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

Single-molecule atomic force microscopy (AFM) was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle. Individual titin molecules were repeatedly stretched, and the applied force was recorded as a function of the elongation. At large extensions, the restoring force exhibited a sawtoothlike pattern, with a periodicity that varied between 25 and 28 nanometers. Measurements of recombinant titin immunoglobulin segments of two different lengths exhibited the same pattern and allowed attribution of the discontinuities to the unfolding of individual immunoglobulin domains. The forces required to unfold individual domains ranged from 150 to 300 piconewtons and depended on the pulling speed. Upon relaxation, refolding of immunoglobulin domains was observed.

The giant sarcomeric protein titin contains a protein kinase domain (TK) ideally positioned to sense mechanical load. We identified a signaling complex where TK interacts with the zinc-finger protein nbr1 through a mechanically inducible conformation. Nbr1 targets the ubiquitin-associated p62/SQSTM1 to sarcomeres, and p62 in turn interacts with MuRF2, a muscle-specific RING-B-box E3 ligase and ligand of the transactivation domain of the serum response transcription factor (SRF). Nuclear translocation of MuRF2 was induced by mechanical inactivity and caused reduction of nuclear SRF and repression of transcription. A human mutation in the titin protein kinase domain causes hereditary muscle disease by disrupting this pathway.During muscle differentiation, a specific program of gene expression leads to the translation of myofibrillar proteins and their assembly into contractile units, the sarcomeres, which are constantly remodeled to adapt to changes in mechanical load. The giant protein titin (also known as connectin) acts as a molecular blueprint for sarcomere assembly by providing specific attachment sites for numerous sarcomeric proteins, as well as acting as a molecular spring (1, 2). Titin also contains a catalytic serine-threonine kinase domain (TK), which is inhibited by a specific dual mechanism (3). However, the upstream elements controlling TK activation, its range of cellular substrates, and particularly the role of TK in mature muscle are largely unknown. Spanning half sarcomeres from Z disk to M band, titin is in a unique position to sense mechanical strain along the sarcomere (1). The elastic properties of the titin molecule and the mechanical deformation of the M band during stretch and contraction (4) suggest that the signaling properties of TK might be modulated by mechanically induced conformational changes. Molecular dynamics simulations suggest that mechanical strain can induce a catalytically active conformation of TK (5).The catalytic kinase domain of titin interacts with nbr1. We searched for further elements of a putative signaling pathway that might recognize mechanically induced conformational intermediates of titin's catalytic domain. In a systematic two-hybrid screening approach with various structure-based open states of the catalytic site [kin1, kin2, and kin3 (6)], we identified the zinc-finger protein nbr1 (7) as a TK ligand, which interacted via its Nterminal PB1 domain with the semiopened construct kin3 (Fig. 1, A and B). This interaction was also seen in precipitation experiments with nbr1 and TK-kin3 ( fig. S1A). Kin1, where the complete regulatory domain closes the active site, and kin2, where the a helix R1 (3) is deleted, did not interact. Thus, aR1 was necessary but not sufficient for nbr1 binding, which also required a semiopened catalyt-