搜索

Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast
使用金属标签透射电子显微技术定位酵母菌中番茄丛矮病毒复制区   

评审
匿名评审
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Journal of Cell Science
Jan 2017

Abstract

Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication organelles. Therefore, efficient virus replication depends on the assembly of specialized membranes where viral macromolecular complexes are turned on and hold a variety of functions. Detailed characterization of viral replication platforms in cells requires sophisticated imaging approaches. Here we present a protocol to visualize the three-dimensional organization of the tombusvirus replicase complex in yeast with MEtal-Tagging Transmission Electron Microscopy (METTEM). This protocol allowed us to image the intracellular distribution of the viral replicase molecules in three-dimensions with METTEM and electron tomography. Our study showed how viral replicase molecules build replication complexes within specialized cell membranes.

Keywords: Metal-tagging transmission electron microscopy (金属标签透射电子显微镜技术), METTEM (METTEM), Clonable tag (可克隆标签), Viral replication complex (病毒复制复合体), Electron tomography (电子断层扫描), 3D electron microscopy (3D电子显微镜技术)

Background

Replication of positive-stranded RNA viruses depends on the remodeling of cellular membranes. Intracellular membranes serve as a structural scaffold for VRC assembly, provide essential lipids and co-factors that modulate the activity of the viral replicase and protect the viral RNA from the antiviral defenses of the host (Miller and Krijnse-Locker, 2008; den Boon et al., 2010; Nagy and Pogany, 2011; Nagy, 2016). The architecture of replication organelles with active VRCs has been observed by electron microscopy. VRCs assemble in single membrane vesicles or ‘spherules’, tubulovesicular cubic membranes, double membrane vesicles (DMV) or planar oligomeric arrays (de Castro et al., 2013). Spherules are often observed in cells infected by RNA viruses. They form by invagination in a variety of organelles and have a narrow opening to the cytosol (den Boon et al., 2010).

Tomato bushy stunt virus (TBSV) is a small (+) RNA virus that belongs to the Tombusviridae, a family of viruses that infect plants. TBSV has recently emerged as a model virus to study viral replication and virus-host interactions using the yeast Saccharomyces cerevisiae as a model host (Nagy and Pogany, 2011). Studies of S. cerevisiae infected with plant viruses have facilitated the identification of numerous factors needed for viral replication (Nagy, 2008). Tombusviruses encode five proteins including two replication proteins, p92pol and p33. p92pol is the RNA-dependent RNA polymerase. The auxiliary protein p33 is an RNA chaperone that facilitates the recruitment of the viral RNA to the site of replication, in the cytosolic face of peroxisome membranes (McCartney et al., 2005; Jonczyk et al., 2007).

Understanding the biogenesis and functional architecture of VRCs in cell membranes is challenging and it requires sophisticated imaging techniques. Transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of macromolecular assemblies in cells. However, methods to unambiguously identify proteins within the environment of the cell are lagging behind. In our lab, we have developed a new labeling method named METTEM from MEtal-Tagging Transmission Electron Microscopy. This method uses the metal-binding protein metallothionein (MT) as a genetically clonable tag for electron microscopy (Diestra et al., 2009; Risco et al., 2012). Mouse MT 1 is a small, 61-amino acid protein with 20 cysteine residues that bind gold atoms very efficiently. MT fused to a protein of interest and treated with gold salts, builds an electron-dense gold nanocluster of around 1 nm diameter, easily visualized by electron microscopy (Mercogliano and DeRosier, 2006 and 2007) (Figure 1). METTEM allows identification and localization of intracellular proteins with high specificity and exceptional sensitivity at molecular-scale resolution ( Diestra et al., 2009; Delebecque et al., 2011; Bouchet-Marquis et al., 2012; Risco et al., 2012; Barajas et al., 2014a; de Castro Martin et al., 2017; Fernandez de Castro et al., 2017).


Figure 1. Imaging viral replicase complexes with METTEM. Viral replicase protein is fused with metallothionein (MT) and expressed in yeast cells (Fernandez de Castro et al., 2017). MT-tagged viral replicase molecules assemble VRCs in peroxisome membranes. Cells are incubated with gold salts in vivo and MT-gold-replicase molecules are visualized by electron microscopy.

Here we described a protocol to visualize TBSV replicase molecules in VRCs using METTEM (Figure 2). The combination of this technology with electron tomography allowed us to study the distribution of replicase molecules in the viral replication compartment in three-dimensions (3D). Due to the high sensitivity of the method we could distinguish different states of aggregation of the viral replicase molecules in situ. This methodology can be used to detect any protein of interest in different subcellular locations of bacteria, yeast and mammalian cells. Furthermore, one advantage of this electron microscopy approach is that it can be used to study many different viruses in a variety of cell types by visualizing the MT tag incorporated in either complete viral particles or their proteins. This method has revealed virus-induced structures not seen before, as reported for Rubella virus, Tombusvirus and influenza virus (Risco et al., 2012; de Castro Martin et al., 2017; Fernandez de Castro et al., 2014 and 2017).


Figure 2. Schematic workflow of the protocol. Pre-grown transformed yeast cells are incubated overnight in YPG. Next day viral replication is induced during 24 h at 23 °C. Cells are treated with zymolyase to obtain spheroplasts. Spheroplasts are incubated with gold salts to build nanoclusters in MT tags. Cell pellets are dehydrated and embedded in resin. Serial sections are transferred to EM grids and imaged by TEM.

Materials and Reagents

  1. Pipette tips
  2. Perfect loop (Electron Microscopy Sciences, catalog number: 70945 )
  3. 50 ml disposable centrifuge tubes
  4. Eppendorf tubes
  5. Sterile transfer pipettes
  6. Serum Acrodisc® 37 mm syringe filter (Pall, catalog number: 4525 )
  7. Gelatin capsule size 1 6.5 mm diameter-0.50 ml (TAAB, catalog number: C089/1 )
  8. GEM® Single Edge Blades 3-Facet 0.009"/0.23 mm (AccuTec Blades, catalog number: 62-0179-0000 )
  9. Saccharomyces cerevisiae yeast strains RS453 (MATa ade2-1 his3, 15 leu2-3, 112 trp1-1 ura3 52) and pah1Δnem1Δ (SwissProt ID for Nem1 is P38757) (pah1Δ::TRP1nem1Δ::HIS3 derivative of RS453) (Choi et al., 2011; Barajas et al., 2014b)
  10. Zymolyase® 20T (Arthrobacter luteus) (Amsbio, catalog number: 120491-1 )
  11. Gold(III) chloride ≥ 99.99% (Sigma-Aldrich, catalog number: 379948 )
  12. Glutaraldehyde 50% (TAAB, catalog number: G015 )
  13. Ethanol Dry (Merck, catalog number: 1009901001 )
  14. LR-White Resin Medium Grade Acrylic resin (TAAB, catalog number: L012 )
  15. BactoTM yeast extract (BD, BactoTM, catalog number: 212750 )
  16. BactoTM peptone (BD, BactoTM, catalog number: 211677 )
  17. D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270 )
  18. Agar (Sigma-Aldrich, catalog number: 05038 )
  19. D-(+)-Galactose (Sigma-Aldrich, catalog number: G0750 )
  20. Lithium acetate 99.95% (Sigma-Aldrich, catalog number: 517992 )
  21. ssDNA (Deoxyribonucleic acid sodium salt from salmon testes) (Sigma-Aldrich, catalog number: D1626 )
  22. PEG MW3350 (Polyethylene glycol) (Sigma-Aldrich, catalog number: P4338 )
  23. Trizma® hemisulfate (Sigma-Aldrich, catalog number: T8379 )
  24. 1,4-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: DTT-RO)
    Manufacturer: Roche Diagnostics, catalog number: 10197777001 .
  25. Yeast nitrogen base without amino acids (Sigma-Aldrich, catalog number: Y0626 )
  26. Yeast Synthetic Drop-out Medium Supplements without tryptophan (Sigma-Aldrich, catalog number: Y1876 )
  27. D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
  28. Tris (Base) (Norgen Biotek, catalog number: 28029 )
  29. Paraformaldehyde EM (TAAB, catalog number: P026 )
  30. NaOH
  31. 10x PBS
  32. 1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N’-bis(2-ethanesulfonic acid) PIPES (Sigma-Aldrich, catalog number: P6757 )
  33. 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375 )
  34. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  35. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  36. YPD (see Recipes)
  37. YPG (see Recipes)
  38. Transformation mix (see Recipes)
  39. TSD reduction buffer (see Recipes)
  40. Spheroplast medium A (see Recipes)
  41. 4% paraformaldehyde (PFA) solution (see Recipes)
  42. PHEM solution, pH 6.9 (see Recipes)

Equipment

  1. 500 ml flask
  2. Pipettes
  3. Diamond Knife 45° DIATOME (Fedelco)
  4. QUANTIFOIL® R 3.5/1 100 Holey Carbon Films Grids Au 300 mesh (Quantifoil)
  5. Oven Memmert UN 55 (Memmert, model: UN55 ) (Genesys) equipped with a shaker
  6. Spectrophotometer 722N (Terra Universal, Laboratory Equipment, model: 722N )
  7. Centrifuge 5810 R (Eppendorf, model: 5810 R )
  8. Centrifuge miniSpin plus (Eppendorf, model: MiniSpin® plus )
  9. Ultrasonic Cleaner 1510 (Branson, model: 1510 )
  10. pH meter Basic 20 (HACH LANGE SPAIN, Crison, model: Basic 20 )
  11. Fume Hood (Flow-Tronic)
  12. Ultramicrotome (Leica Microsystems, model: Leica EM UC6 )
  13. Jeol JEM 1011 electron microscope operating at 100 kV (JEOL, model: JEM-1011 )
  14. FEI Tecnai G2 F20 (200 kV) electron microscope (FEI)
  15. Tecnai Spirit Twin (120 kV) electron microscope (FEI)

Software

  1. IMOD software
  2. Amira software

Procedure

  1. Transformation of Saccharomyces cerevisiae yeast strains
    1. Streak the yeast strain from a glycerol stock on a yeast extract peptone dextrose (YPD) agar plate. Grow cells at 30 °C for 1-2 days. Inoculate 10 ml YPD solution with a single colony with a loop. Grow culture overnight at 30 °C at 250 rpm (OD around 2).
    2. Dilute overnight culture to OD 0.2-0.3 in 50 ml YPD medium.
      Note: Use a 500 ml flask.
    3. Grow cells for 3-4 h at 30 °C at 250 rpm.
    4. Centrifuge cells at 3,590 x g for 5 min at room temperature. Use 50 ml disposable centrifuge tubes.
    5. Discard supernatant and add 1 ml of sterile dH2O, vortex gently, then fill up tube with sterile dH2O.
    6. Centrifuge cells at 3,590 x g for 5 min. Use 50 ml disposable centrifuge tubes.
    7. Discard supernatant and resuspend pellet in 1 ml of 100 mM lithium acetate.
    8. Transfer cells into an Eppendorf tube and centrifuge for 2 min at 3,590 x g. Discard supernatant and dissolve the cell pellet in 0.4 ml of 100 mM lithium acetate. Incubate at room temperature for 10-15 min.
      Note: Use a sterile transfer pipette.
    9. Add 1-5 μl plasmid DNA (5-10 μg) to each fresh Eppendorf tube.
    10. Make up the master mix for transformation (see Recipes).
    11. Add 0.4 ml transformation mix to each Eppendorf tube containing the plasmids. Vortex gently for 2 sec to mix components.
      Note: Make sure that yeast cells are well resuspended.
    12. Incubate cells at 30 °C (no shaking) for 30 min.
    13. Incubate cells at 42 °C (no shaking) for 40 min.
    14. Centrifuge cells at 570 x g for 2 min. Discard supernatant and add 100 μl sterile dH2O. Disperse pellet by gently pipetting up and down, and dispense cells on agar plates with the appropriate selection medium. Incubate plates at 30 °C for 2-3 days.
      Note: Try to spread cells with few strokes.

  2. Cell culture
    1. Pre-grow yeast in 2 ml of yeast extract peptone galactose (YPG) and incubate overnight at 30 °C and 250 rpm.
    2. Culture 50 ml of yeast cells in an Erlenmeyer for 24 h in YPG at 23 °C and 250 rpm.
      Note: These conditions are necessary to induce and maintain viral replication.
    3. Measure and control optical density at 600 nm (OD600) and when OD600 is around 2 harvest cells by centrifuging for 5 min, 4,000 x g, room temperature, and carefully remove supernatant.
    4. Resuspend cells at 5 to 10 OD600 units/ml in Tris-sulfate with dithiothreitol (DTT) (TSD) reduction buffer and incubate for 10 min at room temperature.
      Notes:
      1. DTT facilitates cell wall digestion by breaking disulfide bonds and making β-glucan linkages more accessible to the β-glucanase activity present in the zymolyase.
      2. The results of the experiment will change if cells are processed at different OD.
    5. Incubate yeast cells with 0.1 mg/ml zymolyase 20T for 10 min at 30 °C to obtain spheroplasts
      Notes:
      1. The optimal zymolyase concentration and digestion time depend on yeast strains and growth conditions. Setting zymolyase treatment is critical for an adequate preservation of cell ultrastructure.
      2. Before zymolyase treatment, it is important to sonicate cells for 5 sec in an Ultrasonic cleaner to avoid yeast aggregation. 
    6. Centrifuge spheroplasts at 280 x g for 5 min and remove supernatant.
    7. Wash 3 times with ice-cold spheroplast medium A.

  3. Gold treatment and fixation
    1. Incubate live spheroplasts with 2 mM HAuCl4 in spheroplast medium A for 75 min at room temperature in the dark.
      Notes:
      1. To prepare the HAuCl4 solution, dissolve in distilled and sterile water protected from light.
      2. MT binds gold atoms and builds a nanocluster visible by TEM.
      3. It is important to adjust the gold treatment conditions due to the potential toxicity of gold salts in eukaryotic cells.
    2. Wash cells with spheroplast medium A.
    3. Fix cells with 4% paraformaldehyde and 0.2% glutaraldehyde in PHEM solution for 1 h at room temperature.
    4. After fixation wash spheroplasts 3 times with PHEM.

  4. Embedding
    1. Dehydrate cells in 10 min steps, with increasing concentrations of 1 ml ethanol (30, 50, 70, 90% and twice in 100%) at 4 °C. Centrifugation between steps is not necessary.
    2. Incubate cells in mixtures of ethanol–LR-White resin (2:1, 1:1, 1:2) on a rocking shaker for 1 h each at room temperature and protected from light.
      Note: Do not expose LR-White resin to light and manipulate inside a well ventilated hood.
    3. Remove cells from the ethanol-resin mix and place them in 100% resin for 24 h.
    4. Polymerize samples in gelatin capsules for 48 h at 60 °C.

  5. Ultramicrotomy
    1. Remove gelatin capsules and trim samples with a razor blade to make a trapeze.
    2. Collect 50-60 nm ultrathin sections on 300-mesh Quantifoil holey carbon grids.
    3. Observe sections under a transmission electron microscope at 30,000-50,000x nominal magnifications.
      Note: Sections are observed by TEM without staining to avoid masking the small MT-gold nanoclusters.

  6. Electron tomography and image processing
    1. Obtain semi-thick sections (~300 nm) and collect them on Quantifoil grids.
    2. Acquire tilt series automatically at 1.5° increments over an angular range of -60° to +60° on FEI Tecnai G2 F20 and Tecnai Spirit Twin microscopes with an accelerating voltage of 200 and 120 kV, respectively.
    3. Use IMOD software to align tilt series and for tomographic reconstruction.
    4. For tomogram segmentation and 3D reconstruction, Amira software is used.
      Note: Noise reduction and automated segmentation software helps to highlight membrane visualization.

Data analysis

We have used the method METTEM to visualize the Tombusvirus replicase p33 in yeast cells. Electron microscopy imaging in Figure 3 showed the precise location of p33–MT-gold molecules in a membranous compartment compatible with peroxisome-derived multivesicular body (MVB), which is the Tombusvirus replication organelle in plant and yeast cells (Barajas et al., 2014a). With this approach, we could detect p33-metallothionein-gold nanoparticles in ER membranes inside the replication platform (Figures 3A and 3B).


Figure 3. Visualization of p33–MT-gold molecules by METTEM. Sections were not stained in order to avoid masking the MT-gold nanoclusters. A. Inside the tombusvirus replication compartment p33–MT-gold nanoparticles (~1 nm) delineate membranes (arrows). Single MT clusters are marked with arrowheads. B. Tombusvirus replication platform with p33–MT-gold nanoclusters inside (arrowheads) and in the surrounding endoplasmic reticulum (ER) membranes. Scale bars = 200 nm.

For more precise details, we studied the viral replication organelles in three dimensions with electron tomography (Figure 4). Tomograms showed the internal organization of p33–MT-gold molecules in the viral replication complex. Viral replicase molecules distributed in a variety of aggregation states in different domains of the replication compartment (Figure 4) (Fernandez de Castro et al., 2014 and 2017).


Figure 4. Electron tomography of the viral replication organelles. A. 3D model of the replication platform. Membranes are shown in yellow and p33–MT-gold molecules in red (arrows). B. Computational tomographic slice corresponding to the replication platform showed in A. Arrows point to aggregates of p33–MT-gold molecules inside membranes. Nano-clusters have an approximate diameter of 1 nm. Scale bars = 100 nm.

Notes

  1. Analyze the sequence of the protein of interest in order to identify the best site for inserting the MT tag. In addition, it is relevant to test if the MT-tagged protein activity and function are affected.
  2. Adjust the optimal conditions in your system for expression of the MT-tagged protein.
  3. Make a preliminary EM study to get familiar with the ultrastructure of the cell type. This will facilitate data interpretation.
  4. Check that the gold salts do not precipitate in the medium where cells are going to be treated.
  5. Incubate control cells without MT-tagged proteins with gold salts and visualize electron microscopy to verify lack of nonspecific background.
  6. Use acrylic resin (such as LR White or Lowicryl) in the absence of staining agents to visualize the MT-nanoclusters.
  7. Perform immunogold labeling with antibodies specific for the MT-tagged protein to confirm labeling specificity.

Recipes

  1. YPD
    10 g yeast extract
    20 g peptone
    20 g dextrose (glucose)
    Add dH2O to 1,000 ml and mix well
    Autoclave to sterilize
    For plates add 20 g of agar/L
  2. YPG
    10 g yeast extract
    20 g peptone
    20 g galactose
    Add dH2O to 1,000 ml and mix well
    Autoclave to sterilize
    For plates add 20 g of agar/L
  3. Transformation mix
    0.36 ml 1 M lithium acetate
    0.1 ml 10 mg/ml ssDNA
    0.69 ml dH2O
    2.4 ml 50%PEG MW3350
    Add dH2O up to 10 ml
  4. TSD reduction buffer
    0.1 M Trizma® hemisulfate, pH 9.4
    10 mM DTT (added just before use)
  5. Spheroplast medium A
    1x yeast nitrogen base
    2% (w/v) glucose
    1x amino acids
    1 M sorbitol
    20 mM Tris-HCl, pH 7.5
    Store up to 4 weeks at room temperature
  6. 4% paraformaldehyde (PFA) solution
    1. Dissolve 4 g of PFA in 90 ml of distilled water at 60 °C under a well ventilated hood
    2. Add drops of 1 N NaOH until the solution is transparent and without precipitates
    3. Then remove from heating and cool down on ice
    4. Add 10 ml of 10x PBS and fill up with distilled water to 100 ml
    5. Control the temperature and avoid heating the solution above 70 °C because PFA will break down at higher temperatures
    6. Filter the solution with 37 mm syringe filters
    7. Use the solution immediately or store for 1 day at 4 °C or at -20 °C for months
  7. PHEM solution, pH 6.9
    20 mM PIPES
    50 mM HEPES
    20 mM EGTA
    4 mM MgCl2
    Store for several months at 4 °C

Acknowledgments

We would like to express our gratitude to Drs José Jesús Fernández and Peter Nagy for expert advice and helpful discussions. This work was supported by grant BIO2015-68758-R (to C.R.) from the Spanish Ministry of Economy and Competitiveness (MINECO-AEI/FEDER, EU). The protocol is adapted from Fernandez de Castro et al. (2017). The authors declare no conflicts of interest.

References

  1. Barajas, D., Martin, I. F., Pogany, J., Risco, C. and Nagy, P. D. (2014a). Noncanonical role for the host Vps4 AAA+ ATPase ESCRT protein in the formation of Tomato bushy stunt virus replicase. PLoS Pathog 10(4): e1004087.
  2. Barajas, D., Xu, K., de Castro Martin, I. F., Sasvari, Z., Brandizzi, F., Risco, C. and Nagy, P. D. (2014b). Co-opted oxysterol-binding ORP and VAP proteins channel sterols to RNA virus replication sites via membrane contact sites. PLoS Pathog 10(10): e1004388.
  3. Bouchet-Marquis, C., Pagratis, M., Kirmse, R. and Hoenger, A. (2012). Metallothionein as a clonable high-density marker for cryo-electron microscopy. J Struct Biol 177(1): 119-127.
  4. Choi, H. S., Su, W. M., Morgan, J. M., Han, G. S., Xu, Z., Karanasios, E., Siniossoglou, S. and Carman, G. M. (2011). Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: identification of SER602, THR723, AND SER744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase. J Biol Chem 286(2): 1486-1498.
  5. de Castro, I. F., Volonte, L. and Risco, C. (2013). Virus factories: biogenesis and structural design. Cell Microbiol 15(1): 24-34.
  6. de Castro Martin, I. F., Fournier, G., Sachse, M., Pizarro-Cerda, J., Risco, C., & Naffakh, N. (2017). Influenza virus genome reaches the plasma membrane via a modified endoplasmic reticulum and Rab11-dependent vesicles. Nat Commun 8(1): 1396.
  7. Delebecque, C. J., Lindner, A. B., Silver, P. A. and Aldaye, F. A. (2011). Organization of intracellular reactions with rationally designed RNA assemblies. Science 333(6041): 470-474.
  8. den Boon, J. A., Diaz, A. and Ahlquist, P. (2010). Cytoplasmic viral replication complexes. Cell Host Microbe 8(1): 77-85.
  9. Diestra, E., Fontana, J., Guichard, P., Marco, S. and Risco, C. (2009). Visualization of proteins in intact cells with a clonable tag for electron microscopy. J Struct Biol 165(3): 157-168.
  10. Fernandez de Castro, I., Fernandez, J. J., Barajas, D., Nagy, P. D. and Risco, C. (2017). Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex. J Cell Sci 130(1): 260-268.
  11. Fernandez de Castro, I., Sanz-Sanchez, L. and Risco, C. (2014). Metallothioneins for correlative light and electron microscopy. Methods Cell Biol 124: 55-70.
  12. Jonczyk, M., Pathak, K. B., Sharma, M. and Nagy, P. D. (2007). Exploiting alternative subcellular location for replication: tombusvirus replication switches to the endoplasmic reticulum in the absence of peroxisomes. Virology 362(2): 320-330.
  13. McCartney, A. W., Greenwood, J. S., Fabian, M. R., White, K. A. and Mullen, R. T. (2005). Localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway. Plant Cell 17(12): 3513-3531.
  14. Mercogliano, C. P. and DeRosier, D. J. (2006). Gold nanocluster formation using metallothionein: mass spectrometry and electron microscopy. J Mol Biol 355(2): 211-223.
  15. Mercogliano, C. P. and DeRosier, D. J. (2007). Concatenated metallothionein as a clonable gold label for electron microscopy. J Struct Biol 160(1): 70-82.
  16. Miller, S. and Krijnse-Locker, J. (2008). Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 6(5): 363-374.
  17. Nagy, P. D. (2008). Yeast as a model host to explore plant virus-host interactions. Annu Rev Phytopathol 46: 217-242.
  18. Nagy, P. D. (2016). Tombusvirus-host interactions: Co-opted evolutionarily conserved host factors take center court. Annu Rev Virol 3(1): 491-515.
  19. Nagy, P. D. and Pogany, J. (2011). The dependence of viral RNA replication on co-opted host factors. Nat Rev Microbiol 10(2): 137-149.
  20. Risco, C., Sanmartin-Conesa, E., Tzeng, W. P., Frey, T. K., Seybold, V. and de Groot, R. J. (2012). Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy. Structure 20(5): 759-766.

简介

正链(+)RNA病毒是人,动物和植物中的细胞内病原体。构建病毒复制酶复合物(VRC)病毒操纵脂质流动和重组亚细胞膜。重新设计的膜集中了病毒和宿主因子,并创造了促进复制细胞器内VRC形成的环境。因此,有效的病毒复制取决于病毒大分子复合物开启并具有各种功能的特殊膜的组装。细胞中病毒复制平台的详细特征需要复杂的成像方法。在这里我们提出一个协议,用肉眼标记透射电子显微镜(METTEM)可视化酵母中的tombusvirus复制酶复合物的三维组织。该协议使我们能够用METTEM和电子断层扫描成像三维病毒复制酶分子的细胞内分布。我们的研究显示病毒复制酶分子如何在特化细胞膜内构建复制复合物。

【背景】正链RNA病毒的复制取决于细胞膜的重塑。细胞内膜作为VRC装配的结构支架,提供调节病毒复制酶活性和保护病毒RNA免受宿主抗病毒防御的必需脂质和辅因子(Miller和Krijnse-Locker,2008; den Boon <等,2010; Nagy和Pogany,2011; Nagy,2016)。电子显微镜观察到具有活性VRC的复制细胞器的结构。 VRC以单个膜囊或'小球',管状球形立方体膜,双膜囊泡(DMV)或平面寡聚体阵列装配(de Castro等人,2013)。通常在RNA病毒感染的细胞中观察到小球。它们通过在各种细胞器中内陷而形成,并具有对胞质溶胶的狭窄开口(den Boon et al。,2010)。

番茄丛生特技病毒(TBSV)是一种小型(+)RNA病毒,属于感染植物的病毒家族Tombusviridae 。最近,TBSV作为模型病毒出现,用酵母酿酒酵母作为模型宿主研究病毒复制和病毒 - 宿主相互作用(Nagy和Pogany,2011)。对S的研究。感染了植物病毒的酿酒酵母已经促进了病毒复制所需的许多因子的鉴定(Nagy,2008)。 Tombusviruses编码五种蛋白,包括两种复制蛋白p92pol和p33。 p92pol是RNA依赖性RNA聚合酶。辅助蛋白p33是促进病毒RNA募集到过氧化物酶体膜的胞质表面的复制位点的RNA分子伴侣(McCartney等人,2005; Jonczyk等人。,2007)。

了解细胞膜中VRC的生物发生和功能结构是具有挑战性的,它需要复杂的成像技术。透射电子显微镜(TEM)有助于我们理解细胞中大分子组装的结构和组织。然而,明确识别细胞环境中的蛋白质的方法滞后。在我们的实验室,我们已经开发了一种新的标记方法,名为METTEM从MEtal标记透射电子显微镜。该方法使用金属结合蛋白金属硫蛋白(MT)作为电子显微镜的基因可克隆标签(Diestra等人,2009; Risco等人,2012)。小鼠MT1是一种小型的61个氨基酸的蛋白质,含有20个半胱氨酸残基,可非常有效地与金原子结合。 MT与感兴趣的蛋白质融合并用金盐处理后,可以通过电子显微镜(Mercogliano and DeRosier,2006和2007)(图1)构建一个直径约为1 nm的电子致密金纳米簇。 METTEM允许在分子尺度分辨率下以高特异性和出色的灵敏度鉴定和定位细胞内蛋白质(Diestra等人,2009; Delebecque等人,2011; Bouchet- Marquis et al。,2012; Risco et al。,2012; Barajas et al。,2014a; de Castro Martin et al。 。2017; Fernandez de Castro等人,2017)。

图1.用METTEM成像病毒复制酶复合物病毒复制酶蛋白与金属硫蛋白(MT)融合并在酵母细胞中表达(Fernandez de Castro等人,2017)。 MT标记的病毒复制酶分子在过氧化物酶体膜上装配VRC。细胞与体内的金盐一起温育,MT-金复制酶分子通过电子显微镜观察。

在这里我们描述了使用METTEM可视化VRC中的TBSV复制酶分子的方案(图2)。该技术与电子断层扫描技术的结合使我们能够研究三维(3D)病毒复制区室中复制酶分子的分布。由于该方法的高灵敏度,我们可以区分病毒复制酶分子在原位的不同聚集状态。该方法可用于检测细菌,酵母和哺乳动物细胞的不同亚细胞位置中的任何感兴趣的蛋白质。此外,这种电子显微镜方法的一个优点是,它可以用于通过可视化包含在完整病毒颗粒或其蛋白质中的MT标签来研究多种细胞类型中的许多不同病毒。正如报道的风疹病毒,Tombusvirus和流感病毒(Risco et al。,2012; de Castro Martin等人)所报道的,该方法揭示了以前未见过的病毒诱导结构。 ,2017;费尔南德斯德卡斯特罗等人,2014年和2017年)。


图2.方案的示意性工作流程将预先生长的转化的酵母细胞在YPG中温育过夜。第二天,在23℃下诱导病毒复制24小时。用裂解酶处理细胞以获得原生质球。原生质球与金盐一起温育以在MT标签中构建纳米团簇。将细胞团粒脱水并包埋在树脂中。串行部分转移到EM网格并通过TEM成像。

关键字:金属标签透射电子显微镜技术, METTEM, 可克隆标签, 病毒复制复合体, 电子断层扫描, 3D电子显微镜技术

材料和试剂

  1. 移液器吸头
  2. 完美的循环(电子显微镜科学,目录号:70945)
  3. 50毫升一次性离心管
  4. Eppendorf管
  5. 无菌移液器
  6. 血清Acrodisc 37mm注射器过滤器(Pall,目录号:4525)

  7. 明胶胶囊1号直径6.5毫米-0.5毫升(TAAB,目录号:C089 / 1)
  8. GEM ®单刃刀片3面0.009英寸/0.23毫米(AccuTec刀片,目录号:62-0179-0000)
  9. 酿酒酵母菌株RS453(ema ade2-1 his3,15 leu2-3,112 trp1-1 ura3 52)和pah1Δnem1Δ(Nem1的SwissProt ID是P38757)(pah1Δ ::TRP1nem1Δ:: RS453的HIS3衍生物)(Choi等人,2011; Barajas等人,2014b)
  10. Zymolyase 20T(Arthrobacter luteus)(Amsbio,目录号:120491-1)
  11. 氯化金(III)≥99.99%(Sigma-Aldrich,目录号:379948)
  12. 戊二醛50%(TAAB,目录号:G015)
  13. 乙醇干燥(Merck,目录号:1009901001)
  14. LR-White树脂中等级丙烯酸树脂(TAAB,目录号:L012)
  15. Bacto TM酵母提取物(BD,Bacto TM,目录号:212750)
  16. Bacto TM蛋白胨(BD,Bacto TM,目录号:211677)
  17. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
  18. 琼脂(Sigma-Aldrich,目录号:05038)
  19. D - (+) - 半乳糖(Sigma-Aldrich,目录号:G0750)
  20. 乙酸锂99.95%(Sigma-Aldrich,目录号:517992)
  21. ssDNA(来自鲑鱼睾丸的脱氧核糖核酸钠盐)(Sigma-Aldrich,目录号:D1626)
  22. PEG MW3350(聚乙二醇)(Sigma-Aldrich,目录号:P4338)
  23. Trizma半硫酸盐(Sigma-Aldrich,目录号:T8379)
  24. 1,4-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:DTT-RO)
    制造商:Roche Diagnostics,目录号:10197777001。
  25. 不含氨基酸的酵母氮碱(Sigma-Aldrich,目录号:Y0626)
  26. 不含色氨酸的酵母合成辍学培养基补充剂(Sigma-Aldrich,目录号:Y1876)
  27. D-山梨糖醇(Sigma-Aldrich,目录号:S1876)
  28. Tris(碱)(Norgen Biotek,目录号:28029)
  29. 多聚甲醛EM(TAAB,目录号:P026)
  30. NaOH
  31. 10倍PBS
  32. 1,4-哌嗪二磺酸,哌嗪-1,4-双(2-乙磺酸),哌嗪-N,N'-双(2-乙磺酸)PIPES(Sigma-Aldrich,目录号:P6757)
  33. 4-(2-羟乙基)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)(HEPES)(Sigma-Aldrich,目录号:H3375)
  34. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  35. 乙二醇 - 双(2-氨基乙基醚)-N,N,N',N' - 四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  36. YPD(见食谱)
  37. YPG(见食谱)
  38. 转化组合(见食谱)
  39. TSD还原缓冲液(见食谱)
  40. 原生质球培养基A(见食谱)
  41. 4%多聚甲醛(PFA)溶液(见食谱)
  42. PHEM溶液,pH 6.9(见食谱)

设备

  1. 500毫升烧瓶
  2. 移液器
  3. 钻石刀45°DIATOME(Fedelco)
  4. QUANTIFOIL ® R 3.5 / 1 100多孔碳膜网格Au 300 mesh(Quantifoil)
  5. 配备摇床的Oven Memmert UN 55(Memmert,型号:UN55)(Genesys)
  6. 分光光度计722N(Terra Universal,实验室设备,型号:722N)
  7. 离心机5810 R(Eppendorf,型号:5810 R)
  8. 离心miniSpin plus(Eppendorf,型号:MiniSpin ®)加上)
  9. 超声波清洗机1510(布兰森,型号:1510)
  10. pH计Basic 20(HACH LANGE SPAIN,Crison,型号:Basic 20)
  11. 通风柜(Flow-Tronic)
  12. 超薄切片机(徕卡显微系统,型号:Leica EM UC6)

  13. 在100 kV下运行的Jeol JEM 1011电子显微镜(JEOL,型号:JEM-1011)
  14. FEI Tecnai G2 F20(200 kV)电子显微镜(FEI)
  15. Tecnai Spirit Twin(120 kV)电子显微镜(FEI)

软件

  1. IMOD软件。
  2. Amira软件

程序

  1. 酿酒酵母菌株的转化
    1. 从酵母提取物蛋白胨右旋糖(YPD)琼脂平板上的甘油储备物中筛选酵母菌株。在30°C培养细胞1-2天。接种10 ml YPD溶液,并加入带有环的单菌落。
      在30°C以250转/分钟(OD约2)培养过夜。

    2. 在50ml YPD培养基中过夜培养至OD 0.2-0.3 注意:使用500毫升烧瓶。
    3. 在30℃,250转/分的条件下培养3-4小时。
    4. 在室温下将细胞在3,590×g下离心5分钟。使用50毫升一次性离心管。
    5. 弃去上清液并加入1ml无菌dH 2 O,轻轻涡旋,然后用无菌dH 2 O充满管。
    6. 将细胞在3,590×g下离心5分钟。使用50毫升一次性离心管。
    7. 丢弃上清液,并在1毫升100mM醋酸锂中重悬沉淀。
    8. 将细胞转移到Eppendorf管中并以3,590×gg离心2分钟。弃去上清液并将细胞沉淀溶解在0.4ml 100mM乙酸锂中。在室温下孵育10-15分钟。
      注意:使用无菌移液管。

    9. 添加1-5微升质粒DNA(5-10微克)到每个新鲜的Eppendorf管
    10. 弥补转化的主要组合(见食谱)。
    11. 向含有质粒的每个Eppendorf管中加入0.4ml转化混合物。轻轻涡旋2秒以混合组分。
      注意:确保酵母细胞重新悬浮。

    12. 在30°C孵育细胞(不摇动)30分钟
    13. 在42°C孵育细胞(不摇动)40分钟。
    14. 在570gxg离心细胞2分钟。弃去上清液并加入100μl无菌dH 2 O 2。轻轻地上下吸取分散沉淀,并用合适的选择培养基将细胞分配到琼脂平板上。
      在30°C孵育平板2-3天。
      注意:尝试用几个笔划传播单元格。

  2. 细胞培养
    1. 在2ml酵母提取物蛋白胨半乳糖(YPG)中预培养酵母并在30℃和250rpm下孵育过夜。
    2. 在YPG中于23℃和250rpm下在锥形瓶中培养50ml酵母细胞24小时。
      注意:这些条件对诱导和维持病毒复制是必需的。
    3. 在600nm(OD 600nm)下测量和控制光密度,并且当OD 600约为2个收获细胞时,通过离心5分钟,4,000×gg / ,室温下,小心取出上清液。
    4. 在含有二硫苏糖醇(DTT)(TSD)还原缓冲液的Tris-硫酸盐中以5至10OD 600个单位/ ml重悬细胞并在室温下孵育10分钟。
      注意:
      1. DTT通过破坏二硫键促进细胞壁消化,并使β-葡聚糖键更易接近裂解酶中存在的β-葡聚糖酶活性。
      2. 如果细胞在不同的OD处理,实验结果将会改变。
    5. 在30°C下用0.1mg / ml的酵母裂解酶20T孵育酵母细胞10分钟以获得原生质体
      注意:
      1. 最佳的酶解浓度和消化时间取决于酵母菌株和生长条件。设置酵素裂解酶处理对于充分保存细胞超微结构至关重要。
      2. 在发酵裂解酶处理之前,在超声波清洗器中超声处理细胞5秒以避免酵母聚集是很重要的。
    6. 在280gxg离心原生质球5分钟并除去上清液。
    7. 用冰冷的原生质球培养基A洗涤3次。

  3. 黄金治疗和固定
    1. 在室温下在黑暗中用原生质球培养基A中的2mM HAuCl 4 4孵育活的原生质球75分钟。
      注意:
      1. 4 解决方案,溶解于避光的蒸馏水和无菌水中。
      2. MT结合金原子并构建TEM可见的纳米团簇。
      3. 由于金盐在真核细胞中的潜在毒性,调整黄金处理条件非常重要。
    2. 用原生质球培养基A清洗细胞。
    3. 用PHEM溶液中的4%多聚甲醛和0.2%戊二醛在室温下固定细胞1小时。
    4. 固定后用PHEM洗涤原生质球3次。

  4. 嵌入
    1. 在10分钟步骤中脱水细胞,在4℃下用1ml乙醇(30,50,70,90%和100%两次)浓度递增。
      步骤之间的离心是不必要的。
    2. 在室温下将细胞在乙醇-LR-白色树脂(2:1,1:1,1:2)的混合物中在摇动振荡器中各自孵育1小时并避光。
      注意:请勿将LR-White树脂暴露在光线下,并在通风良好的通风橱内操作。
    3. 从乙醇 - 树脂混合物中取出细胞,并将它们置于100%树脂中24小时。

    4. 在明胶胶囊中于60°C下聚合48小时。

  5. 超薄切片

    1. 除去明胶胶囊并用剃刀修剪样本制作空中飞人。
    2. 在300目Quantifoil多孔碳网格上收集50-60 nm超薄切片。
    3. 在透射电子显微镜下观察标称放大倍数为30,000-50,000倍的切片。
      注意:通过透射电子显微镜观察切片而不染色,以避免掩盖小型MT-金纳米团簇。

  6. 电子断层扫描和图像处理
    1. 获取半厚切片(〜300 nm)并将它们收集到Quantifoil网格上。
    2. 在FEI Tecnai G2 F20和Tecnai Spirit Twin显微镜上分别以-60°至+ 60°的角度范围以1.5°的增量自动获取倾斜系列,加速电压分别为200和120 kV。
    3. 使用IMOD软件对齐倾斜系列和层析重建。
    4. 对于断层扫描分割和三维重建,使用Amira软件。
      注意:降噪和自动分割软件有助于突出膜的可视化。

数据分析

我们使用METTEM方法在酵母细胞中可视化Tombusvirus复制酶p33。图3中的电子显微镜成像显示p33-MT-金分子在与过氧化物酶体衍生的多泡体(MVB)相容的膜室中的精确定位,所述多泡体是植物和酵母细胞中的Tombusvirus复制细胞器(Barajas等人。,2014a)。采用这种方法,我们可以在复制平台内的ER膜中检测p33-金属硫蛋白 - 金纳米颗粒(图3A和3B)。


图3. METTEM可视化p33-MT-金分子切片未被染色,以避免掩盖MT-金纳米簇。 A.在tombusvirus复制区室内,p33-MT-金纳米颗粒(〜1nm)描绘了膜(箭头)。单个MT群集标有箭头。 B.具有p33-MT-金纳米簇内部(箭头)和周围内质网(ER)膜的Tombusvirus复制平台。比例尺= 200纳米。

为了更精确的细节,我们用电子断层扫描研究了三维病毒复制细胞器(图4)。断层图显示了病毒复制复合物中p33-MT-金分子的内部组织。病毒复制酶分子分布在复制隔室不同区域中的多种聚集状态(图4)(Fernandez de Castro等人,2014和2017)。


图4.病毒复制细胞器的电子断层成像A.复制平台的3D模型。以黄色显示膜,红色显示p33-MT-金分子(箭头)。 B.对应于复制平台的计算断层切片显示在A中。箭头指向膜内p33-MT-金分子的聚集体。纳米团簇的近似直径为1纳米。比例尺= 100纳米。

笔记

  1. 分析感兴趣的蛋白质的序列,以确定插入MT标签的最佳位点。另外,测试MT-标记的蛋白质活性和功能是否受到影响是相关的。
  2. 调整系统中表达MT标记蛋白质的最佳条件。
  3. 进行初步的EM研究以熟悉细胞类型的超微结构。这将有助于数据解释。
  4. 检查金盐不会在待处理细胞的培养基中沉淀。
  5. 用金盐孵育没有MT标记蛋白质的对照细胞并可视化电子显微镜以验证缺乏非特异性背景。
  6. 在没有染色剂的情况下使用丙烯酸树脂(如LR White或Lowicryl)以显示MT-纳米簇。
  7. 用特异于MT-标记的蛋白质的抗体进行免疫金标记以确认标记特异性。

食谱

  1. YPD
    10克酵母提取物
    20克蛋白胨
    20克葡萄糖(葡萄糖)
    将dH <2> O加入1000毫升并充分混合
    高压灭菌器消毒
    对于平板添加20克琼脂/ L
  2. YPG
    10克酵母提取物
    20克蛋白胨
    20克半乳糖
    将dH <2> O加入1000毫升并充分混合
    高压灭菌器消毒
    对于平板添加20克琼脂/ L
  3. 转型组合
    0.36毫升1M乙酸锂
    0.1ml 10mg / ml ssDNA
    0.69毫升dH 2 O 0 2.4毫升50%PEG MW3350
    将dH <2> O添加至10 ml
  4. TSD减缓缓冲区
    0.1MTrizma半硫酸盐,pH 9.4
    10 mM DTT(在使用前添加)
  5. 原生质球培养基A

    1x酵母氮基 2%(w / v)葡萄糖
    1x氨基酸
    1 M山梨醇
    20mM Tris-HCl,pH7.5

    在室温下储存4周
  6. 4%多聚甲醛(PFA)溶液

    1. 在60°C下将4 g PFA溶于90 ml蒸馏水中
    2. 加入1N氢氧化钠溶液直至溶液透明并且没有沉淀
    3. 然后取出加热并在冰上冷却。
    4. 加入10毫升的10x PBS,并加入蒸馏水至100毫升
    5. 控制温度并避免将溶液加热到70°C以上,因为PFA会在较高温度下分解。

    6. 用37毫米注射器过滤器过滤溶液
    7. 立即使用该溶液或在4°C或-20°C下储存1天
  7. PHEM溶液,pH 6.9
    20 mM PIPES
    50 mM HEPES
    20 mM EGTA
    4mM MgCl 2 2/2
    在4°C储存数月

致谢

我们要感谢JoséJesúsFernández和Peter Nagy博士提供专家建议和有益的讨论。这项工作得到了西班牙经济和竞争力部(MINECO-AEI / FEDER,EU)授予BIO2015-68758-R(至C.R.)的资助。该协议改编自Fernandez de Castro et。(2017)。作者宣称没有利益冲突。

参考

  1. Barajas,D.,Martin,I.F.,Pogany,J.,Risco,C.和Nagy,P.D。(2014a)。 宿主Vps4 AAA + ATPase ESCRT蛋白在番茄丛矮病毒形成中的非规范作用复制酶
  2. Barajas,D.,Xu,K.,de Castro Martin,I. F.,Sasvari,Z.,Brandizzi,F.,Risco,C.和Nagy,P. D.(2014b)。 选择性氧化甾醇结合的ORP和VAP蛋白通过膜接触位点将甾醇引导至RNA病毒复制位点。 PLoS Pathog 10(10):e1004388。
  3. Bouchet-Marquis,C.,Pagratis,M.,Kirmse,R.和Hoenger,A。(2012)。 金属硫蛋白作为冷冻电子显微镜的可克隆高密度标记。 J Struct Biol 177(1):119-127。
  4. Choi,H. S.,Su,W. M.,Morgan,J. M.,Han,G. S.,Xu,Z.,Karanasios,E.,Siniossoglou,S.and Carman,G.M。(2011)。 磷酸化磷酸酶在酿酒酵母中调节其膜结合和生理功能 :作为由CDC28(CDK1)编码的细胞周期蛋白依赖性激酶磷酸化的位点的SER 602,THR 723和AND SER 744的鉴定 / J> J Biol Chem 286(2):1486-1498。
  5. de Castro,I. F.,Volonte,L.和Risco,C。(2013)。 病毒工厂:生物发生和结构设计 Cell Microbiol 15(1):24-34。
  6. de Castro Martin,I. F.,Fournier,G.,Sachse,M.,Pizarro-Cerda,J.,Risco,C.,&amp; Naffakh,N.(2017)。 流感病毒基因组通过改良的内质网和Rab11依赖性囊泡到达质膜。 / a> Nat Commun 8(1):1396.
  7. Delebecque,C. J.,Lindner,A. B.,Silver,P.A。和Aldaye,F.A。(2011)。 用合理设计的RNA装配来组织细胞内反应。 科学 > 333(6041):470-474。
  8. den Boon,J.A。,Diaz,A。和Ahlquist,P。(2010)。 细胞质病毒复制复合物
  9. Diestra,E.,Fontana,J.,Guichard,P.,Marco,S.和Risco,C。(2009)。 用电子显微镜观察可克隆标记的完整细胞中的蛋白质 J Struct Biol 165(3):157-168。
  10. Fernandez de Castro,I.,Fernandez,J. J.,Barajas,D.,Nagy,P. D.和Risco,C。(2017)。 功能性病毒RNA复制酶复合物细胞内组装的三维成像。 < J Cell Sci 130(1):260-268。
  11. Fernandez de Castro,I.,Sanz-Sanchez,L.和Risco,C。(2014)。 用于相关光和电子显微镜检查的金属硫蛋白。方法细胞生物学 124:55-70。
  12. Jonczyk,M.,Pathak,K.B.,Sharma,M.和Nagy,P.D。(2007)。 利用替代亚细胞定位进行复制:在没有过氧化物酶体的情况下,tombusvirus复制转换到内质网。< / a> 病毒学 362(2):320-330。
  13. McCartney,A.W.,Greenwood,J.S.,Fabian,M.R.,White,K.A。和Mullen,R.T。(2005)。 番茄丛矮病毒复制蛋白p33的定位揭示了过氧化物酶体 - 内质网分选途径。 Plant Cell 17(12):3513-3531。
  14. Mercogliano,C.P。和DeRosier,D.J。(2006)。 使用金属硫蛋白形成金纳米簇:质谱和电子显微镜 J Mol Biol 355(2):211-223。
  15. Mercogliano,C.P。和DeRosier,D.J。(2007)。 串联金属硫蛋白作为电子显微镜的可克隆金标签。 J Struct Biol 160(1):70-82。
  16. Miller,S.和Krijnse-Locker,J。(2008)。 修改病毒复制的细胞内膜结构 Nat Rev Microbiol 6(5):363-374。
  17. Nagy,P.D。(2008)。 作为模型寄主探索植物病毒与宿主的相互作用。 Annu Rev Phytopathol 46:217-242。
  18. Nagy,P.D。(2016)。 Tombusvirus与宿主的相互作用:选择进化上保守的宿主因子将成为中心法庭。 Annu Rev Virol 3(1):491-515。
  19. Nagy,P.D.和Pogany,J。(2011)。 病毒RNA复制依赖于选择的宿主因子 Nat Rev Microbiol em> 10(2):137-149。
  20. Risco,C.,Sanmartin-Conesa,E.,Tzeng,W.P.,Frey,T.K。,Seybold,V。和de Groot,R.J。(2012)。 使用金属标记透射电子显微镜对真核细胞中蛋白质分子的特异性,敏感性,高分辨率检测。结构 20(5):759-766。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:de Castro, I. F. and Risco, C. (2018). Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast. Bio-protocol 8(8): e2822. DOI: 10.21769/BioProtoc.2822.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。