Alternate header for print version


Reconstruction
Display image description
Tomogram of the immunological synapse between a cytotoxic T lymphocyte (CTL, top) and a target cell (bottom). The microtubule organization centre (MTOC) is polarized to the cell-cell contact site. The electron-dense lytic granules are transported along the microtubules to the synaptic cleft. Here they probably fuse with the membrane and excrete the cytotoxic proteins that trigger the death of the target cell.
Full resolution image description
The tomographic reconstruction is stored as an MRC file (int, 8 bit): CTL_ImmunSynapse_rec.mrc.gz The tomogram has been Gauss-filtered with AMIRA (filter paramerters: 3D, Sigma x=y=z=1, Kernel x=y=z=3) as an MRC file (int, 8 bit): CTL_ImmunSynapse_filter_rec.mrc Segmentation was done using the filtered tomogram. The tar file was created on a LINUX machine: tar -cf CTL_ImmunSynapse_vol.tar *mrc
Volume_dimension
2048, 2048, 76
Volume scale
0.00225, 0.00225, 0.00225
Animation description
Movie showing the tomogram of the immunological synapse between a cytotoxic T lymphocyte (CTL, top) and a target cell (bottom). The microtubule organization centre (MTOC) is polarized to the cell-cell contact site. The electron-dense lytic granules are transported along the microtubules to the synaptic cleft. Here they probably fuse with the membrane and excrete the cytotoxic proteins that trigger the death of the target cell.

Image 2D
Display image description
Projection of the untilted specimen: immunological synapse between a human cytotoxix T lymphocyte (top) and a target cell (bottom).
Full resolution image description
The dual tilt axis series are stored as two MRC files (int, 16 bit): 1. CTL_ImmunSynapse_series_a.mrc, 2. CTL_ImmunSynapse_series_b.mrc The gziped tar file was created on a LINUX machine: tar -czf CTL_ImmunSynapse_img.tar.gz *mrc

Segmentation
Display image description
Segmentation of centriole, microtubules, Golgi apparatus and lytic granules within the CTL and of the synaptic cleft.
Segmentation file description
Segmentation has been done with IMOD and AMIRA. The following objects have been segmented: 1. centriole (in the CTL), 2. microtubules (in the CTL), 3. Golgi apparatus (in the CTL), 4. lytic granules (in the CTL), 5. synaptic cleft (between CTL and target cell). The final model is an IMOD model file: CTL_ImmunSynapse_IMOD.mod.gz (it is gziped on a LIMUX mashine: gzip CTL_ImmunSynapse_IMOD.mod). Masks representing the various objects were generated in MRC format (int, 8 bit): 1. CTL_ImmunSynapse_centriole.mrc.gz, 2. CTL_ImmunSynapse_microtubules.mrc.gz, 3. CTL_ImmunSynapse_Golgi.mrc.gz, 4. CTL_ImmunSynapse_granules.mrc.gz, 5. CTL_ImmunSynapse_synapse.mrc.gz (files are gziped on a LIMUX mashine: gzip CTL_ImmunSynapse_*.mrc). The tar file was created on a LINUX machine: tar -cf CTL_ImmunSynapse_seg.tar CTL_ImmunSynapse_*.gz

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

CCDB:3632*
Project: P1694
Project name
Electron Tomography of the CTL Immunological Synapse
Description
Tomogram of the immunological synapse between a human cytotoxic T lymphocyte (CTL) and a target cell
Funding agency
Wellcome Trust
Leader(s)
Stephen Fuller
Endre Majorovits
Collaborator(s)
Gillian Griffiths
Jane Stinchcombe
Giovanna Bossi
Start date
02-02-2004
End date
unspecified
 
Experiment
Experiment ID
3423
Experiment date
11-08-2005
Title
Structure of the immunological synapse between a human CTL and a target
Purpose
Defining the structure of the immunological synapse between a human cytotoxic T lymphocyte (CTL) and a target cell and assessing the rearrangement of the microtubule organization center (MTOC) and attached organelles within the CTL during target killing.
Experimenter(s)
Endre Majorovits
Microscopy product
Microscopy product ID
3632
Instrument
FEI Tecnai F30
Microscopy type
HVEM
Product type
DOUBLE TILT
Image basename
CTL_ImmunSynapse
Spatial Axis Image Size Pixel Size
X 2048px 2.255 nm/pixels
Y 2048px 2.255 nm/pixels
Subject
Species
human
Scientific name
Homo sapiens
Strain
healthy
Age class
adult?
Tissue section
Anatomical location
blood
Microtome
Leica
Tissue product storage
EM grid
Thickness
0.15 µm
Specimen description
Organ
blood
System
blood
Structure
CTL immunological synapse
Tissue
blood
Cell type
Cytotoxic T Lymphocyte
Imaging parameters
Type
Electron microscopy product
Recording medium
Slow scan cooled 2K CCD camera
Magification
6200
Energy filter type
GIF
Energy filter slit
20 eV
Energy filter type
GIF
Accelerating voltage
300 kV
Specimen preparation
Protocol used
CTLs at 13-16 days after stimulation were labelled overnight in the presence of 1-2mg/ml HRP added directly to the growth medium to load the lytic granules then washed 3 times by pelleting and resuspending in RPMI to remove free HRP and serum and the final pellet resuspended at 5x10^6 cells/ml. CTL were mixed 1:1 with targets. 1mug/ml PHA or anti-CD3 (UCHT-1) was added for conjugation of human CTL. Conjugates were left in suspension at RT for 5 min then plated in individual wells of 4-well plastic tissue culture plates (Nunc) and transferred to 37C for a further 30-60 min. Samples were fixed and processed for DAB-cytochemistry, post-fixed with reduced osmium and EPON embedded. semi-thick were stained with lead citrate and coated with 10nm fiducial gold markers for tomograhpic reconstruction.
Imaging product type
Type
Double tilt
X min range
-68 degrees
X max range
68 degrees
X tilt increment
1 degrees
Y min range
-68 degrees
Y max range
68 degrees
Y tilt increment
1 degrees
Description
Gatan flip-flop holder


Ascb_logo
Ccdb_logo_banner
Nif_button2
Dci_button
Labbyrcw