Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

TRPing on the Lung Endothelium: Calcium Channels That Regulate Barrier Function

Rises in cytosolic calcium are sufficient to initiate the retraction of endothelial cell borders and to increase macromolecular permeability. Although endothelial cell biologists have recognized the importance of shifts in cytosolic calcium for several decades, only recently have we gained a rudimentary understanding of the membrane calcium channels that change cell shape. Members of the transient receptor potential family (TRP) are chief among the molecular candidates for permeability-coupled calcium channels. Activation of calcium entry through store-operated calcium entry channels, most notably TRPC1 and TRPC4, increases lung endothelial cell permeability, as does activation of calcium entry through the TRPV4 channel. However, TRPC1 and TRPC4 channels appear to influence the lung extraalveolar endothelial barrier most prominently, whereas TRPV4 channels appear to influence the lung capillary endothelial barrier most prominently. Thus, phenotypic heterogeneity in ion channel expression and function exists within the lung endothelium, along the arterial–capillary-venous axis, and is coupled to discrete control of endothelial barrier function. Antioxid. Redox Signal. 11, 765–776