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.

Read the full review for this F1000Prime recommended article: A rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for normal golgi movement in plants.

Zea mays plasma membrane intrinsic proteins (ZmPIPs) fall into two groups, ZmPIP1s and ZmPIP2s, that exhibit different water channel activities when expressed in Xenopus oocytes. ZmPIP1s are inactive, whereas ZmPIP2s induce a marked increase in the membrane osmotic water permeability coefficient, P _f . We previously showed that, in Xenopus oocytes, ZmPIP1;2 and ZmPIP2;1 interact to increase the cell P _f . Here, we report the localization and interaction of ZmPIP1s and ZmPIP2s in living maize cells. ZmPIPs were fused to monomeric yellow fluorescent protein and/or monomeric cyan fluorescent protein and expressed transiently in maize mesophyll protoplasts. When expressed alone, ZmPIP1 fusion proteins were retained in the endoplasmic reticulum, whereas ZmPIP2s were found in the plasma membrane. Interestingly, when coexpressed with ZmPIP2s, ZmPIP1s were relocalized to the plasma membrane. Using FRET/fluorescence lifetime imaging microscopy, we demonstrated that this relocalization results from interaction between ZmPIP1s and ZmPIP2s. Immunoprecipitation experiments provided additional evidence for the association of ZmPIP1;2 and ZmPIP2;1 in maize roots and suspension cells. These data suggest that PIP1–PIP2 interaction is required for in planta PIP1 trafficking to the plasma membrane to modulate plasma membrane permeability.

TSPO, a stress-induced, posttranslationally regulated, early secretory pathway-localized plant cell membrane protein, belongs to the TspO/MBR family of regulatory proteins, which can bind porphyrins. This work finds that boosting tetrapyrrole biosynthesis enhanced TSPO degradation in Arabidopsis thaliana and that TSPO could bind heme in vitro and in vivo. This binding required the His residue at position 91 (H91), but not that at position 115 (H115). The H91A and double H91A/H115A substitutions stabilized TSPO and rendered the protein insensitive to heme-regulated degradation, suggesting that heme binding regulates At-TSPO degradation. TSPO degradation was inhibited in the autophagy-defective atg5 mutant and was sensitive to inhibitors of type III phosphoinositide 3-kinases, which regulate autophagy in eukaryotic cells. Mutation of the two Tyr residues in a putative ubiquitin-like ATG8 interacting motif of At-TSPO did not affect heme binding in vitro but stabilized the protein in vivo, suggesting that downregulation of At-TSPO requires an active autophagy pathway, in addition to heme. Abscisic acid–dependent TSPO induction was accompanied by an increase in unbound heme levels, and downregulation of TSPO coincided with the return to steady state levels of unbound heme, suggesting that a physiological consequence of active TSPO downregulation may be heme scavenging. In addition, overexpression of TSPO attenuated aminolevulinic acid–induced porphyria in plant cells. Taken together, these data support a role for TSPO in porphyrin binding and scavenging during stress in plants.

The Arabidopsis thaliana multi-stress regulator TSPO is transiently induced by abiotic stresses. The final destination of this polytopic membrane protein is the Golgi apparatus, where its accumulation is strictly regulated, and TSPO is downregulated through a selective autophagic pathway. TSPO-related proteins regulate the physiology of the cell by generating functional protein complexes. A split-ubiquitin screen for potential TSPO interacting partners uncovered a plasma membrane aquaporin, PIP2;7. Pull-down assays and fluorescence imaging approaches revealed that TSPO physically interacts with PIP2;7 at the endoplasmic reticulum and Golgi membranes in planta. Intriguingly, constitutive expression of fluorescently tagged PIP2;7 in TSPO-overexpressing transgenic lines resulted in patchy distribution of the fluorescence, reminiscent of the pattern of constitutively expressed yellow fluorescent protein-TSPO in Arabidopsis. Mutational stabilization of TSPO or pharmacological inhibition of the autophagic pathway affected concomitantly the detected levels of PIP2;7, suggesting that the complex containing both proteins is degraded through the autophagic pathway. Coexpression of TSPO and PIP2;7 resulted in decreased levels of PIP2;7 in the plasma membrane and abolished the membrane water permeability mediated by transgenic PIP2;7. Taken together, these data support a physiological role for TSPO in regulating the cell-surface expression of PIP2;7 during abiotic stress conditions through protein-protein interaction and demonstrate an aquaporin regulatory mechanism involving TSPO.