In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are 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 monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

[Abstract unavailable

S. cerevisiae inherits its vacuole by projecting vacuole-derived membrane vesicles and tubules into the bud, where they fuse to establish the daughter vacuole. This homotypic fusion event can be assayed in vitro. It requires Sec17p and Sec18p, the homologs of the mammalian alpha-SNAP and NSF, which cooperate in multiple steps of membrane trafficking. We now report that Sec17p, Sec18p, and ATP are only needed for an early stage of the reaction that results in Sec17p release. Sec17p and Sec18p actions precede, and are needed for, the step employing the Ras-like GTPase Ypt7p. Sec18p-driven release of Sec17p can even precede vacuole docking, as it can occur prior to mixing of vacuoles and is insensitive to vacuole concentration. Sec17p and Sec18p thus may function in a predocking stage of the reaction, rather than in bilayer fusion per se.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans-complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2+/calmodulin-dependent fashion. V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion.

The basic reaction mechanisms for membrane fusion in the trafficking of intracellular membranes and in exocytosis are probably identical. But in contrast to regulated exocytosis, intracellular fusion reactions are referred to as 'constitutive' as no final Ca2+-dependent triggering step has been observed. Although transport from the endoplasmic reticulum to the Golgi apparatus in the cell depends on Ca2+, as does endosome fusion and assembly of the nuclear envelope, it is unclear whether Ca2+ triggers these events. Membrane fusion involves several subreactions: priming, tethering and docking. Proteins that are needed for fusion include p115, SNAPs, NSF, SNAREs and small GTPases, which operate in these early reactions, but the machinery that catalyses the final mixing of biological membranes is still unknown. Here we show that Ca2+ is released from the vacuolar lumen following completion of the docking step. We have identified calmodulin as the putative Ca2+ sensor and as the first component required in the post-docking phase of vacuole fusion. Calmodulin binds tightly to vacuoles upon Ca2+ release. Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing. Hence, activation of SNAREs is not sufficient to drive bilayer mixing between physiological membranes. We propose that Ca2+ control of the latest phase of membrane fusion may be a conserved feature, relevant not only for exocytosis, but also for intracellular, 'constitutive' fusion reactions. However, the origin of the Ca2+ signal, its receptor and its mode of processing differ.

Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

Autophagy or ''self eating'' is frequently activated in tumor cells treated with chemotherapy or irradiation. Whether autophagy represents a survival mechanism or rather contributes to cell death remains controversial. To address this issue, the role of autophagy in radiosensitive and radioresistant human cancer cell lines in response to ;-irradiation was examined. We found irradiation-induced accumulation of autophagosomes accompanied by strong mRNA induction of the autophagy-related genes beclin 1, atg3, atg4b, atg4c, atg5, and atg12 in each cell line. Transduction of specific targetsiRNAs led to down-regulation of these genes for up to 8 days as shown by reverse transcription-PCR and Western blot analysis. Blockade of each autophagy-related gene was associated with strongly diminished accumulation of autophagosomes after irradiation. As shown by clonogenic survival, the majority of inhibited autophagy-related genes, each alone or combined, resulted in sensitization of resistant carcinoma cells to radiation, whereas untreated resistant cells but not sensitive cells survived better when autophagy was inhibited. Similarly, radiosensitization or the opposite was observed in different sensitive carcinoma cells and upon inhibition of different autophagy genes. Mutant p53 had no effect on accumulation of autophagosomes but slightly increased clonogenic survival, as expected, because mutated p53 protects cells by conferring resistance to apoptosis. In our system, short-time inhibition of autophagy along with radiotherapy lead to enhanced cytotoxicity of radiotherapy in resistant cancer cells. [Cancer Res 2008;68(5):1485-94]

Aims. Our goal is to study the different morphologies associated to the interaction of the stellar winds of AGB stars and red supergiants with the interstellar medium (ISM) to follow the fate of the circumstellar matter injected into the interstellar medium. Methods. Far-infrared Herschel/PACS images at 70 and 160 μm of a sample of 78 Galactic evolved stars are used to study the (dust) emission structures developing out of stellar wind-ISM interaction. In addition, two-fluid hydrodynamical simulations of the coupled gas and dust in wind-ISM interactions are used for comparison with the observations. Results. Four distinct classes of wind-ISM interaction (i.e. "fermata", "eyes", "irregular", and "rings") are identified, and basic parameters affecting the morphology are discussed. We detect bow shocks for ∼40% of the sample and detached rings for ∼20%. The total dust and gas mass inferred from the observed infrared emission is similar to the stellar mass loss over a period of a few thousand years, while in most cases it is less than the total ISM mass potentially swept-up by the wind-ISM interaction. De-projected stand-off distances (R 0 ) -defined as the distance between the central star and the nearest point of the interaction region -of the detected bow shocks ("fermata" and "eyes") are derived from the PACS images and compared to previous results, model predictions, and the simulations. All observed bow shocks have stand-off distances smaller than 1 pc. Observed and theoretical stand-off distances are used together to independently derive the local ISM density. Conclusions. Both theoretical (analytical) models and hydrodynamical simulations give stand-off distances for adopted stellar properties that are in good agreement with the measured de-projected stand-off distance of wind-ISM bow shocks. The possible detection of a bow shock -for the distance-limited sample -appears to be governed by its physical size as set roughly by the stand-off distance. In particular the star's peculiar space velocity and the density of the ISM appear decisive in detecting emission from bow shocks or detached rings. In most cases the derived ISM densities concur with those typical of the warm neutral and ionised gas in the Galaxy, though some cases point towards the presence of cold diffuse clouds. Tentatively, the "eyes" class objects are associated to (visual) binaries, while the "rings" generally do not appear to occur for M-type stars, only for C or S-type objects that have experienced a thermal pulse.