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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Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.Members of the "BH3-domain-only" subset of BCL-2 family proteins connect proximal death signals to the core apoptotic pathway (1-5). After activation of CD95 (Fas) or TNFR1 death receptors, BID is cleaved and activated to p15 tBID (6-8), which, in a model system using purified mitochondria, serves as a death ligand that induces the oligomerization of BAK (9) and BAX (10). tBID does not cause release of cytochrome c from purified Bakdeficient mitochondria, suggesting that interaction of tBID and BAK is a critical event at least in vitro (9).Here we assess whether BAK is required for tBID-induced apoptosis of intact, whole cells by using a retroviral vector to express tBID in murine embryonic fibroblasts (MEFs). Numerous cells with shrunken, apoptotic morphology were detected in Bak-deficient as well † To whom correspondence should be addressed.

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Decades ago Otto Warburg observed that cancers ferment glucose in the presence of oxygen, suggesting that defects in mitochondrial respiration may be the underlying cause of cancer. We now know that the genetic events, which drive aberrant cancer cell proliferation, also alter biochemical metabolism including promoting aerobic glycolysis, but do not typically impair mitochondrial function. Mitochondria supply energy, provide building blocks for new cells, and control redox homeostasis, oncogenic signaling, innate immunity and apoptosis. Indeed, mitochondrial biogenesis and quality control are often upregulated in cancers. While some cancers have mutations in nuclear-encoded mitochondrial tricarboxylic acid (TCA) cycle enzymes that produce oncogenic metabolites, there is negative selection for pathogenic mitochondrial genome mutations. Eliminating mitochondrial DNA limits tumorigenesis and rare human tumors with mutant mitochondrial genomes are relatively benign. Thus, mitochondria play a central and multi-functional role in malignant tumor progression, and targeting mitochondria provides therapeutic opportunities.

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Organismal homeostasis depends on an intricate balance between cell death and renewal. Early pathologists recognized that this balance could be disrupted by the extensive damage observed in internal organs during the course of certain diseases. This form of tissue damage was termed "necrosis", derived from the Greek "nekros" for corpse. As it became clear that the essential building block of tissue was the cell, necrosis came to be used to describe pathologic cell death. Until recently, necrotic cell death was believed to result from injuries that caused an irreversible bioenergetic compromise. The cell dying by necrosis has been viewed as a victim of extrinsic events beyond its control. However, recent evidence suggests that a cell can initiate its own demise by necrosis in a manner that initiates both inflammatory and/or reparative responses in the host. By initiating these adaptive responses, programmed cell necrosis may serve to maintain tissue and organismal integrity.

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