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Jul 2020

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Electron Tomography to Study the Three-dimensional Structure of the Reovirus Egress Pathway in Mammalian Cells
电子断层扫描研究哺乳动物细胞呼肠孤病毒出口途径的三维结构   

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Abstract

Mammalian orthoreoviruses (reoviruses) are nonenveloped, double-stranded RNA viruses that replicate and assemble in cytoplasmic membranous organelles called viral inclusions (VIs). To define the cellular compartments involved in nonlytic reovirus egress, we imaged viral egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs). Electron and confocal microscopy showed that reovirus mature virions are recruited from VIs to modified lysosomes termed sorting organelles (SOs). Later in infection, membranous carriers (MCs) emerge from SOs and transport new virions to the plasma membrane for nonlytic egress. Transmission electron microscopy (TEM) combined with electron tomography (ET) and three-dimensional (3D) reconstruction revealed that these compartments are connected and form the exit pathway. Connections are established by channels through which mature virions are transported from VIs to MCs. In the last step, MCs travel across the cytoplasm and fuse with the plasma membrane, which facilitates reovirus egress. This bio-protocol describes the combination of imaging approaches (TEM, ET, and 3D reconstruction) to analyze reovirus egress zones. The spatial information present in the 3D reconstructions, along with the higher resolution relative to 2D projections, allowed us to identify components of a new nonlytic viral egress pathway.

Keywords: Reovirus (呼肠孤病毒), Nonlytic egress (非解析出口 ), Lysosome (溶酶体), Electron tomography (电子断层扫描), Three-dimensional reconstruction (三维重建), Tomogram processing (断层图像处理), Image analysis (图像分析)

Background

Viruses recruit and transform cellular compartments to build membranous viral factories or inclusions, where genome replication and particle assembly take place (Miller and Krijnse-Locker, 2008; Sachse et al., 2019). Following genome packaging and assembly, progeny virions leave the host cell. Both enveloped and nonenveloped viruses can induce cell death by lysis, resulting in the release of viral particles. However, viral egress pathways can also be mediated by intracellular organelles that transport new virions to the plasma membrane, allowing them to exit cells without lysis (Roth et al., 2020). Therefore, many viruses use cellular membranous organelles for replication and assembly, as well as for egress and cell-to-cell transmission (Altan-Bonnet, 2017; Bird and Kirkegaard, 2015).


Reoviruses belong to the Reoviridae family, which includes several pathogens of plants, animals, and humans. Reoviruses are nonenveloped, double-stranded RNA viruses that assemble membranous cytoplasmic structures termed VIs. Early in infection, reovirus nonstructural proteins σNS and μNS induce the remodeling of ER membranes to form VIs (Tenorio et al., 2018). Viral genome replication, secondary rounds of transcription, and capsid assembly occur in VIs and, as a result, mature virions as well as empty capsids gather in this network of membranes. Reovirus uses different types of egress mechanisms depending on the cell type and culture conditions. In some types of cultured cells, reovirus infection leads to NF-κB activation, inducing apoptotic signaling and eventually lytic cell death (Danthi et al., 2013); however, reovirus can undergo nonlytic egress in other cell types such as HBMECs (Lai et al., 2013). In nonpolarized HBMECs, progeny virions exit from discrete areas at the basal surface. The detailed study of these regions is difficult because of their low frequency and location at the cell base. Due to these constraints, late steps of reovirus infection, including nonlytic egress, are not well understood.


The use of several imaging techniques in combination allowed us to discover that the reovirus egress pathway is composed of two different membranous elements called SOs and MCs. SOs are modified lysosomes that are recruited to the periphery of VIs during late phases of infection. Only mature genome-containing virions are collected in SOs, whereas empty capsids are absent. The smaller MCs are formed by budding from SOs and transport viral progeny to the plasma membrane (Fernandez de Castro et al., 2020). ET and 3D reconstruction were essential in defining the spatial relationships of the different compartments and elucidating the intracellular reovirus egress pathway (a summary of the protocol is shown in Figure 1).


ET can be used to visualize the structure of viruses, cells, and their constituents in three dimensions. The chosen approach defines the volume that can be analyzed as well as the possible resolution that can be obtained. In TEM-ET, micrographs of the sample are obtained from various orientations by tilting the specimen, and the resulting micrographs are computationally combined to form a 3D volume (Ercius et al., 2015, Hoppe 1974). Developments in hardware and software during recent years have allowed automatization of the image acquisition process. These innovations enable the production of tomograms, which allows appreciation of cellular organelle ultrastructure and inter-organelle connections in unprecedented detail in 3D. The 3D volumes are useful for studies to determine how cellular compartments interact since they display the spatial organization of structures within the volume, whereas a single 2D image provides only a projection of this information. Interestingly, 3D-ET data showed connections between VIs, SOs, and MCs, the three main components of the reovirus egress pathway. Furthermore, we were able to determine the size of the channels that connect the egress compartments (around 90-100 nm in diameter), which could not be established by analysis of ultra-thin sections (50-70 nm). We were also able to observe that viral egress from MCs to the extracellular medium is mediated by membrane fusion events at the plasma membrane. These events were visualized using double-tilt ET, which orients the specimen around two orthogonal axes, resulting in two independent tomograms computed from each tilt series. The two tomograms were aligned with each other and combined to obtain a single tomogram. As a result, a dual-axis tomogram shows better resolution at any orientation in the plane of the specimen than a single-axis series, in which the specimen is tilted about only one axis (Mastronarde 1997).


In this Bio-protocol, we describe the use of 3D-ET to study the reovirus nonlytic egress pathway and define the morphology and connections between the cellular organelles that compose the exit route. This methodology provides great potential to characterize many other cellular pathways in which different organelles are involved and connected as well as the complexity of pathogen-cell interactions.



Figure 1. Electron tomography of the reovirus egress pathway workflow. Sections of reovirus-infected cells were imaged by transmission electron microscopy to discern reovirus exit zones. The structures of interest were captured, and tilt series were obtained. After image processing and reconstruction, the 3D tomogram can be analyzed. Created with BioRender.com. © 2020 Fernández de Castro et al., originally published in J Cell Biol https://doi.org/10.1083/jcb.201910131.


Materials and Reagents

  1. Human brain microvascular endothelial cells (HBMECs) (Stins et al., 1997; Stins et al., 2001)

  2. Reovirus strain T1L M1-P208S was recovered from plasmids by reverse genetics as described previously (Kobayashi et al., 2007 and 2009). This strain was engineered by the Terence Dermody Lab.

  3. Roswell Park Memorial Institute (RPMI)-1640 Medium (Merck, catalog number: R8758)

  4. Trypsin-ethylenediaminetetraacetic acid (EDTA) solution 10× (Merck, catalog number: 9002-07-7)

  5. Fetal bovine serum (FBS) (BI Biological Industries, catalog number: 04-007-1A)

  6. Corning® Nu-SerumTM IV Growth Medium Supplement (Corning®, Life Sciences, catalog number: 55004)

  7. Minimum Essential Medium (MEM) vitamin solution 100× (ThermoFisher Scientific, GibcoTM, catalog number: 11120052)

  8. Sodium pyruvate solution (Merck, Sigma-Aldrich, catalog number: 113-24-6)

  9. MEM non-essential amino acid Solution 100× (Merck, catalog number: M7145)

  10. L-glutamine (Merck, catalog number: 56-85-9)

  11. Penicillin-streptomycin (Merck, Sigma-Aldrich, catalog number: P4333)

  12. Amphotericin B solution (Merck, Sigma-Aldrich, catalog number: 1397-89-3)

  13. 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid) HEPES (Merck, Sigma-Aldrich, catalog number: 7365-45-9)

  14. Distilled 50% glutaraldehyde (TAAB Laboratories, catalog number: G015)

  15. Sapphire disks Ø1.4 mm (Martin Wohlwend, catalog number: 454)

  16. NuncTM cell culture-treated multidishes (ThermoFisher Scientific, catalog number: 140675)

  17. Bovine serum albumin (BSA) (Merck, Sigma-Aldrich, catalog number: 9048-46-8)

  18. Phosphate-buffered saline (PBS) (Merck, Sigma-Aldrich, catalog number: P3813)

  19. Ultrapure Milli-Q water

  20. Dumont Tweezers type 4 (TAAB Laboratories, catalog number: T052)

  21. Dumont Tweezers type 7 (TAAB Laboratories, catalog number: T050)

  22. Osmium Tetroxide Electron Microscopy (EM) solution (TAAB Laboratories, catalog number: O015)

  23. Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400)

  24. Methanol dried (Merck, catalog number: 67-56-1)

  25. Acetone dried (Merck, catalog number: 1-00299-0500)

  26. 812 resin (TAAB Laboratories, catalog number: T026)

  27. Dodecenyl succinic anhydride (DDSA) (TAAB Laboratories, catalog number: D027)

  28. Methyl nadic anhydride (MNA) (TAAB Laboratories, catalog number: MG12)

  29. Benzyldimethylamine (BDMA) (TAAB Laboratories, catalog number: B022)

  30. BEEM® embedding capsule 00 (TED PELLA, INC., catalog number: 130)

  31. GEM® stainless-steel blade (TED PELLA, INC., catalog number: 62-0179)

  32. Perfect loop (Electron Microscopy Sciences, catalog number: 70945)

  33. Ultra-diamond knife 45° dry (DIATOME, catalog number: DU4530)

  34. QUANTIFOIL® R 3.5/1 Holey Carbon Film Grids Cu 300 mesh (QUANTIFOIL®, catalog number: Q11394)

  35. Protein A Gold 10 nm (PAG10). The source is the Cell Microscopy Core (Department of Cell Biology, University Medical Center Utrecht)

  36. Ethanol absolute (100% ethanol) (Merck, catalog number: 64-17-5)

  37. Liquid nitrogen

Equipment

  1. Water purification system (Merck, Milli-Q®, model: Advantage A10)

  2. pH meter Basic 20 (HACH LANGE SPAIN, Crison, model: Basic 20)

  3. Carbon coating system (Leica Microsystems, model: Leica EM MED020)

  4. Cell incubator (BINDER, model: CB 170)

  5. High-pressure vitrification system (Leica Microsystems, model: Leica EM PACT2)

  6. Automatic cryosubstitution system (Leica Microsystems, model: Leica EM AFS2)

  7. Fume hood (Flow-Tronic)

  8. Ultramicrotome (Leica Microsystems, model: Leica EM UC6)

  9. Field Electron and Ion Company (FEI) Tecnai electron microscope (FEI, model: G2 F20 (200 kV))

  10. Charge-couple device (CCD) camera (FEI, model: Eagle 4k×4k)

Software

  1. Tomography 3 (https://www.thermofisher.com/) (EM software, ThermoFisher Scientific). This software was used for Tecnai imaging and tilt series acquisitions.

  2. IMOD (Kremer et al., 1996) (https://bio3d.colorado.edu/imod/) was used for the alignment of the raw tilt series and tomogram generation.

  3. Amira (https://www.thermofisher.com/) (2D-5D visualization and analysis software, ThermoFisher Scientific). Amira was used for tomogram segmentation, reconstruction, and visualization.

Procedure

Note: This protocol involves the manipulation of dangerous compounds, namely osmium tetroxide, glutaraldehyde, epoxy resin, and uranyl acetate. Osmium tetroxide, glutaraldehyde, and epoxy resin are a contact and inhalation hazard. Prepare and use these toxic compounds in a chemical fume hood and wear gloves. Uranyl acetate is a uranium salt that does not represent a significant external radiation hazard, as the alpha particles do not penetrate the external layer of skin. There are beta and gamma emitters, but the betas also do not have enough energy to penetrate the skin, and the amount of gamma radiation is minimal. Despite this, uranyl acetate has associated chemical and radiological hazards, and some basic safety precautions must be adopted, with emphasis on avoiding the possibility of inhalation or ingestion of the material. Uranyl acetate has to be stored and disposed of as a low-level radioactive compound. Please read the official guideline of each compound carefully before use and follow the local safety regulations.


  1. Culturing cells on sapphire disks and viral infection

    1. Before plating cells, the 50-µm-thick sapphire disks (1.4 mm in diameter) must be carefully cleaned with 100% ethanol and covered with a carbon layer of 4-7-µm thickness using a coating device. Most cell lines propagate most efficiently using this substrate. To know on which side of the disk the cells are, it is useful to scratch an asymmetric letter or a number in the carbon.

    2. The carbon coating must be stabilized to avoid detachment from the disks. To stabilize before use, place the carbon-coated disks in a glass dish and bake overnight at 120°C. If the disks will not be used right away, they can be stored in a humid atmosphere in a refrigerator at 4°C for 2-4 weeks. After storage, the disks should be glow-discharged before use as a cell substrate. Finally, the disks should be sterilized prior to culturing cells (e.g., using ultraviolet radiation).

      Note: The glow-discharge treatment of TEM grids makes the carbon film surface negatively charged, which allows aqueous solutions to spread easily and facilitates attachment of cells and particles.

    3. Place the sapphire disks in plastic dishes. HBMECs (5 × 105 cells per well) are added and maintained in 2 ml complete RPMI-1640 medium (see Recipes) supplemented with 10% heat-inactivated fetal bovine serum (FBS).

    4. Cells are incubated at 37°C and 5% CO2 for 24 h and adsorbed with reovirus strain T1LM1 P208S at a multiplicity of infection (MOI) of 1 plaque-forming unit (PFU)/cell (stock diluted in culture medium) at 37°C for 1 h.

      Note: The maintenance medium must be aspirated prior to reovirus absorption.

    5. After the inoculum is removed, fresh RPMI medium supplemented with 2% FBS is added, and cells are incubated for 18 h post-infection (hpi).

    6. For biosafety reasons, HBMECs are fixed with 1 ml 1% glutaraldehyde in HEPES buffer (see Recipes) at room temperature (RT) for 1 h.

      Note: Prior to fixation, the culture medium must be aspirated.


      Video 1. Coating of the sapphire disks using the Leica EM MED020

  2. High pressure freezing (HPF)Note: HPF was accomplished using the Leica (EM PACT2) HPF instrument. For other HPF instruments, different sized sapphire discs may be required, and the loading protocol may differ.

    1. Remove the disks from the culture dish and dip them into 20% BSA in PBS.

    2. Place sapphire disks with cells facing into the cavity of the flat specimen holders filled with 20% BSA (see Figure 2A, Verkade, 2008). The BSA functions as a cryoprotectant. Other cryoprotectants can be used, including FBS and normal goat serum [NGS]. Ensure that there are no air bubbles in the liquid present in the sample holder.

    3. The flat specimen holders are placed in a sample holder pod and tightly sealed by pressing them against a surface made of black diamond (see Figure 2).

    4. Load the sample holder in the Leica EM PACT2 with the loading stage (see Figure 2).

    5. To freeze the sample, on the touch screen press “start,” and the system starts the high pressure freezing. After freezing is complete, the pod loaded with the sample falls automatically into a liquid nitrogen bath.

    6. Check the cooling rate and the pressure rise on the screen.

    7. Place the pod holder in the socket bath of the liquid nitrogen and unscrew the pod holder, leaving frozen samples in the bottom of the bath.

    8. Following freezing, the flat specimen holders are transferred under liquid nitrogen from the EM PACT2 instrument to a Leica AFS2 freeze substitution system pre-cooled to -90°C.



      Figure 2. Loading of sapphire disks for high pressure freezing. (A) Diagram showing the sandwich of the sapphire disk with the flat sample holder for HPF (Step B2). Prepared with BioRender.com. (B) A torque screwdriver was used to tighten the flat sample holder containing the sapphire disk in the pod (Step B3). (C) A pod holder was used to pick up and introduce the pod with the sample to the Leica EM PACT2 (Step B3). (D) The pod holder was charged in the loading stage of the Leica EM PACT2 (Step B4).


  3. Freeze substitution and embedding in epoxy resin

    Note: Always maintain the frozen samples in liquid nitrogen before loading into the freeze substitution liquid. All instruments (e.g., forceps to transfer sample to the tube containing the substitution liquid) and solutions should be precooled before use.

    1. Samples are freeze-substituted at -90°C for 24 h in a mixture of 1% osmium tetroxide, 0.1% uranyl acetate, 5% water, and 2% methanol in dry acetone.

    2. The temperature is increased to -25°C at a rate of 2°C/h, and finally to 0°C in increments of 5°C/h.

    3. Following completion of the -90°C to 0°C transition, the machine can be held at 0°C for 1-2 h. This procedure increases contrast of the sample. If the duration is longer than 2 h, membranes will not be as well-preserved.

    4. All of the following steps are conducted at RT. Wash samples three times for 20 min with 1 ml dried-pure acetone.

    5. The samples were gradually infiltrated with mixtures of acetone-epoxy resin (see Recipes), increasing the concentration of resin in steps of 30%, 50%, 70%, and 90% for 1 h each.

    6. For infiltration in pure epoxy resin, the sapphire disks are separated from the flat sample holders using tweezers and placed into flat-bottomed beam capsules.

    7. Capsules are filled with freshly prepared epoxy resin for polymerization at 60°C for 2 days.


  4. Ultramicrotomy

    1. Beam capsules are removed using a stainless-steel blade, and the sapphire disks are separated from the resin blocks with thermal shocks. The samples are loaded into liquid nitrogen for a few seconds and then returned to room temperature. Repeat these two steps until the sapphire disks are detached from the resin blocks.

    2. Samples are cut into small pieces (~1 × 1 mm) using a stainless-steel blade and affixed to a resin block as a holder (see Figure 3).

    3. The sample surface is hand-trimmed to obtain a trapezoidal shape using a new stainless-steel blade.

    4. Collect semi-thick sections (~ 350-400 nm) using a ultramicrotome UC6 with a perfect loop and place the sections in Quantifoil® cooper grids.

      Note: Other types of grid can be used for ET.



      Figure 3. Mounting the samples for ultramicrotomy. (A) After detachment of the sapphire disc, the resin with embedded cell monolayers is cut into small pieces of about 1-mm in size. (B) The small pieces of sample are glued onto cylindrical resin blocks, which allows mounting of the sample in the sample holder of the microtome. Scale bar = 10 mm.


  5. Coating grids with fiducial markers (protein A gold spheres [10 nm diameter], PAG10) on both sides of the section for better alignment and staining with uranyl acetate. The protocol is summarized in Figure 4.

    Note: The fiducial markers are gold particles of 10 nm diameter required for the alignment of the tilt images during tomogram processing prior to image reconstruction.

    1. On a parafilm sheet, place one drop per sample of the protein A gold solution (see Recipes) and three drops per sample of water.

    2. Incubate the grids on the gold drops for 10 min.

    3. Sequentially lay the grids for a few seconds on the three water drops to remove excess gold solution.

    4. Absorb the excess liquid with absorbent filter paper.

    5. Repeat Steps E1-E4, incubating the other side of the grid.

    6. Let the grids dry for at least 2 h.

      Note: To increase the contrast of the images, stain the sections with uranyl acetate.

    7. On a parafilm sheet, place one drop of 2% uranyl acetate (see Recipes) and incubate the grids for 20 min.

    8. Wash the grids with three drops of water for a few seconds to remove the uranyl acetate.

    9. Absorb the excess liquid with filter paper.

    10. Allow the grids to dry for at least 2 h prior to imaging with an electron microscope.



      Figure 4. Schematic of grid preparation prior to electron tomography. Created with BioRender.com.


  6. Electron tomography

    1. Examine the sections using an electron microscope for reovirus egress zones, selecting and saving the regions of interest (ROIs) for the tilt series.

    2. Pre-irradiate the ROIs prior to collection of the tilt series. The electron beam produces damage in resin-embedded samples, resulting in specimen shrinkage, especially along the Z-axis (Luther et al., 1988). To avoid this problem during tomogram acquisition, it is important to pre-irradiate the ROIs, including a larger area than the ROI since outside areas could later enter in the field.

      Notes:

      1. About 2,000 electrons per square Angstrom at a low magnification (2,000×) of EM-irradiation is enough to allow the ROIs to assume sufficient size and stability.

      2. The ROIs should not be close to the grid bars, as the grid bars will block acquisition at high tilt angles.

    3. Single- and dual-axis ET is conducted using a Tecnai G2 F20 transmission electron microscope (FEI) operated at 200 kV.

    4. The specimen is tilted within the electron microscope to produce transmission electron microscopy images from many different angles, resulting in a tilt series.

    5. Acquire tilt series automatically at 1° increments over an angule range of −60° to 60°. The camera parameters should be set to allow optimum resolution and contrast for the sample. In our studies, we acquired the tilt series with a nominal magnification of ×11,500, resulting in a pixel size of 1.01 nm.

    6. Images are recorded using an Eagle 4k×4k slow-scan charge-coupled device (FEI) and Tomography 3 software.

    7. To conduct dual-axis ET, collect a second tilt series after rotation of the specimen by 90° around an axis perpendicular to its support plane.

      Note: Dual-axis or double-tilt ET increases the information collected and reduces artifacts of acquisition and reconstruction (Mastronarde, 1997).


  7. Tomogram processing

    Note: The different perspectives obtained by ET are used to reconstruct a 3D view, which resolves the ultrastructure of the reovirus egress organelles in three dimensions.

    1. Download and install the IMOD software to align tilt series and obtain tomographic reconstructions.

    2. Start eTomo, providing the entries in the Setup Panel. The important parameters regarding the tilt series are:

      1. Axis type: single or dual axis

      2. Frame type

      3. Pixel size and fiducial diameter

      4. Tilt angles

      Note: In this Setup Panel, it is possible to remove excluded views (images that are suboptimal for analysis).

    3. Once the Setup Panel is complete, activate Create Com Scripts.

    4. Preprocessing. Run Ccderaser to erase X-rays and other artifacts from the images. The X-ray model creates a fixed stack that can be saved and used.

    5. Coarse Alignment. Calculate the cross-correlation with Tiltxcorr to determine the X and Y translations required to align each image with the previous one, generating the coarse aligned stack.

    6. Fiducial Model Generation. There are several ways to generate a fiducial model. We selected well-distributed fiducials or gold beads on the zero-degree view and used Beadtrack to find their locations in the rest of the tilt series. Finally, a Seed Model was created. The seed points can be marked manually or automatically. An overview of the alignment process is shown in Figure 5.

    7. Fine Alignment. Perform the alignment using Tiltalign and examine the results in the log file after the process is complete. It is possible to view a model of the Residual Vectors, which gives an idea of the alignment quality (see Figure 5). The smaller the residual vectors, the better the alignment. A typical mean residual value of a good alignment is around 1 pixel.

    8. Tomogram Positioning. Prepare a simple model with horizontal lines across the top and bottom faces of the section at three locations in Y. It is important to delineate the thickness of the sections. After defining the edge of the section, select Find boundary model automatically, creating the Boundary Model (see Figure 5).

    9. Final Aligned Stack. Use Newstack to obtain the aligned stack. As an option, it is possible in this step to correct the microscope CTF (contrast transfer function) and filter the aligned stack.

    10. Tomogram Generation. Use Tilt to generate the tomogram.Note: In double-tilt tomography, both tomograms, called A and B, are processed separately. Tomogram A is first aligned, indicating that it is dual axis. Tomogram B is aligned with reference to tomogram A using its fiducials (matching the tomogram B to A). As a result, a combined tomogram is prepared.



      Figure 5. Screen captures of the IMOD software. Representative models constructed using tomogram alignment are shown.


  8. Tomogram segmentation and 3D visualization

    1. The tomogram segmentation and 3D visualization were conducted with the Amira Software.

    2. Load the tomogram (.rec) in the Amira interface and apply a Gaussian filter (3D type) for noise reduction.

    3. An Orthoslice can be made to visualize and compare the tomogram with the filtered tomogram.

    4. The filtered tomogram was segmented with the function LabelField, which allows the area of the different structures to be marked in the images. Labeling a structure in all images of the tomogram is tedious work, which can be simplified with the Interpolate function. The Interpolate function allows the segmentation to be done automatically after marking the images.

      Note: It is important to verify that the Interpolate function is working well, as occasionally manual corrections of the automatic segmentation must be made to some of the labeled images or structures.

    5. The functions Arithmetic and Isosurface were used to visualize the structures in 3D.

      Note: The threshold values of labeled structures are obtained using histograms for each structure. Adjusting the threshold value using Isosurface improves visualization of the reconstructions.

Data analysis

We used 3D-ET to analyze the reovirus egress pathway. The tomogram shown in Figure 6 was obtained using a semi-thick section (350 nm), which allowed us to identify the spatial relationship between the different structures. The analysis of the VI-SO-MC interfaces by ET revealed the presence of physical connections between these structures (Figure 6). The computational slices of the tomogram in combination with the 3D visualization revealed that there are points of membrane continuity between VIs and SOs (dashed box in Figure 6A) and SOs and MCs (dashed box in Figure 6B), respectively. These interactions are restricted to a few computational slices, suggesting that the association between these compartments occurs through discrete channels observed in the higher-magnification images shown in Figure 6A and B.



Figure 6. Electron tomography of the reovirus egress machinery. (A and B) HBMECs were adsorbed with reovirus T1L M1-P208S at an MOI of 1 PFU/cell and processed at 18 hpi by high-pressure freezing, freeze substitution, and semi-thick sectioning. Single-tilt tomography and 3D reconstruction show the connection between the components of the reovirus egress machinery (dashed boxes). Higher magnifications of the specific membrane fusion points between a sorting organelle (SO) and a viral inclusion (VI) in (A) or a membranous carrier (MC) adherent to an SO through a channel in (B). mi, mitochondrion. Scale bars = 500 nm A and B; 100 nm in the higher magnifications. © 2020 Fernández de Castro et al. originally published in J Cell Biol https://doi.org/10.1083/jcb.201910131.


We used double-tilt ET to study the final steps of the reovirus egress pathway (Figure 7). Examination of the MCs at the plasma membrane by ET and 3D reconstruction revealed details of the carrier morphology before egress and the virion exit mechanism. Mature virions are distributed along arrays of membranes or channels inside the MCs (Figure 7A), which may increase the carrying capacity of the MC or the efficiency of viral transport. The 3D volume depicts the total content of the MC and its association with the plasma membrane (Figures 7B and C). Therefore, progeny reovirus particles exit infected cells through distinct egress zones after virion-filled MCs contact the plasma membrane (Fernandez de Castro et al., 2020).



Figure 7. Electron tomography of a membranous carrier. HBMECs were adsorbed with reovirus T1L M1-P208S at an MOI of 1 PFU/cell and processed at 18 hpi by high-pressure freezing, freeze substitution, and semi-thick sectioning. (A) Computational tomography slice showing a membranous carrier (MC) with membranous channels (white arrowheads) and virions (white arrows) close to the plasma membrane (PM). (B) A 3D reconstruction of the MC overlapping with a 2D computational slice. (C) One view of the 3D volume showing the total content of the MC after segmentation. Mature virions, blue; channels, beige; membranous carrier, light brown; plasma membrane, green. Scale bars = 200 nm. © 2020 Fernández de Castro et al. originally published in J Cell Biol https://doi.org/10.1083/jcb.201910131.


The ET experiments were conducted with at least two biological replicates. Eight tomograms showing different reovirus egress zones were recorded, and the VI-SO-MC interface was analyzed from nine tomograms. Statistical significance was determined using a two-sample unequal variance t-test with two-tailed distribution.

Notes

  1. The reovirus strain T1L M1-P208S is identical to the prototype T1L strain except for a proline-to-serine substitution at position 208 of the μ2 protein (encoded by the M1 gene). This mutation changes inclusion morphology from filamentous to globular (Parker et al., 2002).

  2. In the tilt series alignment, the most critical part is the distribution of the fiducial markers in the sections. Only those fiducials maintained in every section should be marked at the same Z-plane, and they should be well-distributed throughout the sections.

Recipes

  1. Complete RPMI-1640 medium

    10% Nu serum

    1% MEM vitamins

    1% sodium pyruvate

    1% MEM nonessential amino acids

    1% L-glutamine

    1% penicillin/streptomycin

    0.1% amphotericin

    Store at 4°C

  2. HEPES 0.4 M

    9.54 g HEPES

    Add Milli-Q water to 50 ml and mix well until HEPES is dissolved entirely

    Adjust pH with 1N NaOH solution to 7.2-7.4

    Add Milli-Q water to 100 ml

    Store at 4°C

  3. Epoxy resin

    Use plastic Pasteur pipettes to weigh the epoxy resin components.

    Mix the resin components by inverting the tube several times.

    The accelerator (BDMA) has to be added at the moment of use.

    Centrifugate the mixture at 14,100 × g for 10 min to eliminate small bubbles.

    19.2 g 812 resin

    7.6 g DDSA

    13.2 g NMA

    0.8 g (or 20 drops) BDMA

  4. Protein A Gold or fiducial marker solution

    Mix colloidal gold solution in water (1:50 ratio)

    Prepare this solution fresh every time

    Note: The dilution (and size) of the fiducial marker depends on the magnification of the tilt series acquisition. The higher the magnification, the denser the fiducials and the smaller the gold size.

Acknowledgments

This work was supported in part by Public Health Service awards AI032539 (T.S.D. and C.R.). Special thanks to Dr. Martin Sachse for expert technical advice and support throughout this work and for critical review of the manuscript, Dr. Francisco Javier Chichón from the CNB-CIB (CSIC) cryoEM facility for technical advice with ET, and Cristina Patiño, Beatriz Martín, and Pablo Solá from the CNB (CSIC) EM facility. This protocol was adapted from previous work: Fernández de Castro et al., originally published in J Cell Biol https://doi.org/10.1083/jcb.201910131.

References

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简介

[摘要]哺乳动物正呼肠病毒( reoviruses ) 是无包膜的双链 RNA 病毒,可在称为病毒包涵体 (VI) 的细胞质膜细胞器中复制和组装。为了定义参与非溶解呼肠孤病毒出口的细胞区室,我们对受感染的非极化人脑微血管内皮细胞 (HBMEC) 中的病毒出口进行了成像。电子显微镜和共聚焦显微镜显示呼肠孤病毒成熟病毒粒子从 VI 招募到修饰的溶酶体,称为分选细胞器 (SO)。在感染后期,膜载体 (MCs) 从 SO 中出现并将新病毒粒子运输到质膜以进行非裂解性出口。透射电子显微镜 (TEM) 结合电子断层扫描 (ET) 和三维 (3D) 重建显示,这些隔室相互连接并形成出口通路。连接是通过通道建立的,成熟的病毒粒子通过这些通道从 VI 传输到 MC。在最后一步,MCs 穿过细胞质并与质膜融合,从而促进呼肠孤病毒的排出。该生物协议描述了成像方法(TEM、ET 和 3D 重建)的组合来分析呼肠孤病毒出口区。Ť他存在于3D重建的空间信息,以相对于2D投影较高分辨率沿,使我们能够确定一个新的nonlytic病毒流出途径的组分。

[背景]病毒招募和转化细胞区室以构建膜状病毒工厂或内含物,在那里进行基因组复制和粒子组装(Miller和Krijnse-Locker ,2008 年;Sachse等人,2019 年)。在基因组包装和组装之后,子代病毒粒子离开宿主细胞。有包膜和无包膜病毒都可以通过裂解诱导细胞死亡,导致病毒颗粒的释放。然而,病毒流出途径也可以由细胞内细胞器介导,这些细胞器将新病毒粒子运输到质膜,使它们能够在不裂解的情况下离开细胞(Roth等,2020)。因此,许多病毒使用细胞膜细胞器进行复制和组装以及传播和细胞间传播(Altan-Bonnet ,2017 年;Bird和Kirkegaard ,2015 年)。

呼肠孤病毒属于呼肠孤病毒科,包括多种植物、动物和人类病原体。呼肠孤病毒是组装膜质细胞质结构的无包膜双链 RNA 病毒,称为VI。在感染早期,呼肠孤病毒非结构蛋白σ NS 和μ NS 诱导 ER 膜重塑以形成 VI (Tenorio等,2018)。病毒基因组复制、第二轮转录和衣壳组装发生在 VI 中,因此,成熟的病毒粒子和空衣壳聚集在这个膜网络中。呼肠孤病毒根据细胞类型和培养条件使用不同类型的出口机制。在某些类型的培养细胞中,呼肠孤病毒感染会导致 NF- κB激活,诱导凋亡信号传导并最终导致细胞裂解(Danthi等,2013);ħ H但是,呼肠孤病毒可以在其它细胞类型,例如HBMECs经历nonlytic出口(赖等人,2013年)。在非极化 HBMECs 中,子代病毒粒子从基底表面的离散区域退出。这些区域的详细研究是困难的,因为它们的频率低且位于细胞基部。由于这些限制,呼肠孤病毒感染的晚期步骤,包括非裂解性出口,还不是很清楚。

结合使用几种成像技术使我们能够发现呼肠孤病毒出口途径由两种不同的膜元件组成,称为 SOs 和 MCs。SOs 是经过修饰的溶酶体,在感染后期被募集到 VIs 的外围。在 SO 中仅收集包含成熟基因组的病毒粒子,而没有空衣壳。较小的 MC 是通过从 SO 出芽并将病毒后代运输到质膜而形成的(Fernandez de Castro等,2020)。ET及三维重建是必不可少在defin荷兰国际集团的不同隔室的空间关系和elucidat荷兰国际集团的呼肠孤病毒的细胞内出口通路(该协议的概要示于图1)。

ET 可用于在三个维度上可视化病毒、细胞及其成分的结构。所选择的方法定义了可以分析的体积以及可以获得的可能分辨率。在 TEM-ET 中,通过倾斜样品从不同方向获得样品的显微照片,并且通过计算将所得显微照片组合以形成 3D 体积(Ercius等人,2015 年,Hoppe 1974)。近年来硬件和软件的发展已经实现了图像采集过程的自动化。这些创新能够生成断层图像,从而以前所未有的 3D 细节欣赏细胞器超微结构和细胞器间连接。3D 体积对于确定细胞隔室如何相互作用的研究很有用,因为它们显示了体积内结构的空间组织,而单个 2D 图像仅提供此信息的投影。有趣的是,3D-ET 数据显示了呼肠孤病毒出口途径的三个主要组成部分 VI、SO 和 MC 之间的联系。此外,我们能够确定连接出口室的通道的大小(约90 - 100纳米中的直径),其不能通过超薄切片的分析来确定(50 - 70纳米)。我们还能够观察到病毒从 MCs 流出到细胞外介质是由质膜上的膜融合事件介导的。这些事件使用双倾斜 ET 进行可视化,它围绕两个正交轴定位标本,从而从每个倾斜系列计算出两个独立的断层图像。两个断层图像相互对齐并组合以获得单个断层图像。因此,与单轴系列相比,双轴断层图像在样品平面中的任何方向显示出更好的分辨率,其中样品仅围绕一个轴倾斜(Mastronarde 1997)。

在此生物协议中,我们描述了使用 3D-ET 研究呼肠孤病毒非裂解性出口途径并定义构成出口路线的细胞器之间的形态和连接。这种方法为表征许多其他细胞通路提供了巨大的潜力,其中不同的细胞器参与和连接,以及病原体-细胞相互作用的复杂性。





的图1的电子断层扫描的呼肠孤病毒的出口通路的工作流程。呼肠孤病毒感染细胞的切片通过透射电子显微镜成像以辨别呼肠孤病毒出口区。捕获感兴趣的结构,并获得倾斜系列。在图像处理和重建之后,可以分析 3D 断层图像。使用 BioRender.com 创建。© 20 20 Fernández de Castro等。,最初发表于J Cell Biol https://doi.org/10.1083/jcb.201910131。

关键字:呼肠孤病毒, 非解析出口 , 溶酶体, 电子断层扫描, 三维重建, 断层图像处理, 图像分析


材料和试剂

人脑微血管内皮细胞 (HBMEC) (Stins等人,1997 年;Stins等人,2001 年)
呼肠孤病毒株T1L M1-P208S通过如前所述的反向遗传学从质粒中回收(Kobayashi等人,2007和2009) 。该菌株由 Terence Dermody 实验室设计。
罗斯威尔公园纪念研究所(RPMI)-1640 Medium(默克,目录号:R8758)
胰蛋白酶-乙二胺四乙酸(EDTA)š olution 10 × (默克,目录号:9002-07-7)
胎牛血清(FBS)(BI Biological Industries,目录号:04-007-1A)
康宁® NU-血清TM IV生长培养基补充物(康宁® ,生命科学,目录号:55004)
极限必需培养基(MEM)v itamin小号olution 100 × (赛默飞世科学,Gibco公司TM ,目录号:11120052)
丙酮酸钠溶液(默克,Sigma-Aldrich,目录号:113-24-6)
MEM n on - essential a mino a cid Solution 100×(默克,目录号:M7145)
L-克lutamine(默克,目录号:56-85-9)
青霉素小号treptomycin(默克,Sigma-Aldrich公司,目录号:P4333)
两性霉素 B 溶液(Merck,Sigma-Aldrich,目录号:1397-89-3)
4-(2- ħ ydroxyethyl)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)HEPES(默克,Sigma-Aldrich公司,目录号:7365-45-9)
蒸馏的 50% 戊二醛(TAAB Laboratories,目录号:G015)
蓝宝石盘 Ø1.4 毫米(Martin Wohlwend,目录号:454)
NUNC TM Ç ELL Ç ulture-吨reated米ultidishes (赛默飞世科学,目录号:140675)
牛血清白蛋白(BSA)(默克,Sigma-Aldrich,目录号:9048-46-8)
磷酸盐缓冲盐水(PBS)(默克,Sigma-Aldrich,目录号:P3813)
超纯军用升的i-Q水
Dumont Tweezers type 4(TAAB Laboratories,目录号:T052)
Dumont Tweezers type 7(TAAB Laboratories,目录号:T050)
四氧化锇电子显微术(EM)小号olution(TAAB实验室,目录号:O015)
醋酸铀(Electron Microscopy Sciences,目录号:22400)
干燥的甲醇(默克,目录号:67-56-1)
丙酮干燥(默克,目录号:1-00299-0500)
812树脂(TAAB Laboratories,目录号:T026)
十二烯基琥珀酸酐(DDSA)(TAAB Laboratories,目录号:D027)
甲基降冰片烯二酸酐(MNA)(TAAB实验室,目录号:MG12)
苄基二甲胺(BDMA)(TAAB实验室,目录号:B022)
BEEM ® Ë mbedding Ç apsule 00(TED PELLA,INC,目录号:130)
GEM ®小号tainless -s TEEL b拉德(TED PELLA,INC,目录号:62-0179)
完美环(电子显微镜科学,目录号:70945)
超金刚石刀 45°干燥(DIATOME,目录号:DU4530)
QUANTIFOIL ® R 3.5/1 多孔碳膜网格 Cu 300 网(QUANTIFOIL ® ,目录号:Q11394)
蛋白 A 金 10 nm (PAG 10 )。来源是细胞显微镜核心(乌得勒支大学医学中心细胞生物学系)
无水乙醇(100% 乙醇)(默克,目录号:64-17-5)
液氮

设备

净水系统(Merck, Milli-Q ® ,型号:Advantage A10)
pH计Basic 20(HACH LANGE SPAIN,Crison ,型号:Basic 20)
碳涂层系统(Leica Microsystems,型号:Leica EM MED020)
细胞培养箱(BINDER,型号:CB 170)
高压玻璃化系统(Leica Micro systems,型号:Leica EM PACT2)
自动低温置换系统(Leica Microsystems,型号:Leica EM AFS2)
通风柜(Flow-Tronic)
超薄切片机(Leica Microsystems,型号:Leica EM UC6)
Field Electron and Ion Company (FEI) Tecnai电子显微镜(FEI,型号:G2 F20 (200 kV))
电荷耦合器件(CCD)相机(FEI,型号:Eagle 4k×4k)

软件

断层扫描 3 ( https://www.thermofisher.com/ )(EM 软件,ThermoFisher Scientific)。该软件用于Tecnai成像和倾斜系列采集。
IMOD (KREM ER等人,19 96) (https://bio3d.colorado.edu/imod/)被用于对准的原始倾斜系列和断层图像生成的。
Amira ( https://www.thermofisher.com/ )(2D-5D 可视化和分析软件,ThermoFisher Scientific)。阿米拉用于断层图像分割,重建,和可视化。

程序

注意:该协议涉及危险的化合物,即四氧化锇,戊二醛,环氧树脂的操作,和双氧铀乙酸盐。四氧化锇,戊二醛,和环氧树脂是一个接触和吸入的危险。在化学通风橱中准备和使用这些有毒化合物并戴上手套。醋酸铀是一种铀盐,不会造成显着的外部辐射危害,因为 α 粒子不会穿透皮肤的外层。有 β 和 γ 发射器,但 β 也没有足够的能量穿透皮肤,而且 γ 辐射的量很小。尽管如此,醋酸双氧铀具有相关的化学和放射危害,必须采取一些基本的安全预防措施,重点是避免吸入或摄入该材料的可能性。乙酸双氧铀必须被存储和设置的作为低放射性化合物。请仔细阅读每种化合物的官方指南仔细在使用前,并按照当地安全法规。

      文化性荷兰国际集团在蓝宝石上的磁盘和病毒感染的细胞
电镀细胞之前,50微米厚的蓝宝石圆盘(1.4毫米直径)必须被小心地用100%乙醇清洗并覆盖着的4碳层- 7-微米使用涂布装置的厚度。大多数细胞系使用这种底物最有效地繁殖。要知道细胞在磁盘的哪一侧,在碳上划一个不对称的字母或数字很有用。
碳涂层必须稳定以避免与磁盘分离。为了在使用前保持稳定,请将碳涂层磁盘放入玻璃盘中,并在 120°C 下烘烤过夜。如果不立即使用磁盘,可以将它们存放在 4°C 的潮湿环境中的冰箱中 2 - 4 周。贮存后,应将这些磁盘辉光放电之前我们Ë作为细胞底物。最后,应该在培养细胞之前对圆盘进行消毒(例如,使用紫外线辐射)。
注意:TEM 网格的辉光放电处理使碳膜表面带负电,使水溶液易于扩散并促进细胞和颗粒的附着。
将蓝宝石盘放在塑料盘中。HBMEC (5× 10 5 个细胞/孔)加入并维持在补充有10% 热灭活胎牛血清 (FBS) 的2 ml 完全 RPMI-1640 培养基(参见配方)中。
细胞在 37°C 和 5% CO 2下孵育24 小时,并以 1 个噬菌斑形成单位 (PFU)/细胞(在培养基中稀释的原液)的感染复数 (MOI) 吸附于呼肠孤病毒株 T1LM1 P208S ℃ 1 小时。
注意:维持培养基必须在呼肠孤病毒吸收之前吸出。
除去接种物后,加入补充有2% FBS 的新鲜 RPMI 培养基,并将细胞在感染后孵育 18 小时 ( hpi )。
出于生物安全原因,HBMEC 在室温 (RT) 下用 1 ml 1% 戊二醛的 HEPES 缓冲液(参见配方)固定 1 小时。
注意:在固定之前,必须吸出培养基。


视频 1. 使用 Leica EM MED020 对蓝宝石磁盘进行涂层

高压冷冻 (HPF)
注意:HPF 是使用 Leica (EM PACT2) HPF 仪器完成的。对于其他 HPF 仪器,可能需要不同尺寸的蓝宝石光盘,并且加载协议可能不同。
从培养皿中取出磁盘,并将它们浸入 PBS 中的 20% BSA。
将带有细胞的蓝宝石圆盘放入填充有 20% BSA 的扁平样品架的空腔中(参见图 2A,Verkade ,2008 年)。BSA 起到冷冻保护剂的作用。可以使用其他冷冻保护剂,包括 FBS 和正常山羊血清 [NGS]。确保样品架中的液体中没有气泡。
将扁平样品架放置在样品架舱中,并通过将它们压在黑色钻石制成的表面上而紧密密封(见图 2)。
使用加载台将样品架加载到 Leica EM PACT2 中(参见图 2)。
要冷冻样品,在触摸屏上按“开始”,系统开始高压冷冻。冷冻完成后,载有样品的荚果落自动至液氮浴中。
检查冷却速度和屏幕上的压力上升。
将豆荚支架放在液氮的插座浴中,然后拧下豆荚支架,将冷冻样品留在浴槽底部。
冷冻后,扁平样品架在液氮下从 EM PACT2 仪器转移到预冷至 -90°C 的 Leica AFS2 冷冻替代系统。
图 2. 加载用于高压冷冻的蓝宝石磁盘。(A)图中显示了与HPF扁平样本保持器(蓝宝石盘的夹层小号TEP乙2)。使用 BioRender.com 准备。(B)扭矩螺丝刀被用于收紧含有吊舱蓝宝石盘(平坦样品架小号TEP乙3)。(C)的分离舱保持器被用于拾取和介绍与样本荚到徕卡EM PACT2(小号TEP乙3)。(d)该吊舱架装入徕卡EM PACT2(的装载台小号TEP乙4)。

冷冻置换和环氧树脂包埋
注意:在装入冷冻替代液之前,始终将冷冻样品保持在液氮中。所有仪器(例如,将样品转移到含有替代液体的管中的镊子)和溶液在使用前都应预冷。
样品冷冻-在-90℃下代替24小时中的1%四氧化锇,0.1%乙酸双氧铀,5%水和2%甲醇的无水丙酮的混合物。
温度以2°C/h 的速率升至 -25 °C ,最后以 5°C/h 的增量升至 0°C。
完成 -90°C 到 0°C 的转换后,机器可以在 0°C 下保持 1 - 2 小时。此过程增加了样品的对比度。如果持续时间超过2小时长,膜会不会还有-保存。
以下所有步骤均在 RT 中进行。用 1 ml 干燥的纯丙酮洗涤样品 3 次,每次 20 分钟。
用丙酮-环氧树脂混合物(参见配方)逐渐渗透样品,以 30%、50%、70% 和 90% 的步骤增加树脂浓度,每次 1 小时。
对于纯环氧树脂的渗透,使用镊子将蓝宝石圆盘与扁平样品架分开,并放入平底ed光束胶囊中。
胶囊填充新鲜制备的环氧树脂,用于在 60°C 下聚合 2 天。

超薄切片术
光束胶囊用一个不锈钢刀片移除,并且蓝宝石盘一个再从与耐热冲击树脂的块分离。将样品装入液氮中几秒钟,然后恢复到室温。重复这两个步骤,直到蓝宝石盘与树脂块分离。
使用不锈钢刀片将样品切成小块(~1 × 1 毫米),并固定在树脂块上作为支架(见图 3)。
使用新的不锈钢刀片手工修剪样品表面以获得梯形形状。
使用带有完美环的超薄切片机 UC6收集半厚切片(~ 350 - 400 nm),并将切片放置在Quantifoil ®铜网格中。
注意:其他类型的网格可用于 ET。


图 3. 安装超微切片样品。(A)在蓝宝石圆盘的分离之后,用嵌入式细胞单层树脂被切成小块的约1 -毫米大小。(B)样品的小片粘贴在以筒状树脂块,其允许安装的在切片机的试样支架的样品。比例尺 = 10 毫米。

在切片的两侧涂上带有基准标记(蛋白 A 金球 [10 nm 直径],PAG 10 )的网格,以便更好地对齐和用醋酸铀染色。该协议在图 4中进行了总结。
注意:基准标记是 10 nm 直径的金颗粒,用于在图像重建之前的断层扫描处理期间对齐倾斜图像。
在封口膜片上,每个蛋白 A 金溶液样品滴一滴(参见配方),每个水样品滴三滴。
将金滴上的网格孵化 10 分钟。
依次将网格放在三个水滴上几秒钟,以去除多余的金溶液。
用吸水滤纸吸收多余的液体。
重复小号TEPS ë 1 -E 4 ,温育所述网格的另一侧。
让网格干燥至少 2 小时。
注意:Ť Ó增加图像的对比度,用乙酸双氧铀染色切片。
在封口膜片上,放置一滴 2% 醋酸双氧铀(参见配方)并将网格孵育 20 分钟。
用三滴水清洗网格几秒钟以去除醋酸铀。
用滤纸吸收多余的液体。
允许网格到干燥用电子显微镜成像前至少2小时。
图 4. 电子断层扫描前的网格准备示意图。使用 BioRender.com 创建。

电子Ť omography
使用电子显微镜检查呼肠孤病毒出口区域的部分,选择并保存倾斜系列的感兴趣区域 (ROI)。
在收集倾斜系列之前预先照射 ROI。电子束会对树脂嵌入的样品造成损坏,导致样品收缩,尤其是沿Z轴的收缩(Luther等人,1988 年)。为避免在断层扫描过程中出现此问题,重要的是预先照射 ROI,包括比 ROI 更大的区域,因为外部区域稍后可能会进入现场。
注意小号:
约2 ,每平方000电子甲ngstrom在低放大倍数(2 ,000 × EM-照射的)是足以允许的ROI假定足够的尺寸和稳定性。
ROI 不应靠近网格条,因为网格条会在高倾斜角度阻止采集。
单轴和双轴 ET 使用Tecnai G2 F20 透射电子显微镜 (FEI) 在 200 kV 下运行。
样品在电子显微镜内倾斜以从许多不同角度产生透射电子显微镜图像,从而产生倾斜系列。
采集倾斜系列自动以1℃的增量在一个angul Ë范围的-60°〜60°。相机参数应设置为允许样品的最佳分辨率和对比度。在我们的研究中,我们获得了标称放大倍数为 ×11,500 的倾斜系列,导致像素大小为 1.01 nm。
使用 Eagle 4k × 4k 慢扫描电荷耦合器件 (FEI)和断层扫描 3 软件记录图像。
要进行双轴 ET,在样品围绕垂直于其支撑平面的轴旋转90°后收集第二个倾斜系列。
注意:双轴或双倾斜 ET 增加了收集的信息并减少了采集和重建的伪影(Mastronarde ,1997)。

断层图像处理
注意:ET 获得的不同视角用于重建 3D 视图,该视图在三个维度上解析呼肠孤病毒出口细胞器的超微结构。
下载并安装 IMOD 软件以对齐倾斜系列并获得断层扫描重建。
启动eTomo ,提供设置面板中的条目。关于倾斜系列的重要参数是:
轴类型:单轴或双轴
帧类型
像素大小和基准直径
倾斜角度
注意:在此设置面板中,可以删除排除的视图(不适合分析的图像)。
设置面板完成后,激活创建 Com 脚本。
预处理。运行Ccderaser以擦除图像中的 X 射线和其他伪影。X 射线模型创建了一个可以保存和使用的固定堆栈。
粗对准。使用Tiltxcorr计算互相关,以确定将每个图像与前一个图像对齐所需的 X 和 Y 平移,生成粗对齐堆栈。
基准模型生成。有多种方法可以生成基准模型。我们在零度视图中选择了分布均匀的基准点或金珠,并使用Beadtrack在倾斜系列的其余部分中找到它们的位置。最后,创建了一个种子模型。可以手动或自动标记种子点。对齐过程的概述如图 5 所示。
精细对齐。使用Tiltalign执行对齐并在该过程完成后检查日志文件中的结果。可以查看残差向量模型,该模型给出了对齐质量的概念(参见图 5)。残差向量越小,对齐越好。良好对齐的典型平均残差值约为 1 个像素。
断层扫描定位。准备一个简单的模型,在 Y 中三个位置的截面顶面和底面上有水平线。划定截面的厚度很重要。定义截面的边缘后,选择自动查找边界模型,创建边界模型(见图 5)。
最终对齐堆栈。使用Newstack获取对齐的堆栈。作为一个选项,可以在此步骤中校正显微镜 CTF(对比度传递函数)并过滤对齐的堆栈。
断层扫描生成。使用倾斜生成断层图。
注意:在双倾斜断层扫描中,称为 A 和 B 的两个断层扫描图像是分开处理的。断层图A首先对齐,表明它是双轴。使用其基准点(将断层图像 B 与 A 匹配)参考断层图像 A 对齐断层图像 B。结果,准备了组合断层图像。
图 5. IMOD 软件的屏幕截图。显示了使用断层图对齐构建的代表性模型。

断层图像分割和 3D 可视化
断层图像分割和三维可视化用进行的阿米拉软件。
在 Amira 界面中加载断层图 (.rec) 并应用高斯滤波器(3D 类型)以降低噪声。
一个Orthoslice可以进行可视化和比较断层扫描与过滤的X线断层。
过滤后的断层图使用函数LabelField进行分割,它允许在图像中标记不同结构的区域。在断层扫描的所有图像中标记结构是一项繁琐的工作,可以使用插值功能简化。插值功能允许在标记图像后自动完成分割。
注意:验证 Interpolate 功能是否运行良好很重要,因为有时必须对某些标记图像或结构进行自动分割的手动更正。
Arithmetic 和Isosurface函数用于在 3D 中可视化结构。
注意:标记结构的阈值是使用每个结构的直方图获得的。使用Isosurface调整阈值可改善重建的可视化。

数据分析

我们使用 3D-ET 来分析呼肠孤病毒的出口途径。图 6 中所示的断层图是使用半厚切片 (350 nm) 获得的,这使我们能够识别不同结构之间的空间关系。ET 对 VI-SO-MC 界面的分析揭示了这些结构之间存在物理连接(图 6)。断层图的计算切片与 3D 可视化相结合,显示 VI 和 SO(图 6A 中的虚线框)以及 SO 和 MC(图 6B 中的虚线框)之间存在膜连续性点。这些相互作用仅限于几个计算切片,表明这些隔间之间的关联是通过在图 6A 和 B 所示的更高放大倍率图像中观察到的离散通道发生的。


图6.的电子断层扫描的呼肠孤病毒出口机械。(A 和 B)HBMEC 被呼肠孤病毒 T1L M1-P208S 以 1 PFU/细胞的 MOI 吸附,并在 18 hpi通过高压冷冻、冷冻置换和半厚切片进行处理。单倾斜断层扫描和 3D 重建显示呼肠孤病毒出口机器(虚线框)的组件之间的连接。(A)中分选细胞器 (SO) 和病毒包涵体 (VI)或通过 (B) 中的通道附着在 SO 上的膜载体 (MC)之间的特定膜融合点的更高放大倍数。mi,线粒体。比例尺 = 500 nm A 和 B;在更高的放大倍数下为 100 nm。© 2020 费尔南德斯·德卡斯特罗等人。最初发表于J Cell Biol https://doi.org/10.1083/jcb.201910131。

我们使用双倾斜 ET 来研究呼肠孤病毒出口途径的最后步骤(图7 )。通过 ET 和 3D 重建对质膜上的 MC进行检查,揭示了出口前载体形态和病毒粒子出口机制的细节。成熟的病毒粒子沿着 MC 内的膜或通道阵列分布(图 7A),这可能会增加 MC 的承载能力或病毒运输的效率。3D体积描绘了MC和它与细胞膜缔合(图的总含量小号7B和C)。因此,在充满病毒的 MC 接触质膜后,后代呼肠孤病毒颗粒通过不同的出口区离开受感染的细胞(Fernandez de Castro等,2020)。


图 7.膜状载体的电子断层扫描。HBMECs 被呼肠孤病毒 T1L M1-P208S 以 1 PFU/细胞的 MOI 吸附,并在 18 hpi通过高压冷冻、冷冻置换和半厚切片进行处理。(A) 计算机断层扫描切片显示膜载体 (MC) 与膜通道 (白色箭头) 和病毒粒子 (白色箭头) 靠近质膜 (PM)。(B)的3D重建的MC overlapp的ING用二维计算切片。(C) 显示分割后 MC总内容的 3D 卷的一个视图。成熟的病毒粒子,蓝色;通道,米色;膜质载体,浅棕色;质膜,绿色。比例尺 = 200 nm。© 2020 费尔南德斯·德卡斯特罗等人。最初发表于J Cell Biol https://doi.org/10.1083/jcb.201910131。

  该ET实验中有至少两个生物学重复进行。记录了显示不同呼肠孤病毒出口区的八张断层图,并从九张断层图分析了 VI-SO-MC界面。使用两样品不等方差确定统计学显着吨-试验用双尾分布。


笔记

呼肠孤病毒株 T1L M1-P208S 与原型 T1L 株相同,除了 μ2 蛋白(由 M1 基因编码)的第 208 位脯氨酸到丝氨酸的取代。这种突变将包涵体形态从丝状变为球状(Parker等,2002)。
在倾斜系列对准中,最关键的部分是基准标记在截面中的分布。只有在每个部分中维护的那些基准点才应该在同一 Z 平面上进行标记,并且它们应该在整个部分中均匀分布。

食谱

完整的 RPMI-1640 介质
10%女小号erum
1% MEM 维生素
1%小号裂果丙酮酸
1% MEM 非必需氨基酸
1% L-谷氨酰胺
1% 青霉素/链霉素
0.1% 两性霉素
商店在 4° ç
HEPES 0.4 M
9.54 克 HEPES
米尔添加升I-Q水至50ml,直至HEPES完全溶解拌匀
用 1N NaOH 溶液调节 pH 值至 7.2 - 7.4
添加英里升I-Q水至100毫升
储存在 4 °C
环氧树脂
使用塑料巴斯德吸管称量环氧树脂组分。
通过倒转管数次混合树脂组分。
使用时必须添加加速器 (BDMA)。
将混合物以14,100 × g离心10 分钟以消除小气泡。
19.2 克 812 树脂
7.6 克 DDSA
13.2 克 NMA
0.8 克(或 20 滴)BDMA
Protein A Gold 或基准标记溶液
在水中混合胶体金溶液(1:50 比例)
每次都新鲜制备此溶液
注意:基准标记的稀释(和大小)取决于倾斜系列采集的放大倍数。放大倍数越高,基准点越密,黄金尺寸越小。

致谢

这项工作得到了公共卫生服务奖 AI032539(TSD 和 CR)的部分支持。特别感谢马丁Sachse的博士整个工作和手稿的严格审查专家技术咨询和支持,弗朗西斯博士哈维尔Chichón从CNB-CIB(CSIC)CR yoEM与ET技术咨询机构,和克里斯蒂娜·帕蒂诺,比阿特丽斯·马丁,和Pablo机Solá从CNB(CSIC)EM设施。该协议改编自以前的工作:Fernández de Castro等人。,Ó riginally发表在细胞与生物学https://doi.org/10.1083/jcb.201910131。

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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Fernandez de Castro, I., Fernández, J. J., Dermody, T. S. and Risco, C. (2021). Electron Tomography to Study the Three-dimensional Structure of the Reovirus Egress Pathway in Mammalian Cells. Bio-protocol 11(13): e4080. DOI: 10.21769/BioProtoc.4080.
  2. Fernandez de Castro, I., Tenorio, R., Ortega-Gonzalez, P., Knowlton, J. J., Zamora, P. F., Lee, C. H., Fernandez, J. J., Dermody, T. S. and Risco, C. (2020). A modified lysosomal organelle mediates nonlytic egress of reovirus. J Cell Biol 219(7).
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