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

In target tissue extracts, heat shock protein hsp9O has been found associated to aDl unganded steroid receptors. Modulation ofimportant functions ofthese receptors, including prevention of DNA binding and optimiztion of transcriptional activity, has been attributed to hsp9O. However no unequivocal in vivo demonstration of interaction between receptors and hsp9O has been presented. We targeted chicken hsp9O, a mainly cytoplasmic protein, with the nucleoplamin nuclear lozation signal (90NLS). After transfection into COS-7 ceDls, 9ONLS was found in the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cytoplasmic. Coexpression of 9ONLS and of the cytoplasmic human glucocorticosteroid receptor mutant led to complete nuclear localization ofthe receptor, indicating its piggyback transport by 9ONLS and thus physical and functional interaction between the two proteins in the absence of hormone. The same nuclear localization was obtained after cotransfection of 9ONLS and a cytoplasmic rabbit progesterone receptor mutant. Finally, coexpression of wild-type rabbit progesterone receptor (nuclear) and wildtpe hsp9O (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp9O into the nucleus. These experiments lay the groundwork on which to study hsp9O as a chaperone, regulating activities of steroid receptors and possibly participating in their nuclear-cytoplasmic shuttling.Steroid hormone receptors are hormone-dependent transcriptional activators bearing hormone-independent nuclear localization signals (NLSs) whose efficiency is the primary determinant of their subcellular localization in the absence of hormone; thus the glucocorticosteroid receptor is predominantly located in the cytoplasm, whereas estrogen and progesterone receptors are essentially nuclear (1-3). Despite this, however, a puzzling observation is that all unliganded steroid receptors form "8S" complexes with 90-kDa heat shock protein (hsp90) in the cytosol of target cell homogenates (4-7), hsp90 being an abundant and ubiquitous protein described as essentially cytoplasmic. Two functions have been ascribed to receptor-bound hsp90: masking of the receptor DNA binding domain and maintenance of the ligand binding domain in a functional hormone-binding conformation, at least for gluco-and mineralocorticosteroid receptors (reviewed in refs. 8 and 9). The in vivo requirement of appropriate levels of hsp90 for efficient hormonal activation of steroid receptors in yeast has also been reported (10). The lack of direct evidence for the in vivo interaction between the two proteins has, however, led to the suspicion that hsp90-steroid receptor complexes represent an artifactual association occurring during cell homogenization or that theseThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 ...

Hsp90, a molecular chaperone required for the functioning of glucocorticosteroid receptor (GR), ensures, by direct interaction, the conformational competence of the steroid-binding pocket. In addition to having this positive function, Hsp90 maintains steroid receptors in an inactive form in the absence of hormone. However, neither the participation of Hsp90 once the pathway has been activated by the ligand nor the importance of increased Hsp90 levels in determining the amplitude of the response has ever been assessed directly. Here, by increasing the Hsp90͞GR ratio in the nuclear compartment, we found an attenuation of the response to glucocorticosteroids that was not due to a nonspecific or toxic effect of the Hsp90 modified by nuclear targeting. Since this negative effect was more pronounced at high levels of hormone, when receptor and Hsp90 are maximally dissociated, the possibility of an interaction between Hsp90 and GR, already activated to a DNA-binding form, was directly investigated. Indeed GR, after in vivo activation by ligand, was still able to reassociate with Hsp90, suggesting that this interaction plays a role in vivo, possibly in receptor recycling. Moreover, the GR binding to its DNA response element was inhibited by an excess of Hsp90, pointing to a function of Hsp90 in the nuclear compartment. It is thus proposed that an increased Hsp90͞GR ratio inf luences the responsiveness to ligand at a step that is after receptor activation. This increased ratio may be of pathophysiological relevance in the different circumstances that lead to an elevated level of nuclear Hsp90.

Arachidonic acid (AA) is generated via Rac-mediated phospholipase A2 (PLA2) activation in response to growth factors and cytokines and is implicated in cell growth and gene expression. In this study, we show that AA activates the stressactivated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in a time-and dose-dependent manner. Indomethacin and nordihydroguaiaretic acid, potent inhibitors of cyclooxygenase and lipoxygenase, respectively, did not exert inhibitory effects on AA-induced SAPK/JNK activation, thereby indicating that AA itself could activate SAPK/JNK. As Rac mediates SAPK/JNK activation in response to a variety of stressful stimuli, we examined whether the activation of SAPK/JNK by AA is mediated by Rac1. We observed that AA-induced SAPK/JNK activation was significantly inhibited in Rat2-Rac1N17 dominant-negative mutant cells. Furthermore, treatment of AA induced membrane ruffling and production of hydrogen peroxide, which could be prevented by Rac1N17. These results suggest that AA acts as an upstream signal molecule of Rac, whose activation leads to SAPK/JNK activation, membrane ruffling and hydrogen peroxide production.z 1999 Federation of European Biochemical Societies.