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- EMDB-1273: Centrosome polarization delivers secretory granules to the immuno... -

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Entry
Database: EMDB / ID: EMD-1273
TitleCentrosome polarization delivers secretory granules to the immunological synapse.
Map dataTomogram of the immunological synapse between a cytotoxic T lymphocyte (CTL) and a target cell. 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. (See the masks representing models of the synaptic cleft, microtubules, centriole, lytic granules and Golgi apparatus.)
Sample
  • Sample: Immunological synapse between a cytotoxic T lymphocyte and a target cell
  • Organelle or cellular component: human CD8 CTL clone, PK-1
  • Organelle or cellular component: P815 mouse target cell
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodelectron tomography / negative staining / Resolution: 50.0 Å
AuthorsStinchcombe JC / Majorovits E / Bossi G / Fuller SD / Griffiths GM
CitationJournal: Nature / Year: 2006
Title: Centrosome polarization delivers secretory granules to the immunological synapse.
Authors: Jane C Stinchcombe / Endre Majorovits / Giovanna Bossi / Stephen Fuller / Gillian M Griffiths /
Abstract: Cytotoxic T lymphocytes (CTLs) destroy virally infected and tumorigenic cells by releasing the contents of specialized secretory lysosomes--termed 'lytic granules'--at the immunological synapse ...Cytotoxic T lymphocytes (CTLs) destroy virally infected and tumorigenic cells by releasing the contents of specialized secretory lysosomes--termed 'lytic granules'--at the immunological synapse formed between the CTL and the target. On contact with the target cell, the microtubule organizing centre of the CTL polarizes towards the target and granules move along microtubules in a minus-end direction towards the polarized microtubule organizing centre. However, the final steps of secretion have remained unclear. Here we show that CTLs do not require actin or plus-end microtubule motors for secretion, but instead the centrosome moves to and contacts the plasma membrane at the central supramolecular activation cluster of the immunological synapse. Actin and IQGAP1 are cleared away from the synapse, and granules are delivered directly to the plasma membrane. These data show that CTLs use a previously unreported mechanism for delivering secretory granules to the immunological synapse, with granule secretion controlled by centrosome delivery to the plasma membrane.
History
DepositionOct 9, 2006-
Header (metadata) releaseOct 10, 2006-
Map releaseOct 10, 2006-
UpdateDec 26, 2012-
Current statusDec 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

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Structure viewerEM map:
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Supplemental images

1273.gif

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Map

FileDownload / File: emd_1273.map.gz / Format: CCP4 / Size: 296.9 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationTomogram of the immunological synapse between a cytotoxic T lymphocyte (CTL) and a target cell. 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. (See the masks representing models of the synaptic cleft, microtubules, centriole, lytic granules and Golgi apparatus.)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
22.5 Å/pix.
x 76 pix.
= 1710. Å		22.5 Å/pix.
x 2048 pix.
= 46080. Å		22.5 Å/pix.
x 2048 pix.
= 46080. Å

Projections

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Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 22.5 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-125.0 - 127.0
Average (Standard dev.)12.024714469999999 (±13.401159290000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions2048204876
Spacing2048204876
CellA: 46080.0 Å / B: 46080.0 Å / C: 1710.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z22.522.522.5
M x/y/z2048204876
origin x/y/z0.0000.0000.000
length x/y/z46080.00046080.0001710.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS2048204876
D min/max/mean-125.000127.00012.025

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Supplemental data

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Segmentation: Microtubule (MT) network within the CTL. The MTs...

AnnotationMicrotubule (MT) network within the CTL. The MTs radiate out from the polarized centrosomal area, running both away from the plasma membrane into the cell and along the cell membrane to the periphery of the synapse. Both lytic granules (see mask granules_mask4) and the Golgi apparatus (see mask golgi_mask5) are linked to and transported along the MTs to the synaptic cleft
Fileemd_1273_msk_1.map
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Segmentation: One of the two CTL centrioles, sitting close...

AnnotationOne of the two CTL centrioles, sitting close to the synaptic cleft. The two centrioles form part of the centrosome that is polarized to the synaptic cleft. Microtubules run from here into the cell and along the cell membrane to the periphery of the synapse (see mask MTs_mask)
Fileemd_1273_msk_2.map
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Segmentation: The lytic granules are the "cytotoxic organelles" of...

AnnotationThe lytic granules are the "cytotoxic organelles" of the CTL. They are transported along the MTs in a minus-end fashion towards the polarized centrosome. After docking to the cell membrane they probably fuse with the membrane at the secretory domain and excrete the cytotoxic proteins into the synaptic cleft
Fileemd_1273_msk_3.map
Projections & Slices
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Histogram (log scale)

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Segmentation: A CTL Golgi apparatus, sitting close to the...

AnnotationA CTL Golgi apparatus, sitting close to the synaptic cleft. The Golgi apparatus is polarized to the cell-cell contact together with the centrosome
Fileemd_1273_msk_4.map
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Segmentation: Synaptic cleft, i.e. space between the CTL and...

AnnotationSynaptic cleft, i.e. space between the CTL and the target cell as defined by the opposing membranes. Areas of close membrane-membrane contact (adhesion/signaling domains) and pocket-like areas with larger membrane-membrane distances (secretion domain) are clearly distinguishable
Fileemd_1273_msk_5.map
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Others

Details[README_masks.txt]
Information about the masks which have been taken from the tomogram map emd_1273.map
emd_1273_msk_1.map:mask1_synapse:
Synaptic cleft, i.e. space between the CTL and the target cell as
defined by the opposing membranes. Areas of close membrane-membrane
contact (adhesion/signaling domains) and pocket-like areas with larger
membrane-membrane distances (secretion domain) are clearly
distinguishable.
emd_1273_msk_2.map:mask2_MTs:
Microtubule (MT) network within the CTL. The MTs radiate out from the
polarized centrosomal area, running both away from the plasma membrane
into the cell and along the cell membrane to the periphery of the
synapse. Both lytic granules (see mask granules_mask4) and the Golgi
apparatus (see mask golgi_mask5) are linked to and transported along the
MTs to the synaptic cleft.
emd_1273_msk_3.map:mask3_centriole:
One of the two CTL centrioles, sitting close to the synaptic cleft. The
two centrioles form part of the centrosome that is polarized to the
synaptic cleft. Microtubules run from here into the cell and along the
cell membrane to the periphery of the synapse (see mask MTs_mask).
emd_1273_msk_4.map:mask4_granules:
The lytic granules are the "cytotoxic organelles" of the CTL. They are
transported along the MTs in a minus-end fashion towards the polarized
centrosome. After docking to the cell membrane they probably fuse with
the membrane at the secretory domain and excrete the cytotoxic proteins
into the synaptic cleft.
emd_1273_msk_5.map:mask5_golgi:
A CTL Golgi apparatus, sitting close to the synaptic cleft. The Golgi
apparatus is polarized to the cell-cell contact together with the
centrosome.

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Sample components

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Entire : Immunological synapse between a cytotoxic T lymphocyte and a targ...

EntireName: Immunological synapse between a cytotoxic T lymphocyte and a target cell
Components
  • Sample: Immunological synapse between a cytotoxic T lymphocyte and a target cell
  • Organelle or cellular component: human CD8 CTL clone, PK-1
  • Organelle or cellular component: P815 mouse target cell

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Supramolecule #1000: Immunological synapse between a cytotoxic T lymphocyte and a targ...

SupramoleculeName: Immunological synapse between a cytotoxic T lymphocyte and a target cell
type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: human CD8 CTL clone, PK-1

SupramoleculeName: human CD8 CTL clone, PK-1 / type: organelle_or_cellular_component / ID: 1 / Name.synonym: human CD8 T cell / Details: human CD8 CTL clone derived from healthy donor / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human

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Supramolecule #2: P815 mouse target cell

SupramoleculeName: P815 mouse target cell / type: organelle_or_cellular_component / ID: 2 / Name.synonym: mouse target cell / Details: 0 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / Strain: P815 mouse cell line / synonym: house mouse

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography

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Sample preparation

BufferDetails: RPMI medium
StainingType: NEGATIVE
Details: CTLs were labelled overnight with 1-2mg/ml HRP to load the lytic granules. After 30 min. conjugation to P815 target cells with anti-CD3 antibodies the sample was fixed and processed for DAB- ...Details: CTLs were labelled overnight with 1-2mg/ml HRP to load the lytic granules. After 30 min. conjugation to P815 target cells with anti-CD3 antibodies the sample was fixed and processed for DAB-cytochemistry and post-fixed with reduced osmium. The serial section was stained with lead citrate for 8 min.
VitrificationCryogen name: NONE

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Electron microscopy

MicroscopeFEI TECNAI F30
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Detailsdouble tilt series
DateNov 8, 2005
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (2k x 2k)
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 6200 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 5.2 µm / Nominal defocus min: 4.8 µm / Nominal magnification: 5600
Sample stageSpecimen holder: flip-flop holder / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -68.0 ° / Tilt series - Axis1 - Max angle: 68.0 ° / Tilt series - Axis1 - Angle increment: 1 °
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Detailsdouble tilt series
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 50.0 Å / Resolution method: OTHER / Software - Name: IMOD
Details: dual-axis tomograms were matched and merged to give a final map with reduced missing "cone"
Number images used: 1

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