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

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A set of growth arrest-specific genes (gas) whose expression is negatively regulated after serum induction has previously been described (C. Schneider, R. M. King, and L. Philipson, Cell 54:787-793, 1988). The detailed analysis of one of them, gas6, is reported here. gas6 mRNA (2.6 kb) is abundantly expressed in serum-starved (48 h in 0.5% fetal calf serum) NIH 3T3 cells but decreases dramatically after fetal calf serum or basic fibroblast growth factor stimulation. The human homolog ofgas6 was also cloned and sequenced, revealing a high degree of homology and a similar pattern of expression in IMR90 human fibroblasts. Computer analysis of the protein encoded by murine and human gas6 cDNAs showed significant homology (43 and 44% amino acid identity, respectively) to human protein S, a negative coregulator in the blood coagulation pathway. By using an anti-human Gas6 monospecific affinity-purified antibody, we show that the biosynthetic level of human Gas6 fully reflects mRNA expression in IMR9O human fibroblasts. This finding thus defines a new member of vitamin K-dependent proteins that is expressed in many human and mouse tissues and may be involved in the regulation of a protease cascade relevant in growth regulation.Interactions between serine proteases, their substrates, and their inhibitors have largely been exploited during evolution. Protease cascades are not confined to the classical blood coagulation or complement cascade. A network of proteases that control the synthesis or activity of a ligand appears an ideal and finely regulatable mechanism to trigger a rapid response to an extracellular event, with the inherent advantage of powerful amplification. Thrombin, in addition to catalyzing fibrin polymerization, can act as a novel ligand for the recently identified thrombin receptor (61), a member of the seven-transmembrane domain receptor family, possibly mediating other known effects of thrombin, including its role as a mitogen for lymphocytes and fibroblasts (8, 9). Hepatocyte growth factor (scatter factor), which promotes cell division (53) and epithelial morphogenesis (47), is similar in structure to serine proteases (38% amino acid sequence identity with plasminogen), although it lacks proteolytic activity as a result of mutation of two residues in the catalytic triad (31,48). Hepatocyte growth factor is the ligand for the c-met proto-oncogene product (5, 49), a transmembrane 190-kDa heterodimer with tyrosine kinase activity that is widely expressed in normal epithelial tissues (20).Recently, a protease pathway has been shown to play a crucial role in the dorsoventral patterning of Drosophila embryos (36). At least three genes (snake, gastrulation defective, and easter) appear to encode extracellular proteases (7,36). Easter appears to be the ultimate protease that processes spatzle that binds and activates its receptor Toll

Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early factors in the failure of the microtubule network that occurs during neuronal apoptosis. Furthermore, this study introduces new insights into the mechanism(s) that generate the truncated forms of tau present in Alzheimer's disease.

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.