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

[Abstract unavailable

The c-Jun NH2-terminal kinase (JNK) is activated when cells are exposed to ultraviolet (UV) radiation. However, the functional consequence of JNK activation in UV-irradiated cells has not been established. It is shown here that JNK is required for UV-induced apoptosis in primary murine embryonic fibroblasts. Fibroblasts with simultaneous targeted disruptions of all the functional Jnk genes were protected against UV-stimulated apoptosis. The absence of JNK caused a defect in the mitochondrial death signaling pathway, including the failure to release cytochrome c. These data indicate that mitochondria are influenced by proapoptotic signal transduction through the JNK pathway.

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is activated by the exposure of cells to multiple forms of stress. A putative scaffold protein was identified that interacts with multiple components of the JNK signaling pathway, including the mixed-lineage group of MAP kinase kinase kinases (MLK), the MAP kinase kinase MKK7, and the MAP kinase JNK. This scaffold protein selectively enhanced JNK activation by the MLK signaling pathway. These data establish that a mammalian scaffold protein can mediate activation of a MAP kinase signaling pathway.

Mitogen-activated protein kinases (MAPKThe mitogen-activated protein kinases (MAPKs) represent an evolutionarily conserved signaling mechanism that is used by cells to respond to changes in their environment (Schaeffer and Weber 1999). The activation of MAPK is mediated by dual phosphorylation on a ThrXaa-Tyr motif located in the kinase activation loop. This phosphorylation is increased in stimulated cells by members of a group of MAPK kinases. These enzymes have dual substrate specificity and can phosphorylate both Thr and Tyr. Studies of a large number of MAPKs demonstrate that this mechanism of activation is conserved in many organisms, including plants, yeast, nematodes, insects, and mammals.Two different MAPK kinases (MKK4 and MKK7) are implicated in the activation of the c-Jun NH 2 -terminal kinase (JNK) group of MAPK (Davis 2000). The presence of two MAPK kinases in a single MAPK signaling module is striking because genetic analysis indicates only a single MAPK kinase in each of the MAPK signaling modules of yeast (Schaeffer and Weber 1999). However, the organization of the JNK signaling pathway is similar to that of other mammalian MAPK modules. Thus, the ERK group of MAPK is activated by MKK1/MKK2 and the p38 group of MAPK is activated by MKK3/MKK6 (Schaeffer and Weber 1999). The presence of two MAPK kinases is therefore a common feature of the organization of mammalian MAPK signaling modules. The functional significance of the dual MAPK kinases found in mammalian MAPK signaling pathways is unclear.Studies of mice demonstrate that both the Mkk4 (Yang et al. 1997;Ganiatsas et al. 1998;Nishina et al. 1999) and Mkk7 genes (Dong et al. 2000) are required for embryonic viability. This observation indicates that the MKK4 and MKK7 protein kinases serve nonredundant functions in vivo. Genetic analysis of the Mkk4 (Han et al. 1998) and Mkk7 (Glise et al. 1995) genes in Drosophila supports this conclusion. However, the molecular basis for the difference in signaling by MKK4 and MKK7 is unclear. Differences in the expression pattern of MKK4 and MKK7 in tissues may be a contributing factor. Alternatively, the distinct biochemical properties of MKK4 and MKK7 may be critical for the nonredundant functions of these protein kinases in vivo.Comparison of the biochemical properties of MKK4 and MKK7 indicates that these protein kinases have different substrate specificities. Thus, in vitro assays demonstrate that MKK4 can activate both JNK and p38 MAPK (Derijard et al. 1995;Lin et al. 1995). In contrast, MKK7 selectively activates only JNK (Holland et al.

Summary Axonal death disrupts functional connectivity of neural circuits and is a critical feature of many neurodegenerative disorders. Pathological axon degeneration often occurs independently of known programmed death pathways, but the underlying molecular mechanisms remain largely unknown. Using traumatic injury as a model, we systematically investigate mitogen-activated protein kinase (MAPK) families, and delineate a MAPK cascade that represents the early degenerative response to axonal injury. The adaptor protein Sarm1 is required for activation of this MAPK cascade, and this Sarm1-MAPK pathway disrupts axonal energy homeostasis, leading to ATP depletion before physical breakdown of damaged axons. The protective cytoNmnat1/Wlds protein inhibits activation of this MAPK cascade. Further, MKK4, a key component in the Sarm1-MAPK pathway, is antagonized by AKT signaling, which modulates the degenerative response by limiting activation of downstream JNK signaling. Our results reveal a regulatory mechanism that integrates distinct signals to instruct pathological axon degeneration.

The c-Jun NH 2 -terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is activated by phosphorylation on Thr and Tyr. Here we report the molecular cloning of a new member of the mammalian MAP kinase kinase group (MKK7) that functions as an activator of JNK. In vitro protein kinase assays demonstrate that MKK7 phosphorylates and activates JNK, but not the p38 or extracellular signal-regulated kinase groups of MAP kinase. Expression of MKK7 in cultured cells causes activation of the JNK signal transduction pathway. MKK7 is therefore established to be a novel component of the JNK signal transduction pathway.

The hallmark of T-cell activation is the production of interleukin 2 (IL-2). c-Jun amino-terminal kinase (JNK), a MAP kinase that phosphorylates c-Jun and other components of the AP-1 group of transcription factors, has been implicated in the activation of IL-2 expression. Previously, we found that T cells from mice deficient in the Jnk1 or Jnk2 gene can be activated and produce IL-2 normally, but are deficient in functional differentiation into Th1 or Th2 subsets. However, studies of mice with compound mutations indicate that JNK1 and JNK2 are redundant during mouse development. Here we use three new mouse models in which peripheral T cells completely lack JNK proteins or signalling, to test whether the JNK signalling pathway is crucial for IL-2 expression and T-cell activation. Unexpectedly, these T cells made more IL-2 and proliferated better than wild-type cells. However, production of effector T-cell cytokines did require JNK. Thus, JNK is necessary for T-cell differentiation but not for naive T-cell activation.