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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When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explae by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a stiking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublngs per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent slglcantiy fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker ofsomatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age ofthe donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.The cellular senescence model of aging was founded by landmark experiments of Hayflick and Moorhead (1), who firmly established that normal human fibroblasts have a finite life-span in vitro. Although much evidence supports this model (2-8), the mechanism accounting for the finite division capacity of normal somatic cells remains a mystery. Olovnikov (9, 10) suggested that the cause of cellular senescence is the gradual loss of telomeres due to the end-replication problem-i.e., the inability of DNA polymerase to completely replicate the 3' end of linear duplex DNA (11) (for review, see refs. 12 and 13).Telomeres play a critical role in chromosome structure and function. They prevent aberrant recombination (14-16) and apparently function in the attachment ofchromosome ends to the nuclear envelope (17 (31)(32)(33). These observations have led to the telomere hypothesis of cellular aging (13), in which loss of telomeres due to incomplete DNA replication and absence of telomerase provides a mitotic clock that ultimately signals cell cycle exit, limiting the replicative capacity of somatic cells. To further explore this hypothesis, we have examined the relationship between telomere length, in vivo age, and replicative capacity of fibroblasts from normal donors and subjects with the Hutchinson-Gilford syndrome of premature aging (34,35). We have also determined the relationship between telomere length in sperm DNA and donor age.MATERIALS AND MET...

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To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.