Rab27a is required for regulated secretion in cytotoxic T lymphocytes.
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.
Stinchcombe
J C
JC
Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, United Kingdom.
Barral
D C
DC
Mules
E H
EH
Booth
S
S
Hume
A N
AN
Machesky
L M
LM
Seabra
M C
MC
Griffiths
G M
GM
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000302:isolation & purification; D000818:Animals; D002402:Cathepsin D; D002462:Cell Membrane / Q000648:ultrastructure; D016764:Cell Polarity; D003594:Cytoplasmic Granules / Q000648:ultrastructure; D003598:Cytoskeletal Proteins; D006056:Golgi Apparatus / Q000648:ultrastructure; D053804:Granzymes; D022861:Hermanski-Pudlak Syndrome; D017496:Hypopigmentation; D007153:Immunologic Deficiency Syndromes; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D012697:Serine Endopeptidases / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000648:ultrastructure; D016608:Talin / Q000302:isolation & purification; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; C495927:Actr2 protein, mouse; C495929:Actr3 protein, mouse; D003598:Cytoskeletal Proteins; D016608:Talin; D000075745:rab27 GTP-Binding Proteins; D001619:beta-N-Acetylhexosaminidases; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human; D002402:Cathepsin D; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins