Autophagy research: Lessons from metabolismMeijer, Alfred J.
doi: 10.4161/auto.5.1.7207pmid: 19115480
Autophagy research continues to expand exponentially. Clearly autophagy and metabolism are intimately connected; however, the rapid expansion of research into this topic inevitably brings the risk that important basic knowledge of metabolism will be overlooked when considering experimental data. Unfortunately, unawareness of possible metabolic complications may sometimes lead to misinterpretation of the data obtained.
The autophagic pathway is a key component in the lysosomal dependent entry of Trypanosoma cruzi into the host cellRomano, Patricia S.; Arboit, María A.; Vázquez, Cristina L.; Colombo, María Isabel
doi: 10.4161/auto.5.1.7160pmid: 19115481
The etiologic agent of Chagas disease, Trypanosoma cruzi, infects mammalian cells activating a signal transduction cascade that leads to the formation of its parasitophorous vacuole. Previous works have demonstrated the crucial role of lysosomes in the establishment of T. cruzi infection. In this work we have studied the possible relationship between this parasite and the host cell autophagy. We show, for the first time, that the vacuole containing T. cruzi (TcPV) is decorated by the host cell autophagic protein LC3. Furthermore, live cell imaging experiments indicate that autolysosomes are recruited to parasite entry sites. Interestingly, starvation or pharmacological induction of autophagy before infection significantly increased the number of infected cells whereas inhibitors of this pathway reduced the invasion. In addition, the absence of Atg5 or the reduced expression of Beclin1, two proteins required at the initial steps of autophagosome formation, limited parasite entry and reduced the association between TcPV and the classical lysosomal marker Lamp-1. These results indicate that mammalian autophagy is a key process that favors the colonization of T. cruzi in the host cell.
Autophagy-assisted glycogen catabolism regulates asexual differentiation in Magnaporthe oryzaeDeng, Yi Zhen; Ramos-Pamplona, Marilou; Naqvi, Naweed I.
doi: 10.4161/auto.5.1.7175pmid: 19115483
Autophagy, a conserved pathway for bulk cellular degradation and recycling in eukaryotes, regulates proper turnover of organelles, membranes and certain proteins. Such regulated degradation is important for cell growth and development particularly during environmental stress conditions, which act as key inducers of autophagy. We found that autophagy and MoATG8 were significantly induced during asexual development in Magnaporthe oryzae. An RFP-tagged MoAtg8 showed specific localization and enrichment in aerial hyphae, conidiophores and conidia. We confirmed that loss of MoATG8 results in dramatically reduced ability to form conidia, the asexual spores that propagate rice-blast disease. Exogenous supply of glucose or sucrose significantly suppressed the conidiation defects in a MoATG8-deletion mutant. Comparative proteomics based identification and characterization of Gph1, a glycogen phosphorylase that catalyzes glycogen breakdown, indicated that autophagy-assisted glycogen homeostasis is likely important for proper aerial growth and conidiation in Magnaporthe. Loss of Gph1, or addition of G6P significantly restored conidiation in the Moatg8Δ mutant. Overproduction of Gph1 led to reduced conidiation in wild-type Magnaporthe strain. We propose that glycogen autophagy actively responds to and regulates carbon utilization required for cell growth and differentiation during asexual development in Magnaporthe.
Autophagy contributes to degradation of Hirano bodiesKim, Dong-Hwan; Davis, Richard C.; Furukawa, Ruth; Fechheimer, Marcus
doi: 10.4161/auto.5.1.7228pmid: 18989098
Hirano bodies are actin-rich inclusions reported most frequently in the hippocampus in association with a variety of conditions including neurodegenerative diseases, and aging. We have developed a model system for formation of Hirano bodies in Dictyostelium and cultured mammalian cells to permit detailed studies of the dynamics of these structures in living cells. Model Hirano bodies are frequently observed in membrane-enclosed vesicles in mammalian cells consistent with a role of autophagy in the degradation of these structures. Clearance of Hirano bodies by an exocytotic process is supported by images from electron microscopy showing extracellular release of Hirano bodies, and observation of Hirano bodies in the culture medium of Dictyostelium and mammalian cells. An autophagosome marker protein Atg8-GFP, was co-localized with model Hirano bodies in wild type Dictyostelium cells, but not in atg5- or atg1-1 autophagy mutant strains. Induction of model Hirano bodies in Dictyostelium with a high level expression of 34 kDa ΔEF1 from the inducible discoidin promoter resulted in larger Hirano bodies and a cessation of cell doubling. The degradation of model Hirano bodies still occurred rapidly in autophagy mutant (atg5-) Dictyostelium, suggesting that other mechanisms such as the ubiquitin-mediated proteasome pathway could contribute to the degradation of Hirano bodies. Chemical inhibition of the proteasome pathway with lactacystin, significantly decreased the turnover of Hirano bodies in Dictyostelium providing direct evidence that autophagy and the proteasome can both contribute to degradation of Hirano bodies. Short term treatment of mammalian cells with either lactacystin or 3-methyl adenine results in higher levels of Hirano bodies and a lower level of viable cells in the cultures, supporting the conclusion that both autophagy and the proteasome contribute to degradation of Hirano bodies.
High-throughput functional screening for autophagy-related genes and identification of TM9SF1 as an autophagosome-inducing geneHe, Pengfei; Peng, Zhi; Luo, Ye; Wang, Lan; Yu, Peng; Deng, Weiwei; An, Yunqing; Shi, Taiping; Ma, Dalong
doi: 10.4161/auto.5.1.7247pmid: 19029833
Autophagy is a tightly regulated process responsible for the bulk degradation of most long-lived proteins and some organelles, which is associated with several forms of human disease including cancer, neurodegenerative disease, and cardiomyopathies. However, the molecular machinery involved in autophagy in mammalian cells remains poorly understood. Here, we describe a high-throughput, cell-based functional screening platform based on automated fluorescence microscopy system, that enable acquiring and quantitatively analyzing images of GFP-LC3 dots in cotransfected cells. From a library of 1,050 human cDNA clones, we identified three genes (TM9SF1, TMEM166, and TMEM74) whose overexpression induced high levels of autophagosome formation. In particular, overexpression of TM9SF1, which colocalized with LC3 according to the confocal assay, led to a significant increase in the number of GFP-LC3 dots. The results of transmission electron microscopy and immunoblotting to examine LC3-II levels further confirmed the ability of TM9SF1 to induce autophagy. Furthermore, knockdown of TM9SF1 expression by RNA interference could hamper starvation-induced autophagy. The functional screening platform therefore can be applied for high-throughput genomic screening candidate autophagy-related genes, which would provide new insights into underlying molecular mechanisms that may regulate autophagy in mammalian cells.
Role of ULK-FIP200 complex in mammalian autophagy: FIP200, a counterpart of yeast Atg17?Hara, Taichi; Mizushima, Noboru
doi: 10.4161/auto.5.1.7180pmid: 18981720
The yeast serine threonine kinase Atg1 appears to be a key regulator of autophagy and its kinase activity is crucial for autophagy induction. Recent reports have indicated that a mammalian Atg1 homolog, UNC-51-like kinase (ULK) 1, is required for autophagy. We found that ULK1 localizes to the autophagic isolation membrane and its kinase activity is important for autophagy induction. Furthermore, we identified a focal adhesion kinase (FAK) family interacting protein of 200 kD (FIP200) as a ULK-interacting protein. FIP200 also localizes to the isolation membrane together with ULK. Using FIP200-deficient cells, we found that FIP200 is essential for autophagosome formation and the proper function of ULK. Here, we discuss the role of the ULK-FIP200 complex in autophagy and the possibility that FIP200 functions as a mammalian counterpart of Atg17.
On the level: IRGM gene function is all about expressionHuett, Alan; McCarroll, Steven A.; Daly, Mark J.; Xavier, Ramnik J.
doi: 10.4161/auto.5.1.7263pmid: 19029815
Crohns disease is a complex, multigenic, chronic inflammatory bowel disease of uncertain etiology. Recent advances in genetics, including high-throughout single-nucleotide polymorphism typing platforms and deep sequencing technologies have begun to shed light upon disease predisposition and pathogenesis. Autophagy is emerging as a key player in both innate and adaptive immunity, as well as tissue homeostasis and development in the gut. Here we describe our recent studies into the Crohns disease-associated Immunity-Related GTPase family, M (IRGM) gene and our discovery of a large risk-conferring upstream deletion. We discuss the effects of this deletion upon expression levels of IRGM alleles and how tissue-specific expression might be affected by the promoter polymorphism. In addition, we comment upon the potential roles of IRGM in autophagy of intracellular pathogens, and the challenges ahead for further elucidating IRGM function.