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

SUMMARY Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.

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Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic lossof-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagyrelated 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.M acroautophagy is a catabolic pathway that shuttles cytoplasmic components via double-membrane vesicles (autophagosomes) into lysosomes for degradation and recycling. Autophagosome formation and elongation are facilitated by ubiquitin-like molecules such as MAP1LC3A/B (herein referred to as "LC3") and its homologs which are directly conjugated to phosphatidylethanolamine (PE), a reaction which requires the ubiquitin E1-like activity of autophagy-related 7 (ATG7), the E2-like activity of ATG3, and the E3-like activity of the ATG5-ATG12-ATG16L1 complex (1). Autophagy cargo receptors such as p62/ SQSTM1 bind both LC3 and ubiquitinated cargo, enabling cargo recruitment into autophagosomes and delivery to lysosomes (2, 3).Basal levels of macroautophagy control cellular homeostasis by clearing misfolded proteins or damaged organelles (4, 5). Upon starvation, macroautophagy can be induced above basal levels to supply the cell with nutrients (6, 7). This prosurvival function of macroautophagy is also used by cancer cells under conditions of metabolic stress (8). However, the role of autophagy in cancer is complex and context dependent, because the pathway has been reported to have tumor-suppressing as well as tumor-promoting properties (9-11). Liver-specific deletion of ATG7 results in increased formation of liver tumors through the activation of the Nrf2 pathway (12). Furthermore, the essential autophagy component beclin-1 inhibits tumorigenesis of breast carcinoma cells, and monoallelic deletion of beclin-1 is associated with an enhanced risk of breast cancer (13-15). I...

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-tobackground ratio to identify endogenously expressed G␣s proteincoupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive ␤-2 adrenergic-but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.G protein-coupled receptor ͉ complementation assays ͉ protein-protein interactions ͉ protein fragment G protein-coupled receptors (GPCRs) represent the largest family of cell-surface molecules involved in signal transmission. GPCRs play roles in a broad range of biological processes through regulating the majority of cell-to-cell and cell-toenvironment communication, and, consequently, their dysfunction manifests in numerous diseases (1, 2). The GPCR family has enormous pharmacological importance, as demonstrated by the fact that Ͼ30% of approved drugs elicit their therapeutic effect by selectively acting on known members of this family (3). The human genome harbors Ͼ800 putative GPCRs including a considerable number with unknown physiological function or ligands. GPCR cascades hence remain a major focus of molecular pharmacology (4, 5).Signal transduction by GPCRs is mediated by activation of protein kinases (4), among which the most intensively studied is the cAMP-dependent protein kinase A (PKA) (6). Various extracellular signals converge on the cAMP/PKA pathway through ligand binding to GPCRs. The adenylyl cyclase then converts ATP to the ubiquitous second messenger cAMP. Intrac...