cells, we can observe that a small portion of both cargos enter into different ERES.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Image acquisitionFluorescent proteins have a wide range of emission. Thus, for example, when GFP is expressed at a very high level, the GFP fluorescence signal can be detected even by a camera with a longer-wavelength emission filer. Such a leakage of fluorescence signals is called as bleed-through or crosstalk. It is critical to avoid such bleed-through signals as much as possible. The powers of 3 excitation lasers and the amplification gains of 3 image intensifiers should be independently controlled to minimize bleed-through.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
2D confocal fluorescence data are separated in the custom-made spectroscopic unit. GFP, mCherry, and iRFP fluorescence images finally pass through the spectral windows defined by 2 band pass filters (GFP; 490-545 nm, and mCherry; 580-660 nm) and 1 long pass filter (iRFP; 680 nm-) and are simultaneously collected by synchronized 3 EMCCD cameras.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Collect Z slice of 3-color images. An appropriate range of Z-axis (e.g., 3 μm cover about 80% of yeast cell volume) is scanned into optical slices 100 nm apart by the piezo actuator equipped to the objective lens.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Raw data sets (Z slice 3-color images) are once stored in the TIFF format in the hard-disk memory of the image acquisition computer.
?. [3D image reconstruction and deconvolution using Volocity software]
Transfer raw data sets to the computer installed with Volocity software.
?. [3D image reconstruction and deconvolution using Volocity software]
Reconstruct 3D images from raw data sets using Volocity software.
?. [3D image reconstruction and deconvolution using Volocity software]
Select the option "Create new" from Actions menu in Volocity to make the Image sequence.
?. [3D image reconstruction and deconvolution using Volocity software]
Select the option "Remove Noise" from the Tools menu and remove noise from all 3D images using a fine filter.
?. [3D image reconstruction and deconvolution using Volocity software]
Select the option "Change Colors" from the Tools menu and change thecolors of 3 channels (e.g., GFP as green, mCherry as red, and iRFP as blue).
?. [3D image reconstruction and deconvolution using Volocity software]
Select the option "Properties" from the Edit menu and change information of XYZ pixel sizes in 3D images. Pixels of (X) and (Y) are 0.06 µm and pixel of (Z) is 0.1 µm when we use 100x objective lens and 4x amplification lens and scan Z-axis with optical slices 100 nm apart.
?. [Analysis and presentation of 3D images ]
Select manually regions of interest in deconvolved 3D images. Present the images as XYZ mode and outline the region of interest using the selection tool. Select the option "Crop to Selection" from the Actions menu to create a new deconvolved 3D image. Present 3D images shown in 3D opacity mode at any desired angles.
?. [Analysis and presentation of 3D images ]
Perform measurement of "ERES including cargo" by colocalization analysis with automatic thresholding of Volocity software.
?. [Yeast growth and culture]
The next day, pellet yeast cells by centrifugation at 3000 x g for 5 min, discard the supernatant and resuspend in 4 ml of YPG medium.
?. [Yeast growth and culture]
Pellet yeast cells again by centrifugation at 3000 x g for 5 min, discard the supernatant, resuspend in 3 ml of YPG and incubate at 24 °C for 1 h to induce galactose-regulated expression.
?. [Yeast growth and culture]
Pellet yeast cells by centrifugation at 3000 x g for 5 min, discard the supernatant, resuspend in 1 ml of SG medium and transfer to a 1.5 ml tube.
?. [Yeast growth and culture]
Pellet yeast cells again by centrifugation at 3000 x g for 5 min, resuspend in 1ml of SG medium and incubate at 37 °C for 30 min in a thermoshaker to accumulate galactose-induced protein cargos in the ER by inhibiting COPII-coated vesicle formation.
?. [Yeast sample preparation]
Apply 25 µl of 0.2 mg/ml Concanavalin A on the wells of the chamber slide for about 2 min.
?. [Yeast sample preparation]
Remove the excess Concanavalin A solution.
?. [Yeast sample preparation]
Let the Concanavalin A dry at room temperature for about 10 min.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Stop the rotation of the spinning-disk of the confocal scanner.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Apply a weak light from a halogen lamp light source to objective lens.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Obtain the halogen light images passed through the pinhole with 3 cameras.Note: images with practically the same intensities in 3 cameras can be acquired since the gain of amplification in 3 Image intensifiers are controlled independently.
?. [Live cell imaging by super-resolution confocal live imaging microscopy ]
Adjust the angles of dichroic millers and reflection millers of the custom-made spectroscopic unit to correct the XY positions of the images of the 3 cameras.
?. [3D image reconstruction and deconvolution using Volocity software]
Read out raw data sets in 3 channels into Image sequence to reconstruct 3D images at each time point
?. [3D image reconstruction and deconvolution using Volocity software]
Select the option “Correct Photobleaching” from the Tools menu and correct for photobleaching of 3D images to maintain intensities that are reduced by photobleaching during the observation time.