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Monitoring macroautophagy by major histocompatibility complex class II presentation of targeted antigens


Gannagé, M; Münz, C (2009). Monitoring macroautophagy by major histocompatibility complex class II presentation of targeted antigens. Methods in Enzymology, 452:403-421.

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.

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.

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3 citations in Web of Science®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Experimental Immunology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2009
Deposited On:10 Jan 2010 11:30
Last Modified:05 Apr 2016 13:42
Publisher:Elsevier
ISSN:0076-6879
Publisher DOI:10.1016/S0076-6879(08)03624-0
PubMed ID:19200895
Permanent URL: http://doi.org/10.5167/uzh-26774

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