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1.
We have developed a method for monitoring autophagy using Rosella, a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Its mode of action relies upon differences in pH between different cellular compartments and the vacuole. Here we demonstrate its utility in yeast (Saccharomyces cerevisiae) by expression in the cytosol, and targeting to mitochondria or to the nucleus. When cells were cultured in nitrogen depleted medium, uptake of the compartment labelled with the biosensor (i.e., cytosol, mitochondria, or nucleus) into the vacuole was observed. We showed that this vacuolar uptake was, for cytosol and mitochondria, an ATG8-dependent process while the uptake of the nucleus was significantly reduced in the absence of Atg8p and can be said to be partially ATG8-dependent. We further demonstrated the value of the biosensor as a reporter of autophagy by employing fluorescence-activated cell sorting of discrete populations of cells undergoing autophagy.  相似文献   

2.
Autophagy is a highly conserved eukaryotic degradation process during which bulk cytoplasmic materials are transported by double-membrane autophagosomes into the vacuole for degradation. Methods of monitoring autophagy are indispensable in studying the mechanism and functions of autophagy. AuTophaGy-related protein 8 (ATG8) functions in autophagosome assembly by decorating on autophagic membranes, and the inner membrane-bound ATG8 proteins enter the vacuole via active autophagy flux. Fluorescence protein (FP)-tagged forms of ATG8 have been explored as visual markers to monitor autophagy in animals and several plant species. Here, we evaluated and modified this FP-ATG8-based autophagy monitoring method in wheat (Triticum aestivum L.) by fluorescence observation of green fluorescence protein (GFP)-tagged and Discosoma red fluorescent protein (DsRED)-tagged forms of one wheat ATG8, TaATG8h, in wheat mesophyll protoplasts. Under a nutrient-starvation condition, punctate GFP/DsRED- TaATG8h fluorescence representing autophagosomes was clearly observed in the cytoplasm. The accumulation of GFP-TaATG8h-labeled autophagosomes was impaired by the autophagosome biogenesis inhibitor 3-methyladenine but enhanced by the vacuolar degradation inhibitor concanamycin A. In addition, accumulated spreading fluorescence was observed in the vacuole, indicating active autophagy fluxes which led to continuous degradation of GFP/DsRED-TaATG8h fusions and release of protease-tolerant free GFP/DsRED proteins in the vacuole. To observe FP-tagged TaATG8h in other types of wheat cell, we also expressed GFP-TaATG8h in leaf epidermal cells. Consistent with its performance in protoplasts, GFP-TaATG8h showed punctate fluorescence representing autophagosomes in leaf epidermal cells. Taken together, our results proved the feasibility of using FP-tagged ATG8 to monitor both autophagosome accumulation and autophagy flux in living wheat cells.  相似文献   

3.
4.
《Autophagy》2013,9(8):1466-1467
Autophagy is essential for nutrient recycling and intracellular housekeeping in plants by removing unwanted cytoplasmic constituents, aggregated polypeptides, and damaged organelles. The autophagy-related (ATG)1-ATG13 kinase complex is an upstream regulator that integrates metabolic and environmental cues into a coherent autophagic response directed by other ATG components. Our recent studies with Arabidopsis thaliana revealed that ATG11, an accessory protein of the ATG1-ATG13 complex, acts as a scaffold that connects the complex to autophagic membranes. We showed that ATG11 encourages proper behavior of the ATG1-ATG13 complex and faithful delivery of autophagic vesicles to the vacuole, likely through its interaction with ATG8. In addition, we demonstrated that Arabidopsis mitochondria are degraded during senescence via an autophagic route that requires ATG11 and other ATG components. Together, ATG11 appears to be an important modulator of the ATG1-ATG13 complex and a multifunctional scaffold required for bulk autophagy and the selective clearance of mitochondria.  相似文献   

5.
During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H(+)-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.  相似文献   

6.
ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2  相似文献   

7.
Autophagy encompasses several processes by which cytosol and organelles can be delivered to the vacuole/lysosome for breakdown and recycling. We sought to investigate autophagy of the nucleus (nucleophagy) in the yeast Saccharomyces cerevisiae by employing genetically encoded fluorescent reporters. The use of such a nuclear reporter, n-Rosella, proved the basis of robust assays based on either following its accumulation (by confocal microscopy), or degradation (by immunoblotting), within the vacuole. We observed the delivery of n-Rosella to the vacuole only after prolonged periods of nitrogen starvation. Dual labeling of cells with Nvj1p-EYFP, a nuclear membrane reporter of piecemeal micronucleophagy of the nucleus (PMN), and the nucleoplasm-targeted NAB35-DsRed.T3 allowed us to detect PMN soon after the commencement of nitrogen starvation whilst delivery to the vacuole of the nucleoplasm reporter was observed only after prolonged periods of nitrogen starvation. This later delivery of nuclear components to the vacuole has been designated LN (late nucleophagy). Only a very few cells showed simultaneous accumulation of both reporters (Nvj1p-EYFP and NAB35-DsRed.T3) in the vacuole. We determined, therefore, that delivery of the two respective nuclear reporters to the vacuole is temporally and spatially separated. Furthermore, our data suggest that LN is mechanistically distinct from PMN because it can occur in nvj1Δ and vac8Δ cells, and does not require ATG11. Nevertheless, a subset of the components of the core macroautophagic machinery is required for LN as it is efficiently inhibited in null mutants of several autophagy-related genes (ATG) specifying such components. Moreover, the inhibition of LN in some mutants is accompanied by alterations in nuclear morphology.  相似文献   

8.
Autophagy defends the mammalian cytosol against bacterial invasion. Efficient bacterial engulfment by autophagy requires cargo receptors that bind (a) homolog(s) of the ubiquitin-like protein Atg8 on the phagophore membrane. The existence of multiple ATG8 orthologs in higher eukaryotes suggests that they may perform distinct functions. However, no specific role has been assigned to any mammalian ATG8 ortholog. We recently discovered that the autophagy receptor CALCOCO2/NDP52, which detects cytosol-invading Salmonella enterica serovar Typhimurium (S. Typhimurium), preferentially binds LC3C. The CALCOCO2/NDP52-LC3C interaction is essential for cell-autonomous immunity against cytosol-exposed S. Typhimurium, because cells lacking either protein fail to target bacteria into the autophagy pathway. The selectivity of CALCOCO2/NDP52 for LC3C is determined by a novel LC3C interacting region (CLIR), in which the lack of the key aromatic residue of canonical LIRs is compensated by LC3C-specific interactions. Our findings provide a new layer of regulation to selective autophagy, suggesting that specific interactions between autophagy receptors and the ATG8 orthologs are of biological importance.  相似文献   

9.
Autophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin‐fold proteins Autophagy‐related (ATG)‐8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8‐PE adduct, we also show that ATG8 lipidation requires the ATG12–ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12–ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12–ATG5 adduct is essential for ATG8‐mediated autophagy in plants by promoting ATG8 lipidation.  相似文献   

10.
Autophagy is essential for nutrient recycling and intracellular housekeeping in plants by removing unwanted cytoplasmic constituents, aggregated polypeptides, and damaged organelles. The autophagy-related (ATG)1-ATG13 kinase complex is an upstream regulator that integrates metabolic and environmental cues into a coherent autophagic response directed by other ATG components. Our recent studies with Arabidopsis thaliana revealed that ATG11, an accessory protein of the ATG1-ATG13 complex, acts as a scaffold that connects the complex to autophagic membranes. We showed that ATG11 encourages proper behavior of the ATG1-ATG13 complex and faithful delivery of autophagic vesicles to the vacuole, likely through its interaction with ATG8. In addition, we demonstrated that Arabidopsis mitochondria are degraded during senescence via an autophagic route that requires ATG11 and other ATG components. Together, ATG11 appears to be an important modulator of the ATG1-ATG13 complex and a multifunctional scaffold required for bulk autophagy and the selective clearance of mitochondria.  相似文献   

11.
《Autophagy》2013,9(2):288-289
Autophagy functions as a cell-autonomous effector mechanism of innate immunity by separating bacteria from cytosolic resources and delivering them for lysosomal destruction. How cytosolic bacteria are targeted for autophagy is incompletely understood. We recently discovered that Salmonella enterica serotype Typhimurium and Streptococcus pyogenes are detected by NDP52 (nuclear dot protein 52kDa), after these bacteria enter the cytosol of human cells and become decorated with poly-ubiquitinated proteins. NDP52 binds the bacterial ubiquitin coat as well as ATG8/LC3 and delivers cytosolic bacteria into autophagosomes. In the absence of NDP52 ubiquitin-coated bacteria accumulate outside ATG8/LC3+ autophagosomes. Cells lacking NDP52 fail to restrict bacterial proliferation, as do cells depleted of TBK1, an IKK family kinase colocalizing with NDP52 at the bacterial surface. Our findings demonstrate the existence of a receptor for the selective autophagy of cytosolic bacteria, suggesting that cells are able to differentiate between anti-bacterial and other forms of autophagy.  相似文献   

12.
《Autophagy》2013,9(12):2126-2139
We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.  相似文献   

13.
Ishida H  Yoshimoto K 《Autophagy》2008,4(7):961-962
Excluding the central vacuole, chloroplasts constitute the largest compartment within the leaf cells of plants and contain approximately 80 percent of the total leaf nitrogen, mainly as proteins. Much of this nitrogen is allocated to the carbon-fixing enzyme in photosynthesis, Rubisco. During senescence, plants can mobilize nitrogen from chloroplasts in older leaves to other organs, such as developing seeds. Whereas bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have recently demonstrated that stroma-targeted green fluorescent protein (GFP), DsRed, and GFP-labeled Rubisco can be mobilized to the vacuole of living cells via Rubisco-containing bodies, in an ATG gene-dependent manner. Our results indicate the presence of a specific autophagic pathway for chloroplast stromal proteins, which does not cause chloroplast lysis. Here, we also discuss the involvement of stroma-filled tubules, stromules, which are important for the structural flexibility of the organelle, on the autophagic transfer of stromal proteins to the vacuole.  相似文献   

14.
Xin Wen 《Autophagy》2020,16(2):193-194
ABSTRACT

Xenophagy, a unique type of selective macroautophagy/autophagy, targets invading pathogens as part of the host immune response. In order to survive within the host, bacteria have established various self-defense mechanisms. In a recent paper from Feng Shao’s lab, the Salmonella effector protein SopF has been demonstrated to block xenophagy by interrupting the vacuolar type H+-translocating (v-) ATPase-ATG16L1 axis, which is important for antibacterial autophagy initiation. SopF can specifically ADP-ribosylate Gln124 on ATP6V0C, a v-ATPase component, thus influencing recruitment of ATG16L1 onto the bacteria-containing vacuole within the host cytosol.

Abbreviations: ATG: autophagy-related; S. Typhimurium: Salmonella enterica serovar Typhimurium; T3SS: type III secretion system  相似文献   

15.
Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. In yeast, this encapsulation employs a set of autophagy (ATG) proteins that direct the conjugation of two ubiquitin-like protein tags, ATG8 and ATG12, to phosphatidylethanolamine and the ATG5 protein, respectively. Using an Arabidopsis (Arabidopsis thaliana) atg7 mutant unable to ligate either tag, we previously showed that the ATG8/12 conjugation system is important for survival under nitrogen-limiting growth conditions. By reverse-genetic analyses of the single Arabidopsis gene encoding ATG5, we show here that the subpathway that forms the ATG12-ATG5 conjugate also has an essential role in plant nutrient recycling. Similar to plants missing ATG7, those missing ATG5 display early senescence and are hypersensitive to either nitrogen or carbon starvation, which is accompanied by a more rapid loss of organellar and cytoplasmic proteins. Multiple ATG8 isoforms could be detected immunologically in seedling extracts. Their abundance was substantially elevated in both the atg5 and atg7 mutants, caused in part by an increase in abundance of several ATG8 mRNAs. Using a green fluorescent protein-ATG8a fusion in combination with concanamycin A, we also detected the accumulation of autophagic bodies inside the vacuole. This accumulation was substantially enhanced by starvation but blocked in the atg7 background. The use of this fusion in conjunction with atg mutants now provides an important marker to track autophagic vesicles in planta.  相似文献   

16.
Autophagy is an efficient way of degradation and removal of unwanted or damaged intracellular components in plant cells. It plays an important role in recycling of intracellular structures (during starvation, removal of cell components formed during plant development or damaged by various stress factors) and in programmed cell death. Morphologically, autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which are essential for the isolation and degradation of cytoplasmic components. Among autophagic (ATG) proteins, ATG8 from the ubiquitinlike protein family plays a key role in autophagosome formation. ATG8 is also involved in selective autophagy, fusion of autophagosome with the vacuole, and some other intracellular processes not associated with autophagy. In contrast to yeasts that carry a single ATG8 gene, plants have multigene ATG8 families. The reason for such great ATG8 diversity in plants remains unclear. It is also unknown whether all members of the ATG8 family are involved in the formation and functioning of autophagosomes. To answer these questions, the identification of the structure and the possible functions of plant proteins from ATG8 family is required. In this review, we analyze the structures of ATG8 proteins from plants and their homologs from yeast and animal cells, interactions of ATG8 proteins with functional ligands, and involvement of ATG8 proteins in different metabolic processes in eukaryotes.  相似文献   

17.
Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell‐autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP‐tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9‐GFP‐containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9‐deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild‐type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.  相似文献   

18.
19.
ATG8(自噬相关蛋白8)结合蛋白通过ATG8相互作用基序(ATG8 interaction motif,AIM)或泛素相互作用基序(ubiquitin interaction motif,UIM)与ATG8相互作用,在自噬、选择性自噬和非自噬过程中起关键作用。ATG8结合蛋白在酵母和哺乳动物研究中取得了巨大进展,但在植物领域仍然滞后。本文首先概括了植物ATG8蛋白结构及特征,其次,重点阐述了作为植物选择性自噬受体的ATG8结合蛋白的结构和功能,最后,总结了参与自噬小体闭合、转运和人工合成ATG8结合蛋白研究状况。本文结合最新研究,系统总结了目前发现的植物ATG8结合蛋白结构和功能,以期为植物选择性自噬和自噬的研究提供新思路。  相似文献   

20.
Selective autophagy has been extensively studied in various organisms, but knowledge regarding its functions in plants, particularly in organelle turnover, is limited. We have recently discovered ATG8-INTERACTING PROTEIN1 (ATI1) from Arabidopsis thaliana and showed that following carbon starvation it is localized on endoplasmic reticulum (ER)-associated bodies that are subsequently transported to the vacuole. Here, we show that following carbon starvation ATI1 is also located on bodies associating with plastids, which are distinct from the ER ATI bodies and are detected mainly in senescing cells that exhibit plastid degradation. Additionally, these plastid-localized bodies contain a stroma protein marker as cargo and were observed budding and detaching from plastids. ATI1 interacts with plastid-localized proteins and was further shown to be required for the turnover of one of them, as a representative. ATI1 on the plastid bodies also interacts with ATG8f, which apparently leads to the targeting of the plastid bodies to the vacuole by a process that requires functional autophagy. Finally, we show that ATI1 is involved in Arabidopsis salt stress tolerance. Taken together, our results implicate ATI1 in autophagic plastid-to-vacuole trafficking through its ability to interact with both plastid proteins and ATG8 of the core autophagy machinery.  相似文献   

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