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

Development of nanotechnology calls for a comprehensive understanding of the impact of nanomaterials on biological systems. Autophagy is a lysosome-based degradative pathway which plays an essential role in maintaining cellular homeostasis. Previous studies have shown that nanoparticles from various sources can induce autophagosome accumulation in treated cells. However, the underlying mechanism is still not clear. Gold nanoparticles (AuNPs) are one of the most widely used nanomaterials and have been reported to induce autophagosome accumulation. In this study, we found that AuNPs can be taken into cells through endocytosis in a size-dependent manner. The internalized AuNPs eventually accumulate in lysosomes and cause impairment of lysosome degradation capacity through alkalinization of lysosomal pH. Consistent with previous studies, we found that AuNP treatment can induce autophagosome accumulation and processing of LC3, an autophagosome marker protein. However, degradation of the autophagy substrate p62 is blocked in AuNP-treated cells, which indicates that autophagosome accumulation results from blockade of autophagy flux, rather than induction of autophagy. Our data clarify the mechanism by which AuNPs induce autophagosome accumulation and reveal the effect of AuNPs on lysosomes. This work is significant to nanoparticle research because it illustrates how nanoparticles can potentially interrupt the autophagic pathway and has important implications for biomedical applications of nanoparticles.

A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3′ overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3′ end but also the 5′ end, with the cleavage site determined mainly by the distance (~22 nucleotides) from the 5′ end (5′ counting rule). This cleavage requires a 5′-terminal phosphate group. Further, we identify a novel basic motif (5′ pocket) in human Dicer that recognizes the 5′-phosphorylated end. The 5′ counting rule and the 5′ anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5′ pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5′-pocket mutant. Thus, 5′-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.

Autophagy is a lysosome-based degradation pathway. During autophagy, lysosomes fuse with autophagosomes to form autolysosomes. Following starvation-induced autophagy, nascent lysosomes are formed from autolysosomal membranes through an evolutionarily conserved cellular process, autophagic lysosome reformation (ALR), which is critical for maintaining lysosome homeostasis. Here we report that clathrin and phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) regulate ALR. Combining a screen of candidates identified through proteomic analysis of purified ALR tubules, and large-scale RNAi knockdown, we unveiled a tightly regulated molecular pathway that controls lysosome homeostasis, in which clathrin and PtdIns(4,5)P(2) are the central components. Our functional study demonstrates the central role of clathrin and its associated proteins in cargo sorting, phospholipid conversion, initiation of autolysosome tubulation, and proto-lysosome budding during ALR. Our data not only uncover a molecular pathway by which lysosome homeostasis is maintained through the ALR process, but also reveal unexpected functions of clathrin and PtdIns(4,5)P(2) in lysosome homeostasis.

The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization, translation efficiency1,2, and has been proposed to provide a layer of “epitranscriptomic” gene regulation3. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate “cap” in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria3–6. It has been proposed that NAD+, reduced NAD+ (NADH), and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps6–8. Here, we show instead that NAD+, NADH, and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated “ab initio capping” may occur in all organisms