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Reconstruction
Display image description
Virological synapse formed between HTLV-1 infected CD4+ T-cell (PBMC) and an autologous uninfected CD4+ T-cell as a target cell. These cells were stained against HTLV-1 Gag p19 matrix protein with a specific monoclonal antibody (GIN7). A large immunolabeled HTLV-1 particle is visible between the two cell membranes. See Figure 4D of Majorovits et al., 2008.
Full resolution image description
Tomographic reconstruction in MRC format. Filename = map_3948.ccp4
Volume_dimension
1736, 760, 243
Volume scale
0.00129, 0.00129, 0.00129
Animation description
Animation through computed slices of tomographic reconstruction of a virilogical synapse formed between HTLV-1 infected CD4+ T-cell (PBMC) and an autologous uninfected CD4+ T-cell as a target cell. These cells were stained against HTLV-1 Gag p19 matrix protein with a specific monoclonal antibody (GIN7). A large immunolabeled HTLV-1 particle is visible between the two cell membranes.

License
Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:3948*
Project: P1720
Project name
Electron tomography of the HTLV-1 virological synapse
Description
Tomography of the virological synapse formed between an HTLV-1 infected cells and target cells.
Funding agency
Wellcome Trust
Leader(s)
Endre Majorovits
Collaborator(s)
Charles Bangham
Stephen Fuller
Mohamed Nejmeddine
Start date
02-02-2004
End date
02-02-2004
 
Experiment
Experiment ID
3472
Title
Tomography of naturally infected HTLV-1 virological synapse
Purpose
Study of the HTLV-1 virological synapse in naturally infected T cells using electron tomography and immunocytochemistry of viral protein
Experimenter(s)
Endre Majorovits
Mohamed Nejmeddine
Microscopy product
Microscopy product ID
3948
Instrument
FEI Tecnai F30 FEG
Microscopy type
TEM
Product type
SINGLE TILT
Image basename
HTLV1_VS_human_3
Spatial Axis Image Size Pixel Size
X 690px 3.36 nm/pixels
Y 607px 3.36 nm/pixels
Subject
Species
human
Scientific name
homo sapiens
Strain
sapiens
Group by
viral infection
Treatment
HTLV-1 seropositive patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP)
Age class
adult
Tissue section
Anatomical location
blood
Microtome
ultramicrotome
Thickness
0.2 µm
Specimen description
System
blood
Structure
virological synapse
Cell type
CD4+ T cell
Imaging parameters
Type
Electron microscopy product
Recording medium
Slow scan cooled 2K CCD camera
Magification
10
Energy filter type
Gatan Energy Filter
Energy filter slit
20-25 eV
Energy filter type
Gatan Energy Filter
Accelerating voltage
300 KeV
Notes
Did not specify in column or end of column for GIF
Specimen preparation
Protocol used
The PBMCs were isolated by density gradient centrifugation on Histopaque-1077 (Sigma-Aldrich Company Ltd, Dorset, UK), washed twice with phosphate buffered saline (PBS) and washed once in PBS/10% FCS. CD4+T cells were negatively selected from the PBMCs using the CD4+ T cell isolation Kit from Miltenyi Biotech, Surrey, UK, following the manufacturer's instructions. This procedure yielded CD4+ cells at a purity of greater than 90%, ascertained by flow cytometry (data not shown). Before use, the isolated CD4+ T cells were cultured overnight in 10 cm diameter tissue culture dishes (106 cells/ml), to allow spontaneous expression of HTLV-1 proteins [30]. During this incubation, the cells were widely dispersed to minimize cell-cell contact. Before processing for EM the cells were gently centrifuged for 5 minutes at 300 g to induce cell-cell conjugates and then incubated for another 1 h at 37uC and 5% CO2. T cells were cultured in RPMI 1640 medium (Sigma-Aldrich Company Ltd, Dorset, UK) supplemented with 2 mM glutamine (Invitrogen Ltd, Paisley, UK), 100 IU/ml penicillin (Invitrogen Ltd, Paisley, UK), 100 IU/ml streptomycin (Invitrogen Ltd, Paisley, UK) and 10% heat-inactivated fetal calf serum (PAA Laboratories Ltd, Somerset, UK).Samples were prepared with different buffers and different amounts of fixatives, depending on whether they were destined to be immunostained with the mAb anti-Gag p19 (GIN7) or to be stained with magnesium uranyl acetate (Sigma-Aldrich Company Ltd, Dorset, UK). In general, the fixation of the samples was performed sequentially in two distinct fixative solutions: Solution A, 2% paraformaldehyde (PFA) (Electron Microscopy Science, Hatfield, PA, USA) and 0.5% glutaraldehyde (Agar Scientific Ltd, Essex, UK) in sodium cacodylate buffer 0.1 M, pH 7.2 (Sigma-Aldrich Ltd, Dorset, UK); and Solution B, 1% osmium tetroxide (OsO4) in PBS (Sigma-Aldrich Ltd, Dorset, UK).After incubating the samples to induce cell-cell conjugates, they were pre-fixed in the 10X diluted PFA/glutaraldehyde (Solution A) for 10 minutes before centrifugation. After centrifuging for 5 minutes at 1000 g the samples were fixed for another hour with the undiluted fixative (Solution A). The samples were washed 3 times either with PBS 1% BSA or with sodium cacodylate buffer 0.1 M, pH 7.2, and then processed for antibody staining as described below or post-fixed with 1% OsO4 (Solution B) and stained in magnesium uranyl acetate overnight, respectively.The fixed samples were dehydrated through a series of ethanol exchanges and embedded in Agar 100 resin (Agar Scientific Ltd., Stansted, UK). The samples were baked at 60uC overnight to give solid blocks that were sectioned with an ultramicrotome (Leica Ultracut UCT, Leica Microsystems GmbH, Wetzlar, Germany) at a thickness of 60 to 300 nm. Thin samples (60 to 100 nm) were floated onto 200/300 mesh square Ni grids (Agar Scientific Ltd., Stansted, UK). Thick samples for tomography (200 to 300 nm) were either floated onto 200 mesh square Ni grids or onto formvar film coatedCu 162 mm slot grids (Agar Scientific Ltd., Stansted, UK) to allow for the collection of serial sections. Samples that were not antibody labelled were stained with lead citrate (Leica Ultrostain2) for 3 to 10 min depending on the thickness of the sections, by floating the grids on drops of the staining solution. For electron tomography the grids were covered with 10 nm or 15 nm fiducial gold beads (Sigma-Aldrich Company Ltd, Dorset, UK) to facilitate image processing.To detect HTLV-1 Gag matrix protein in infected cells the samples were labelled against HTLV-1 Gag p19 (Tanaka et al., 1986) using the DAB kit from Molecular Probes (Invitrogen Ltd, Paisley, UK ). After the PFA/glutaraldehyde fixation, the samples were incubated in PBS containing 1% BSA, 0.1% Triton X-100 and 2% H2O2, for 1 h at 37uC to block non-specific antibody binding and to quench the endogenous peroxidase activity of the cells. To detect the HTLV-1 complexes, the cells were incubated with 5 mg/ml of anti-Gag p19 (GIN7 mAb) [33] diluted in PBS 1% BSA, for 1 h at 37uC. Then the second antibody, anti-Mouse mAb conjugated to HRP (Invitrogen Ltd, Paisley, UK ), was added at a final dilutionof 1 mg/ml, and incubated for 30 minutes at 37uC. The cells were washed three times with PBS containing 1% BSA between each step.The sample was centrifuged and embedded into a 1% agarose gel (Sigma-Aldrich Ltd, Dorset, UK). A 1 mg/ml DAB solution (Invitrogen Ltd, Paisley, UK ) diluted in PBS/1%BSA was added to the samples and incubated for 15 minutes at room temperature with gentle shaking. The staining was detected by adding to the samples 0.03% H2O2 diluted in PBS/1%BSA. The samples turned brown within 30 s to 5 min and the reaction was then stopped by washing extensively with PBS. Between each procedure the cells were centrifuged at 1000 g and washed thoroughly at least three times in PBS. The samples were finally fixed for at least 5 minutes in a 1% OsO4 solution (samples turned black) and washed 5 times before further processing.
Imaging product type
Type
Single tilt
Description
singlet_desc
Min range
-65 degrees
Max range
65 degrees
Tilt increment
1 degrees


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