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

Basic information

• Entry: Database: EMDB / ID: EMD-1273
• Title: Centrosome polarization delivers secretory granules to the immunological synapse.
• Map data: Tomogram 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 species: Homo sapiens (human) / Mus musculus (house mouse)
• Method: electron tomography / negative staining / Resolution: 50.0 Å
• Authors: Stinchcombe JC / Majorovits E / Bossi G / Fuller SD / Griffiths GM
• Citation: Journal: 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 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

Deposition	Oct 9, 2006	-
Header (metadata) release	Oct 10, 2006	-
Map release	Oct 10, 2006	-
Update	Dec 26, 2012	-
Current status	Dec 26, 2012	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_1273.map.gz / Format: CCP4 / Size: 296.9 MB / Type: IMAGE STORED AS SIGNED BYTE
• Annotation: Tomogram 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.)
• Voxel size: X=Y=Z: 22.5 Å

Density

Minimum - Maximum	-125.0 - 127.0
Average (Standard dev.)	12.024714469999999 (±13.401159290000001)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 2048 2048 76 Spacing 2048 2048 76
Cell	A: 46080.0 Å / B: 46080.0 Å / C: 1710.0 Å α=β=γ: 90.0 °

CCP4 map header:

mode	envelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z	22.5	22.5	22.5
M x/y/z	2048	2048	76
origin x/y/z	0.000	0.000	0.000
length x/y/z	46080.000	46080.000	1710.000
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-184	-184	-183
NX/NY/NZ	368	368	368
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	2048	2048	76
D min/max/mean	-125.000	127.000	12.025

Supplemental data

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

• Annotation: 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
• File: emd_1273_msk_1.map

Segmentation: One of the two CTL centrioles, sitting close...

• Annotation: 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)
• File: emd_1273_msk_2.map

Segmentation: The lytic granules are the "cytotoxic organelles" of...

• Annotation: 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
• File: emd_1273_msk_3.map

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

• Annotation: A CTL Golgi apparatus, sitting close to the synaptic cleft. The Golgi apparatus is polarized to the cell-cell contact together with the centrosome
• File: emd_1273_msk_4.map

Segmentation: Synaptic cleft, i.e. space between the CTL and...

• Annotation: 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
• File: emd_1273_msk_5.map

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.

Sample components

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

• Entire: Name: 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

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

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

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

• Supramolecule: Name: 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

Supramolecule #2: P815 mouse target cell

• Supramolecule: Name: 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

Experimental details

Structure determination

• Method: negative staining
• Processing: electron tomography

Sample preparation

• Buffer: Details: RPMI medium
• Staining: Type: 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-cytochemistry and post-fixed with reduced osmium. The serial section was stained with lead citrate for 8 min.
• Vitrification: Cryogen name: NONE

Electron microscopy

• Microscope: FEI TECNAI F30
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 6200 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 5.2 µm / Nominal defocus min: 4.8 µm / Nominal magnification: 5600
• Specialist optics: Energy filter - Name: GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
• Sample stage: Specimen 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 °
• Details: double tilt series
• Date: Nov 8, 2005
• Image recording: Category: CCD / Film or detector model: GENERIC GATAN (2k x 2k)
• Experimental equipment: Model: Tecnai F30 / Image courtesy: FEI Company

Image processing

• Final reconstruction: Algorithm: 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
• Details: double tilt series