Linked Life Data

11707514

Source:http://linkedlifedata.com/resource/pubmed/id/11707514

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
Pt 20
pubmed:dateCreated
2001-11-14
pubmed:abstractText
Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/3-methyladenine, http://linkedlifedata.com/resource/pubmed/chemical/Adenine, http://linkedlifedata.com/resource/pubmed/chemical/Amino Acids, http://linkedlifedata.com/resource/pubmed/chemical/Androstadienes, http://linkedlifedata.com/resource/pubmed/chemical/Cadaverine, http://linkedlifedata.com/resource/pubmed/chemical/Cycloheximide, http://linkedlifedata.com/resource/pubmed/chemical/Enzyme Inhibitors, http://linkedlifedata.com/resource/pubmed/chemical/Ethylmaleimide, http://linkedlifedata.com/resource/pubmed/chemical/Fluorescent Dyes, http://linkedlifedata.com/resource/pubmed/chemical/Protein Synthesis Inhibitors, http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Fusion Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Vinblastine, http://linkedlifedata.com/resource/pubmed/chemical/monodansylcadaverine, http://linkedlifedata.com/resource/pubmed/chemical/wortmannin
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0021-9533
pubmed:author
pubmed:issnType
Print
pubmed:volume
114
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3619-29
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2001
pubmed:articleTitle
A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation.
pubmed:affiliation
Laboratorio de Biología Celular y Molecular-Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza, 5500, Argentina.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't