Showing papers on "Autolysosome published in 2008"

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Abstract: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms Recent reviews have described the range of assays that have been used for this purpose(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi) Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response

Does bafilomycin A1 block the fusion of autophagosomes with lysosomes

Abstract: Bafilomycin A1 is a specific inhibitor of the vacuolar type H+-ATPase (V-ATPase) in cells, and inhibits the acidification of organelles containing this enzyme, such as lysosomes and endosomes. Recently, while editing and reviewing chapters on autophagy for Methods in Enzymology, we noticed repeated references to the effect of bafilomycin A1 in blocking the fusion of autophagosomes with lysosomes. Of course we have seen this in various research papers as well, but reading this routinely in chapters written by various people over a short period of time really caused this to stand out. Every one of these chapters referred to the paper by Yamamoto et al. In that paper, treatment with 100 nM bafilomycin A1 for 1 h blocks the fusion of autophagosomes with lysosomes in the rat hepatoma H-4-II-E cell line, based on data from electron microscopy. However, data from one of our labs noted an apparently different result in a relatively recent manuscript. Therefore, we decided to look into this more carefully.

Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways

Abstract: It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosome...

ApoL1, a BH3-only lipid-binding protein, induces autophagic cell death.

Abstract: Macroautophagy (hereafter referred to as autophagy) is a lysosome-dependent mechanism of intracellular degradation that is used for the turnover and recycling of cytoplasmic constituents. It involves the sequestration of a fraction of cytosol and organelles within a de novo synthesized, double-membraned organelle called the autophagosome, which then fuses with the endosome and/or lysosome to create the amphisome and autolysosome, respectively. Eventually, the inner membrane of the autophagosome, together with the enclosed cargo, is degraded by the lysosomal hydrolases.1 Autophagy in mammals serves multiple purposes: it functions as a survival mechanism during periods of starvation and stress, it is directly involved in eliminating aberrant protein aggregates, removing damaged/aged organelles, and defending against pathogens, and it serves an essential role in early neonatal development.2 Evidently, autophagy also functions as type II programmed cell death (PCD), or autophagic cell death (ACD), in multi-cellular organisms.3,4 It has been reported that autophagy is involved in physiological cell death during development of Drosophila and Dictyostelium discoideum, however, ACD has not yet been observed/demonstrated in mammals in vivo.5,6 In the past two decades, yeast has been used as an experimental model in dissecting the molecular bases of autophagy. Currently 31 ATG (autophagy-related) genes have been identified in yeast.3,6 Recent identification and characterization of human orthologs/homologs of yeast ATG genes and the link between autophagy and human diseases further emphasize the importance of autophagy in health and disease. Deficiency of autophagy correlates with diseases such as cancer, cardiomyopathies, inflammatory bowel diseases and neurodegenerative diseases.5,7 In addition, autophagy involves unique membrane dynamics and biosynthesis, requiring de novo synthesis of double-membrane autophagosomes, in which both protein and lipid components are required. Regarding protein components, aside from Atg proteins, Vps34, a class III phosphatidylinositol-3-phosphate kinase (PI3K), the proteins that are part of its complex, and the Bcl-2 family members play critical roles in regulating autophagosome formation. Interestingly, Beclin 1, the mammalian ortholog of yeast Atg6 that is required for autophagy, is a BH3-only protein. In addition, other Bcl-2 family members, such as Bcl-2, Bax, Bid and BNIP3, regulate autophagy.6,8 By contrast, the lipid composition of autophagosomal membranes remains unknown because it is technically difficult to isolate pure autophagosomes. It is evident however that some lipid species, for example, phosphatitylinositol-3-phosphate or PI3P, are required for autophagosomal membranes.1,9 In an effort to identify and characterize novel BH3-only proteins in ACD, and discover molecular regulators that dictate autophagy for death or survival, we examined a novel BH3-only protein, apolipoprotein L1 (ApoL1). We show that wild-type ApoL1 induces ACD in a variety of cells, whereas a BH3 deletion allele of ApoL1 fails to induce cell death, indicating that ApoL1 is a bona fide BH3-only pro-death protein. We further demonstrate that ApoL1 is a potent and specific inducer of ACD that stimulates time-dependent accumulation and translocation of lipidated LC3-II, formation of autophagosomes and subsequently cell death. We also show that ApoL1 is inducible by p53 during p53-induced cell death. p53, a well-known tumor suppressor, regulates both apoptosis and ACD.4 Interestingly, a recent study shows that p53 plays a dual role in the control of autophagy: on the one hand, nuclear p53 can induce autophagy through transcriptional effects, whereas on the other hand, cytoplasmic p53 may act as a master repressor of autophagy.10 Our results suggest that ApoL1 is a novel BH3-only protein and is a p53 downstream effector functioning as a molecular determinant that determines whether autophagy plays a role in cell death.4 Importantly, ApoL1 has been found only in humans and African green monkeys (NCBI Entrez Gene, ApoL1), suggesting that ApoL1-induced ACD is primate-specific. Employing a protein-lipid overlay assay, we demonstrate that ApoL1 is an intracellular lipid-binding protein that binds strongly with phosphatidic acid (PA) and cardiolipin (CL), and with less affinity with different species of phosphoinositides (PIPs) in the following order PI(3,5)P2>PI4P>PI5P>PI3P>PI(4,5)P2>PI(3,4,5)P3>PI(3,4)P2.4 PIPs are phosphorylated derivatives of the membrane phospholipids phosphatidylinositols and play critical roles in regulating receptor signaling, cytoskeleton function and membrane trafficking. They are highly concentrated and localized in distinct pools in the plasma membrane, endosomes, nucleus and autophagosomal membranes, where they function as ligands, adaptors or docking sites for PIP-binding proteins. In addition, the homeostatic equilibrium of PIPs determines cellular proliferation, differentiation and PCD.11,12 Vps34 is required for autophagy, implying an essential role of its product PI3P in this process.13 Recently, Obara and colleagues9 showed that PI3P is highly enriched and delivered to autophagosomal membranes but not as a cargo enclosed in autophagosomes, implying direct involvement of PI3P in autophagosome formation. They also report a possible enrichment of PI3P on the inner autophagosomal membranes compared to the outer membrane.9 Regarding binding with PIPs, we show that ApoL1 possesses higher affinity for PI(3,5)P2, PI4P, PI5P, and PI3P, implying that intracellular accumulation of ApoL1 may alter the homeostasis of PIPs leading to ACD. In addition, we conducted a protein domain homology search and found no homology between ApoL1 and any known PIP-binding domains, including FYVE (Fab1, YOTB, Vac1 and EEA1), PX (Phox homology), PH (Pleckstrin homology), ENTH (Epsin N-terminal homology), FERM (Band4.1, Ezrin, Radixin, and Moesin), GRAM (Glucosyltransferase, Rab-like GTPase Activator, and Myotubularins), ANTH (AP180/CALM), and WD-repeats of WIPIs, human orthologs of yeast Atg18 and/or Atg20.7,14,15 In fact, no specific binding domain for PI(3,5)P2 has been identified thus far. Taken together, ApoL1 may represent a novel class of PIP-binding protein. Importantly, in autophagy, at least 2 Atg proteins are involved in PIP synthesis (Atg6 and Atg14) and 5 Atg proteins bind PIP (Atg18, Atg20, Atg21, Atg24 and Atg27).1,7 Evidently, yeast mutant strains deficient in PIP binding will be useful for functional complementation and biochemical analysis of human ApoL1. Interestingly, ApoL1 also binds PA and CL. PA is a mitogenic messenger that serves as a critical component of mTOR signaling. PA directly interacts with the domain in mTOR that is targeted by rapamycin, and this interaction positively correlates with mTOR's ability to activate downstream effectors. mTOR is a negative regulator of autophagy;16,17 thus, binding of PA by ApoL1 might inhibit mTOR activation and subsequently induce autophagy. In addition, CL, a negatively charged phospholipid abundant in the mitochondrial inner membrane, is important in maintaining protein (for example cytochrome c) and membrane integrity of mitochondria. During apoptosis, CL functions as the docking site for t-Bid and Bad and induces mitochondrial membrane permeability and cytochrome c release.18,19 In addition, CL relocates to the plasma membrane and other subcellular organelles during cell death.20 Sequestering of PA and CL with ApoL1 may alter the homeostasis between survival and death leading to ACD. To our knowledge, this is the first BH3-only protein with lipid binding activity that, when overproduced intracellularly, induces ACD.

In the autophagy trenches: The early research career of Russell L. Deter

Abstract: Christian de Duve is considered to be one of the founding fathers of autophagy. After all, he coined the term, and he played an instrumental role in the discovery of the lysosome, for which he rece...