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.
- MeSH
- autofagie * fyziologie MeSH
- autofagozomy MeSH
- biologické markery MeSH
- biotest normy MeSH
- lidé MeSH
- lyzozomy MeSH
- proteiny spojené s autofagií metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- směrnice MeSH
At the subcellular level, the cytoskeleton regulates cell structure, organelle movement, and cytoplasmic streaming. Autophagy is a process to remove unwanted biomaterials or damaged organelles through double membrane compartments known as autophagosomes. Autophagosome biogenesis requires vesicle trafficking between donor and acceptor compartments, membrane expansion, and fusion, which is very likely to be regulated by the cytoskeleton. Recent studies have demonstrated that by knocking out key actin-regulating proteins, autophagosome biogenesis is inhibited. However, the formation of ATG8 positive structures are not affected when the entire actin network is disrupted. Here, we discuss this paradox and propose the function of the actin cytoskeleton in plant autophagy.
The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1 Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.
- MeSH
- alely MeSH
- Arabidopsis genetika metabolismus MeSH
- cytoplazmatické vezikuly genetika metabolismus MeSH
- endoplazmatické retikulum genetika metabolismus MeSH
- Golgiho aparát genetika metabolismus MeSH
- kineziny genetika metabolismus MeSH
- mikrotubuly genetika metabolismus MeSH
- multivezikulární tělíska genetika metabolismus MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- transport proteinů genetika MeSH
- vakuoly genetika metabolismus MeSH
- vezikulární transportní proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1.
- MeSH
- aktiny metabolismus MeSH
- Arabidopsis metabolismus ultrastruktura MeSH
- autofagie MeSH
- autofagozomy metabolismus ultrastruktura MeSH
- biologické modely MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- endocytóza * MeSH
- endoplazmatické retikulum metabolismus ultrastruktura MeSH
- fylogeneze MeSH
- komplex proteinů 2-3 souvisejících s aktinem metabolismus MeSH
- mikrofilamenta metabolismus MeSH
- mikrofilamentové proteiny metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
