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Rab27a is required for regulated secretion in cytotoxic T lymphocytes.

Stinchcombe JC, Barral DC, Mules EH, Booth S, Hume AN, Machesky LM, Seabra MC, Griffiths GM - J. Cell Biol. (2001)

Bottom Line: In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity.Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A.These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, United Kingdom.

ABSTRACT
Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the alpha subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

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Lytic granules polarize with the Golgi complex but do not dock with the plasma membrane in ash/ash CTLs. Semithick (150–200 nm; a and e) and thin (50–60 nm; b–d and f) stained sections of +/ash (a–c) and ash/ash (d–e) CTLs (left) conjugated to P815 target cells (right). In both cells the Golgi complex (G) polarizes to the CTL plasma membrane at the immunological synapse. In both cell types the lytic granules (LG) have a heterologous appearance. In +/ash CTLs the lytic granules are either closely associated with the plasma membrane (e.g., a) or are absent (e.g., c) because the cells have already exocytosed, whereas in ash/ash CTLs (d–f) they accumulate behind the Golgi complex but fail to dock with the membrane. Note in b–c and e–f that in both cell types the CTLs and P815 cell plasma membranes are tightly aligned in some regions of the contact membrane (arrows) but that there is a gap between the two membranes directly opposite the polarized Golgi complex, probably corresponding to the talin hole. This gap contains crystalline and membranous material similar to that stored in lytic granules in +/ash cells but lacks it in ash/ash cells. Note in the sections shown in a and d in the target cells, endocytic structures (e) also appear to polarize to the contact sites. Bar, 500 nm.
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Figure 5: Lytic granules polarize with the Golgi complex but do not dock with the plasma membrane in ash/ash CTLs. Semithick (150–200 nm; a and e) and thin (50–60 nm; b–d and f) stained sections of +/ash (a–c) and ash/ash (d–e) CTLs (left) conjugated to P815 target cells (right). In both cells the Golgi complex (G) polarizes to the CTL plasma membrane at the immunological synapse. In both cell types the lytic granules (LG) have a heterologous appearance. In +/ash CTLs the lytic granules are either closely associated with the plasma membrane (e.g., a) or are absent (e.g., c) because the cells have already exocytosed, whereas in ash/ash CTLs (d–f) they accumulate behind the Golgi complex but fail to dock with the membrane. Note in b–c and e–f that in both cell types the CTLs and P815 cell plasma membranes are tightly aligned in some regions of the contact membrane (arrows) but that there is a gap between the two membranes directly opposite the polarized Golgi complex, probably corresponding to the talin hole. This gap contains crystalline and membranous material similar to that stored in lytic granules in +/ash cells but lacks it in ash/ash cells. Note in the sections shown in a and d in the target cells, endocytic structures (e) also appear to polarize to the contact sites. Bar, 500 nm.

Mentions: To address this possibility, we examined the localization of granules in +/ash and ash/ash CTLs conjugated to targets by EM. Fig. 5 shows the immunological synapse in +/ash (Fig. 5, a–c) and ash/ash (Fig. 5, d–f) conjugates. The Golgi complex is seen polarized very close to the site of membrane contact (Fig. 5b, Fig. c, Fig. e, and Fig. f). Intriguingly, a central cleft is formed between two areas of tight membrane contact in both ash/ash and +/ash conjugates. Two important differences are observed between wild-type and mutant conjugates. First, in +/ash conjugates, granules are found docked at the plasma membrane (Fig. 5 a), whereas in ash/ash CTL conjugates, the granules do not dock at the membrane but rather line up behind the Golgi complex (Fig. 5 d). Second, the cleft at the contact site is filled with electron-dense and vesicular material in the +/ash conjugates consistent with the secretion of granule contents into this space. However, the cleft is devoid of content in the ash/ash conjugates (Fig. 5e and Fig. f), indicating that the granule contents are not secreted. Curiously, endosomes from the target cell also appear to be recruited to the target side of the contact site in both strains (Fig. 5, a and d).


Rab27a is required for regulated secretion in cytotoxic T lymphocytes.

Stinchcombe JC, Barral DC, Mules EH, Booth S, Hume AN, Machesky LM, Seabra MC, Griffiths GM - J. Cell Biol. (2001)

Lytic granules polarize with the Golgi complex but do not dock with the plasma membrane in ash/ash CTLs. Semithick (150–200 nm; a and e) and thin (50–60 nm; b–d and f) stained sections of +/ash (a–c) and ash/ash (d–e) CTLs (left) conjugated to P815 target cells (right). In both cells the Golgi complex (G) polarizes to the CTL plasma membrane at the immunological synapse. In both cell types the lytic granules (LG) have a heterologous appearance. In +/ash CTLs the lytic granules are either closely associated with the plasma membrane (e.g., a) or are absent (e.g., c) because the cells have already exocytosed, whereas in ash/ash CTLs (d–f) they accumulate behind the Golgi complex but fail to dock with the membrane. Note in b–c and e–f that in both cell types the CTLs and P815 cell plasma membranes are tightly aligned in some regions of the contact membrane (arrows) but that there is a gap between the two membranes directly opposite the polarized Golgi complex, probably corresponding to the talin hole. This gap contains crystalline and membranous material similar to that stored in lytic granules in +/ash cells but lacks it in ash/ash cells. Note in the sections shown in a and d in the target cells, endocytic structures (e) also appear to polarize to the contact sites. Bar, 500 nm.
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Related In: Results  -  Collection

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Figure 5: Lytic granules polarize with the Golgi complex but do not dock with the plasma membrane in ash/ash CTLs. Semithick (150–200 nm; a and e) and thin (50–60 nm; b–d and f) stained sections of +/ash (a–c) and ash/ash (d–e) CTLs (left) conjugated to P815 target cells (right). In both cells the Golgi complex (G) polarizes to the CTL plasma membrane at the immunological synapse. In both cell types the lytic granules (LG) have a heterologous appearance. In +/ash CTLs the lytic granules are either closely associated with the plasma membrane (e.g., a) or are absent (e.g., c) because the cells have already exocytosed, whereas in ash/ash CTLs (d–f) they accumulate behind the Golgi complex but fail to dock with the membrane. Note in b–c and e–f that in both cell types the CTLs and P815 cell plasma membranes are tightly aligned in some regions of the contact membrane (arrows) but that there is a gap between the two membranes directly opposite the polarized Golgi complex, probably corresponding to the talin hole. This gap contains crystalline and membranous material similar to that stored in lytic granules in +/ash cells but lacks it in ash/ash cells. Note in the sections shown in a and d in the target cells, endocytic structures (e) also appear to polarize to the contact sites. Bar, 500 nm.
Mentions: To address this possibility, we examined the localization of granules in +/ash and ash/ash CTLs conjugated to targets by EM. Fig. 5 shows the immunological synapse in +/ash (Fig. 5, a–c) and ash/ash (Fig. 5, d–f) conjugates. The Golgi complex is seen polarized very close to the site of membrane contact (Fig. 5b, Fig. c, Fig. e, and Fig. f). Intriguingly, a central cleft is formed between two areas of tight membrane contact in both ash/ash and +/ash conjugates. Two important differences are observed between wild-type and mutant conjugates. First, in +/ash conjugates, granules are found docked at the plasma membrane (Fig. 5 a), whereas in ash/ash CTL conjugates, the granules do not dock at the membrane but rather line up behind the Golgi complex (Fig. 5 d). Second, the cleft at the contact site is filled with electron-dense and vesicular material in the +/ash conjugates consistent with the secretion of granule contents into this space. However, the cleft is devoid of content in the ash/ash conjugates (Fig. 5e and Fig. f), indicating that the granule contents are not secreted. Curiously, endosomes from the target cell also appear to be recruited to the target side of the contact site in both strains (Fig. 5, a and d).

Bottom Line: In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity.Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A.These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, United Kingdom.

ABSTRACT
Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the alpha subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

Show MeSH
Related in: MedlinePlus