title: Monitoring autophagy in Dictyostelium.
abstract: Autophagy is an intracellular degradation mechanism essential for cell survival and maintenance of cellular homeostasis, differentiation, and development. Recent research has highlighted the impact of autophagy in neurodegenerative diseases and aging. We are still far from fully understanding the molecular mechanism of autophagy and its regulation, essential for its future use as a therapeutic target. In the last years many different techniques have been developed to study this process and some of them have been successfully used in Dictyostelium. We describe here the use of confocal microscopy to detect the pattern of different autophagic markers and the differences expected in strains deficient in autophagy. Autophagy dysfunction might also lead to the formation of intracellular ubiquitinated protein aggregates that can be easily detected by immunofluorescence. In addition, we describe two different techniques that allow the assessment of the so-called autophagic flux, the progression of autophagosomes until their fusion with lysosomes. The first one is a proteolytic cleavage assay of cytosolic markers such as GFP-PgkA and the second the visualization of the RFP-GFP-Atg8 marker by confocal microscopy.
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Mesquita
authors.forename: Javier
authors.initials: J
authors.lastname: Calvo-Garrido
authors.forename: Sergio
authors.initials: S
authors.lastname: Carilla-Latorre
authors.forename: Ricardo
authors.initials: R
authors.lastname: Escalante
mesh_terms: D001343:Autophagy; D015415:Biomarkers / Q000378:metabolism; D002021:Buffers; D004023:Dictyostelium / Q000166:cytology* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D050296:Microbial Viability; D008840:Microfilament Proteins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D059748:Proteolysis; D015800:Protozoan Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D057149:Ubiquitinated Proteins / Q000378:metabolism
chemical_list: D015415:Biomarkers; D002021:Buffers; D008840:Microfilament Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D057149:Ubiquitinated Proteins; D049452:Green Fluorescent Proteins