Monitoring macroautophagy by major histocompatibility complex class II presentation of targeted antigens.
Methods Enzymol
; 452: 403-21, 2009.
Article
em En
| MEDLINE
| ID: mdl-19200895
ABSTRACT
Major histocompatibility complex (MHC) class I and II molecules can both present cytosolic and nuclear antigens to CD8(+) and CD4(+) T cells, respectively. However, MHC class I displays proteasomal, whereas MHC class II molecules display lysosomal, degradation products. One pathway by which intracellular antigens gain access to lysosomal degradation is macroautophagy. Therefore, MHC class II presentation of antigens that are targeted to autophagosomes can be used to investigate regulation events of the macroautophagy pathway. We fuse antigens to Atg8/LC3 for targeting to autophagosomes, because this ubiquitin-like protein is selectively coupled to autophagosome membranes, and the portion that is coupled to the inner autophagosome membrane is degraded with this membrane in lysosomes. The localization of these fusion antigens in MHC class II loading compartments can be visualized by immunofluorescence and electron microscopy, and used as a measure of autophagic amphisome generation. In addition, MHC class II presentation of autophagosome-targeted antigens can be monitored by CD4(+) T cell recognition and indicates completion of macroautophagy. Together these immunological assays are well suited to investigate autophagic flux and analyze experimental conditions and physiological perturbations for their influence on macroautophagy.
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Autofagia
/
Antígenos de Histocompatibilidade Classe II
/
Proteínas de Fluorescência Verde
/
Proteínas Associadas aos Microtúbulos
Limite:
Humans
Idioma:
En
Revista:
Methods Enzymol
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Suíça