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

Hsp70s, ubiquitous molecular chaperones, function in a myriad of biological processes, modulating polypeptides' folding, degradation and translocation across membranes, as well as protein-protein interactions. This multitude of roles is not easily reconciled with the near conformity of biochemical activity of Hsp70s, an ATP-dependent client protein binding/release cycle. Much of the functional diversity of Hsp70s is driven by a diverse class of cofactors, Jproteins (also called Hsp40s). Often, multiple J-proteins function with a single Hsp70. Some target Hsp70 activity to clients at precise locations in cells; others bind client proteins directly, thereby delivering specific clients to Hsp70, directly determining their fate.In their native cellular environment, polypeptides are constantly at risk of attaining conformations that prevent them from functioning properly and/or cause them to aggregate into large, potentially cytotoxic complexes. Molecular chaperones guide the conformation of proteins throughout their lifetime, preventing their aggregation by protecting interactive surfaces against non-productive interactions. Through such interactions, they aid in the folding of nascent proteins as they are synthesized by ribosomes, drive protein transport across membranes, and modulate protein-protein interactions by controlling conformational changes1 , 2 . In addition to these roles under optimal conditions, stresses can exacerbate protein conformational problems (for example, heat shock causing protein unfolding; oxygen radicals causing oxidation and nitrosylation). Although in some cases chaperones can facilitate (re)folding, often such rejuvenation is not possible. In such cases, chaperones can facilitate degradation, either by simply preventing aggregation and thus keeping clients susceptible to proteolysis or by actively facilitating their transfer to proteolytic systems. These diverse functions of molecular chaperones typically involve iterative client binding and release cycles until the client has reached its final active conformation, or has entered the proteolytic system (Figure 1). Strikingly, Hsp70s, one of the most ubiquitous classes of chaperones, has been implicated in all of the biological processes mentioned above2 , 3 . This Review focuses on the means by which Hsp70 molecular chaperone machinery participates in such diverse cellular functions. Their functional diversity is remarkable considering that within and across species, Hsp70s have very high sequence identity. They share a single biochemical activity, an ATPdependent substrate binding and release cycle combined with client protein recognition, which is typically rather promiscuous. The answer to this apparent conundrum lies in the fact that Hsp70s do not work alone, but rather as "Hsp70 machines", collaborating with and h.h.kampinga@med.umcg.nl, ecraig@wisc.edu. regulated by a number of (co)chaperones and cofactors. Here, using examples from yeast and human, we discuss several such factors, particularly concentrating on how the ar...

The expanding number of members in the various human heat shock protein (HSP) families and the inconsistencies in their nomenclature have often led to confusion. Here, we propose new guidelines for the nomenclature of the human HSP families, HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), and HSPB (small HSP) as well as for the human chaperonin families HSPD/E (HSP60/HSP10) and CCT (TRiC). The nomenclature is based largely on the more consistent nomenclature assigned by the HUGO Gene Nomenclature Committee and used in the National Center of Biotechnology Information Entrez Gene database for the heat shock genes. In addition to this nomenclature, we provide a list of the human Entrez Gene IDs and the corresponding Entrez Gene IDs for the mouse orthologs.