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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Mature adipocytes and myocytes are derived from a common mesenchymal precursor. While IGF-1 promotes the differentiation of both cell types, the signaling pathways that specify the distinct cell fates are largely unknown. Here, we show that the Rho GTPase and its regulator, p190-B RhoGAP, are components of a critical switch in the adipogenesis-myogenesis "decision." Cells derived from embryos lacking p190-B RhoGAP exhibit excessive Rho activity, are defective for adipogenesis, but undergo myogenesis in response to IGF-1 exposure. In vitro, activation of Rho-kinase by Rho inhibits adipogenesis and is required for myogenesis. The activation state of Rho following IGF-1 signaling is determined by the tyrosine-phosphorylation status of p190-B RhoGAP and its resulting subcellular relocalization. Moreover, adjusting Rho activity is sufficient to alter the differentiation program of adipocyte and myocyte precursors. Together, these results identify the Rho GTPase as an essential modulator of IGF-1 signals that direct the adipogenesis-myogenesis cell fate decision.

Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes. Atg9 is the only integral membrane protein among autophagy-related (Atg) proteins thought to carry the membrane source for forming autophagosomes. Here we show that Drosophila Atg9 interacts with Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2) to regulate the c-Jun N-terminal kinase (JNK) signaling pathway. Significantly, depletion of Atg9 and dTRAF2 compromised JNK-mediated intestinal stem cell proliferation and autophagy induction upon bacterial infection and oxidative stress stimulation. In mammalian cells, mAtg9 interacts with TRAF6, the homolog of dTRAF2, and plays an essential role in regulating oxidative stress-induced JNK activation. Moreover, we found that ROS-induced autophagy acts as a negative feedback regulator of JNK activity by dissociating Atg9/mAtg9 from dTRAF2/TRAF6 in Drosophila and mammalian cells, respectively. Our findings indicate a dual role for Atg9 in the regulation of JNK signaling and autophagy under oxidative stress conditions.

FF domains are poorly understood protein motifs found in all eukaryotes but in a very small number of proteins. They typically occur in tandem arrays and appear predominantly in splicing and transcription factors. Curiously, they are also present in the p190 family of cytoplasmic Rho GTPase activating proteins (GAPs). We identified the serum-responsive transcriptional regulator TFII-I as a specific interactor with the p190 RhoGAP FF domains. p190 sequesters TFII-I in the cytoplasm via the FF domains, but upon PDGF receptor-mediated phosphorylation of an FF domain, TFII-I is released from p190 and translocates to the nucleus where it can activate transcription of serum-inducible genes including c-fos. These findings reveal a pathway by which mitogens promote gene transcription and indicate a role for FF domains in phosphorylation-mediated signal transduction.

Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. We identified an ortholog of mammalian paxillin in Drosophila (Dpax) and have undertaken a genetic analysis of paxillin function during development. Overexpression of Dpax disrupted leg and wing development, suggesting a role for paxillin in imaginal disc morphogenesis. These defects may reflect a function for paxillin in regulation of Rho family GTPase signaling as paxillin interacts genetically with Rac and Rho in the developing eye. Moreover, a gain-of-function suppressor screen identified a genetic interaction between Dpax and cdi in wing development. cdi belongs to the cofilin kinase family, which includes the downstream Rho target, LIM kinase (LIMK). Significantly, strong genetic interactions were detected between Dpax and Dlimk, as well as downstream effectors of Dlimk. Supporting these genetic data, biochemical studies indicate that paxillin regulates Rac and Rho activity, positively regulating Rac and negatively regulating Rho. Taken together, these data indicate the importance of paxillin modulation of Rho family GTPases during development and identify the LIMK pathway as a critical target of paxillin-mediated Rho regulation.

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