Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.     

Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.     

Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes

Autophagy is a membrane trafficking pathway that carries cytosolic components to the lysosome for degradation. During this process, the autophagosome, a double-membraned organelle, is generated de novo, sequesters cytoplasmic proteins and organelles, and delivers them to lysosomes. However, the mechanism by which autophagosomes are targeted to lysosomes has not been determined. Here, we observed the real-time behavior of microtubule-associated protein light chain 3 (LC3), which localizes to autophagosomes, and showed that autophagosomes move in a microtubule- and dynein-dynactin motor complex-dependent manner. After formation, autophagosomes show a rapid vectorial movement in the direction of the centrosome, where lysosomes are usually concentrated. Microinjection of antibodies against LC3 inhibited this movement; furthermore, using FRAP, we showed that anti-LC3 antibody injection caused a defect in targeting of autophagosomes to lysosomes. Collectively, our data demonstrate the functional significance of autophagosome movement that enables effective delivery from the cytosol to lysosomes.     

Using LC3 to monitor autophagy flux in the retinal pigment epithelium

Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of aged or damaged intracellular organelles and their delivery to lysosomes for degradation.^1,2 Autophagy begins with the formation of membranes arising in part from the endoplasmic reticulum, that elongate and fuse engulfing cytoplasmic constituents into a classic double-membrane bound nascent autophagosome. These early autophagosomes undergo a stepwise maturation process to form the late autophagosome or amphisome that ultimately fuses with a lysosome. Efficient autophagy is dependent on an equilibrium between the formation and elimination of autophagosomes; thus, a deficit in any part of this pathway will cause autophagic dysfunction. Autophagy plays a role in aging and age-related diseases. ^1,2,7 However, few studies of autophagy in retinal disease have been reported.

Recent studies show that autophagy and changes in lysosomal activity are associated with both retinal aging and age-related macular degeneration (AMD).^3,4 This article describes methods which employ the target protein LC3 to monitor autophagic flux in retinal pigment epithelial cells. During autophagy, the cytosolic form of LC3 (LC3-I) is processed and recruited to the phagophore where it undergoes site specific proteolysis and lipidation near the C terminus to form LC3-II.⁵ Monitoring the formation of cellular autophagosome puncta containing LC3 and measuring the ratio of LC3-II to LC3-I provides the ability to monitor autophagy flux in the retina.     

BORC coordinates encounter and fusion of lysosomes with autophagosomes

Whereas the mechanisms involved in autophagosome formation have been extensively studied for the past 2 decades, those responsible for autophagosome-lysosome fusion have only recently begun to garner attention. In this study, we report that the multisubunit BORC complex, previously implicated in kinesin-dependent movement of lysosomes toward the cell periphery, is required for efficient autophagosome-lysosome fusion. Knockout (KO) of BORC subunits causes not only juxtanuclear clustering of lysosomes, but also increased levels of the autophagy protein LC3B-II and the receptor SQSTM1. Increases in LC3B-II occur without changes in basal MTORC1 activity and autophagy initiation. Instead, LC3B-II accumulation largely results from decreased lysosomal degradation. Further experiments show that BORC KO impairs both the encounter and fusion of autophagosomes with lysosomes. Reduced encounters result from an inability of lysosomes to move toward the peripheral cytoplasm, where many autophagosomes are formed. However, BORC KO also reduces the recruitment of the HOPS tethering complex to lysosomes and assembly of the STX17-VAMP8-SNAP29 trans-SNARE complex involved in autophagosome-lysosome fusion. Through these dual roles, BORC integrates the kinesin-dependent movement of lysosomes toward autophagosomes with HOPS-dependent autophagosome-lysosome fusion. These findings reveal a requirement for lysosome dispersal in autophagy that is independent of changes in MTORC1 signaling, and identify BORC as a novel regulator of autophagosome-lysosome fusion.     

Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein,Tandem Fluorescent-Tagged LC3

During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric Red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.     

Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3

During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.     

Plasma membrane helps autophagosomes grow

The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.Key words: autophagy, plasma membrane, endocytosis, phagophore, originWhere do autophagosomes get their membrane from? Although the field of autophagy has grown tremendously since its discovery a few decades ago, the origin(s) of the membranes that contribute to autophagosome biogenesis has been a mystery among autophagy researchers until recently. Mammalian autophagosomes are formed randomly throughout the cytoplasm via a process that involves elongation and fusion of phagophores to form double-membraned autophagosomes. This process involves two ubiquitin-like conjugation systems: conjugation of Atg12 to Atg5 that later forms a macromolecular complex with Atg16L1, and conjugation of phosphatidylethanolamine (PE) with Atg8/LC3-I. The Atg12-Atg5-Atg16L1 complex is targeted to the preautophagic structures, which then acquire Atg8. Atg12-Atg5-Atg16L1 dissociates from completed autophagosomes, while LC3-PE (LC3-II) is associated both with pre-autophagic structures and completed autophagosomes.Some recent studies have explored the contribution of membranes from different organelles supporting the general idea that autophagosomes derive membranes from pre-existing organelles. It is quite possible that there may be multiple membrane sources involved. A few groups have revisited the hypothesis that the endoplasmic reticulum (ER) may be one of the membrane donors. High-resolution 2D electron microscopy (EM) and 3D EM-tomography studies have revealed connections between the ER and the growing autophagosomes. Whether the ER contributes to general autophagy or a specific form of autophagy, reticulophagy, remains to be determined. In addition, it has not been shown if ER membrane is required for autophagosome formation. Recently another study has reported that autophagosomes receive lipids from the outer mitochondrial membrane, but only under starvation conditions, again fueling the multiple-membrane source hypothesis.We have now found evidence for plasma membrane contribution to pre-autophagic structures via endocytosis. Unlike the previous studies, which have focused on LC3- positive structures, we looked specifically at the Atg5-, Atg12- and Atg16-positive pre-autophagic structures, an idea that stemmed from our finding that clathrin heavy-chain immunoprecipitates with Atg16L1. We think that this interaction is partly mediated by the adaptor protein AP2, since knockdown of AP2 decreases the clathrin heavy-chain-Atg16L1 interaction. Immunogold EM also shows clathrin localization on Atg16L1-labeled vesicles close to the plasma membrane.These findings led us to test whether knockdown of proteins involved in clathrin-mediated endocytosis affected Atg16L1-positive pre-autophagic structures. Indeed, knockdown of key proteins in the clathrin-mediated endocytic pathway results in a decrease in the formation of Atg16L1-positive structures both under basal or autophagy-induced conditions (starvation or trehalose treatment). This correlates with a decrease in the number of LC3-labeled autophagosomes. When we directly analyzed vesicle fusion by livecell microscopy, we observed that vesicles endocytosed from the plasma membrane fuse to the Atg16L1-positive vesicles close to the plasma membrane. This was confirmed by immuno-EM when we found cholera toxin B-labeling (used to label plasma membrane that is subsequently internalized by endocytosis) on Atg16L1-vesicles. We noticed that overexpression of an Atg16L1 mutant that does not bind clathrin heavy-chain does not form Atg16L1-vesicular structures in the way we see with wild-type Atg16L1, suggesting that the binding of Atg16L1 to AP2/clathrin is required for the subsequent formation of the Atg16L1 vesicles.When we blocked endocytic vesicle scission (using both genetic and chemical inhibitors) we found that Atg16L1 strongly immunoprecipitates with clathrin-heavy chain probably due to the accumulation of clathrin-Atg16L1 structures at the plasma membrane that failed to pinch off. This was strongly supported by our fluorescence microscopy and immuno-EM studies that showed what we predicted—accumulation of Atg16L1 at the plasma membrane. This suggests that Atg16L1 in a complex with AP2/clathrin is targeted to the plasma membrane and subsequently internalized as Atg16L1-positive structures. Thus, our data strongly suggest that plasma membrane contributes to early autophagic precursors that subsequently mature to form phagophores (Fig. 1).Open in a separate windowFigure 1Plasma membrane contributes to the formation of early autophagic precursors. Previous studies show that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes to form amphisomes, which are Atg16L1-negative, LC3-positive and are also positive for endosomal markers. We show that blocking clathrin-mediated endocytosis inhibits formation of Atg16L1-positive structures that mature to form phagophores and later autophagosomes. These Atg16L1-vesicles are positive for other early autophagosomal markers like Atg5 and Atg12, but are negative for early endosomal markers like EEA1, suggesting that they are high up in the autophagosome biogenesis cascade. Inhibition of dynamin with Dynsasore or the use of a dominant negative K44A mutant blocks scission and results in Atg16L1 accumulation on the plasma membrane, suggesting that endosomal scission is critical for this process.Although previous studies suggest that completely formed autophagosomes need to fuse with early or late endosomes in order for subsequent autophagosomelysosome fusion to occur, they did not look at the formation of pre-autophagic structures. Our study shows that active endocytosis is required both for the formation of autophagosomes, when very early endocytic intermediates immediately pinching off the plasma membrane (not early endosomes) fuse with Atg16L1-positive structures to form phagophores, and also for maturation of autophagosomes when early or late endosomes fuse with Atg16L1-negative but LC3-positive autophagosomes to form amphisomes. Since blocking clathrin-mediated endocytosis does not completely abrogate autophagosome formation, we believe that other endocytic pathways may have a similar role. Depending on the cell type or the physiological conditions, the contributions from the different endocytic pathways may vary accordingly. It will be interesting to know if the endocytic pathway continuously delivers membrane for early steps in autophagy as the preautophagic structures grow and mature to form autophagosomes, deriving membrane from other sources.     

Analysis of macroautophagy by immunohistochemistry

(Macro)Autophagy is a phylogenetically conserved membrane-trafficking process that functions to deliver cytoplasmic cargoes to lysosomes for digestion. The process is a major mechanism for turnover of cellular constituents and is therefore critical for maintaining cellular homeostasis. Macroautophagy is characteristically distinct from other forms of autophagy due to the formation of double-membraned vesicles termed autophagosomes which encapsulate cargoes prior to fusion with lysosomes. Autophagosomes contain an integral membrane-bound form (LC3-II) of the microtubule-associated protein 1 light chain 3 β (MAP1LC3B), which has become a gold-standard marker to detect accumulation of autophagosomes and thereby changes in macroautophagy. Due to the role played by macroautophagy in various diseases, the detection of autophagosomes in tissue sections is frequently desired. To date, however, the detection of endogenous LC3-II on paraffin-embedded tissue sections has proved problematic. We report here a simple, optimized and validated method for the detection of LC3-II by immunohistochemistry in human and mouse tissue samples that we believe will be a useful resource for those wishing to study macroautophagy ex vivo.     

Analysis of macroautophagy by immunohistochemistry

(Macro)Autophagy is a phylogenetically conserved membrane-trafficking process that functions to deliver cytoplasmic cargoes to lysosomes for digestion. The process is a major mechanism for turnover of cellular constituents and is therefore critical for maintaining cellular homeostasis. Macroautophagy is characteristically distinct from other forms of autophagy due to the formation of double-membraned vesicles termed autophagosomes which encapsulate cargoes prior to fusion with lysosomes. Autophagosomes contain an integral membrane-bound form (LC3-II) of the microtubule-associated protein 1 light chain 3 β (MAP1LC3B), which has become a gold-standard marker to detect accumulation of autophagosomes and thereby changes in macroautophagy. Due to the role played by macroautophagy in various diseases, the detection of autophagosomes in tissue sections is frequently desired. To date, however, the detection of endogenous LC3-II on paraffin-embedded tissue sections has proved problematic. We report here a simple, optimized and validated method for the detection of LC3-II by immunohistochemistry in human and mouse tissue samples that we believe will be a useful resource for those wishing to study macroautophagy ex vivo.     

Dissecting the involvement of LC3B and GATE-16 in p62 recruitment into autophagosomes

Autophagy is a major intracellular trafficking pathway that delivers proteins and organelles from the cytoplasm into lysosomes for consequential degradation and recycling. Mammalian Atg8s are key autophagic factors that undergo a unique ubiquitin-like conjugation to the lipid phase of the autophagosomal membrane. In addition to their activity in autophagosome formation, several Atg8s directly bind p62/SQSTM1. Here we show that LC3 and GATE-16 differ in their mode of p62 binding. While the soluble form of both LC3 and GATE-16 bind p62, only the lipidated form of LC3 is directly involved in p62 recruitment into autophagosomes. Moreover, by utilizing chimeras of LC3 and GATE-16 where their N-terminus was swapped, we determined the regions responsible for this differential binding. Accordingly, we found that the chimera of GATE-16 containing the LC3 N-terminal region acts similarly to wild-type LC3 in recruiting p62 into autophagosomes. We therefore propose that LC3 is responsible for the final stages of p62 incorporation into autophagosomes, a process selectively mediated by its N-terminus.     

Dissecting the involvement of LC3B and GATE-16 in p62 recruitment into autophagosomes

Autophagy is a major intracellular trafficking pathway that delivers proteins and organelles from the cytoplasm into lysosomes for consequential degradation and recycling. Mammalian Atg8s are key autophagic factors that undergo a unique ubiquitin-like conjugation to the lipid phase of the autophagosomal membrane. In addition to their activity in autophagosome formation, several Atg8s directly bind p62/SQSTM1. Here we show that LC3 and GATE-16 differ in their mode of p62 binding. While the soluble form of both LC3 and GATE-16 bind p62, only the lipidated form of LC3 is directly involved in p62 recruitment into autophagosomes. Moreover, by utilizing chimeras of LC3 and GATE-16 where their N-terminus was swapped, we determined the regions responsible for this differential binding. Accordingly, we found that the chimera of GATE-16 containing the LC3 N-terminal region acts similarly to wild-type LC3 in recruiting p62 into autophagosomes. We therefore propose that LC3 is responsible for the final stages of p62 incorporation into autophagosomes, a process selectively mediated by its N-terminus.     

Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

Background

Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (ΔN-ErbB2).

Methodology/Principal Findings

Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic ΔN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm.

Conclusions/Significance

Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formation.     

The promoting role of lysosome-localized c-Src in autophagosome-lysosome fusion

Src-family kinases (SFKs), such as c-Src, Lyn and Fyn, belong to non-receptor-type tyrosine kinases and play key roles in cell proliferation, adhesion, and migration. SFKs are anchored to the plasma membrane, Golgi membranes and lysosomal membranes through lipid modifications. Although the functions of SFKs being localized to the plasma membrane are intensively studied, those of SFKs being localized to organelle membranes are poorly understood. Here, we show that, among SFKs, c-Src in particular is involved in a decrease in the amount of LC3-II. c-Src and non-palmitoylated Lyn [Lyn(C3S) (cysteine-3 → serine-3)], which are localized onto lysosomes, decrease the amount of LC3-II and treatment with SFK inhibitors increases the amount of LC3-II, suggesting the importance of SFKs' lysosomal localization for a change of autophagic flux in a kinase activity-dependent manner. Colocalization of LC3-II with the lysosome-associated membrane protein LAMP1 shows that lysosome-localized SFKs promote the fusion of autophagosomes with lysosomes. Lysosome-localized SFKs play a positive role in the maintenance of cell viability under starvation conditions, which is further supported by knockdown of c-Src. Therefore, our results suggest that autophagosome-lysosome fusion is promoted by lysosome-localized c-Src, leading to cell survival under starvation conditions.     

Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.     

Lysosomal Turnover,but Not a Cellular Level,of Endogenous LC3 is a Marker for Autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D, and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.     

LC3‐association with the parasitophorous vacuole membrane of Plasmodium berghei liver stages follows a noncanonical autophagy pathway

Eukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with P. berghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in P. berghei‐infected ATG5^?/? host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3‐independent pathway.     

Autophagosome precursor maturation requires homotypic fusion

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.     

Dual function of CALCOCO2/NDP52 during xenophagy

During xenophagy, pathogens are selectively targeted by autophagy receptors to the autophagy machinery for their subsequent degradation. In infected cells, the autophagy receptor CALCOCO2/NDP52 targets Salmonella Typhimurium to the phagophore membrane by concomitantly interacting with LC3C and binding to ubiquitinated cytosolic bacteria or to LGALS8/GALECTIN 8 adsorbed on damaged vacuoles that contain bacteria. We recently reported that in addition, CALCOCO2 is also necessary for the maturation step of Salmonella Typhimurium-containing autophagosomes. Interestingly, the role of CALCOCO2 in maturation is independent of its role in targeting, as these functions rely on distinct binding domains and protein partners. Indeed, to mediate autophagosome maturation CALCOCO2 binds on the one hand to LC3A, LC3B, or GABARAPL2, and on the other hand to MYO6/MYOSIN VI, whereas the interaction with LC3C is dispensable. Therefore, the autophagy receptor CALCOCO2 plays a dual function during xenophagy first by targeting bacteria to nascent autophagosomes and then by promoting autophagosome maturation in order to destroy bacteria.Xenophagy is the process referring to the selective degradation of intracellular microorganisms by autophagy. Xenophagy is a very potent intrinsic cellular line of defense to fight pathogens and requires first the detection and targeting of microorganisms to growing phagophores prior to autophagosome maturation leading to microbial destruction. The targeting step can be achieved by cytosolic autophagy receptors, which bind on the one hand to the pathogen and on the other hand to LC3, a phagophore membrane-anchored protein. Once entrapped within an autophagosome, bacteria can survive or escape, unless they are rapidly destroyed. Therefore, autophagosome maturation allows the discharge of lysosomal enzymes in autolysosomes, allowing destruction of the bacteria. It is, however, not well known how autophagosomes mature, especially in the context of xenophagy. Recently, the endosomal membrane-bound protein TOM1 and the dynein motor MYO6 have been both shown to be implicated in the transport of endosomes into the vicinity of autophagosomes in order to ensure fusion of autophagosomes with vesicles of the endo/lysosomal pathway. Moreover, the concomitant absence of 3 autophagy receptors, CALCOCO2, TAX1BP1/T6BP, and OPTN/OPTINEURIN, impairs autophagosome biogenesis and maturation. As CALCOCO2 was already shown to have a MYO6 binding domain, we wondered whether CALCOCO2 could also be implicated in autophagosome maturation per se to promote bacterial degradation.We first observed that the binding site of CALCOCO2 to MYO6 was required for cells to control Salmonella Typhimurium intracellular growth. Nevertheless, when the binding of CALCOCO2 to MYO6 was abolished, bacteria were still efficiently targeted to autophagosomes, but yet still able to replicate to levels similar to the one observed in CALCOCO2-depleted cells. Strikingly, in noninfected cells the absence of CALCOCO2 perturbs the autophagy flux, resulting in a strong accumulation of autophagosomes, suggesting a positive role for CALCOCO2 in the autophagosome-lysosome fusion process. Surprisingly, we found that CALCOCO2 binding to LC3C, through its noncanonical LC3 interacting region (CLIR), is not involved in the maturation of autophagosomes. Instead, we identified another motif in the primary sequence of CALCOCO2, which mediates binding to at least LC3A, LC3B, and GABARAPL2 (but not LC3C). We referred to this motif as “LIR-like” as it differs from the canonical LIR motif by the absence of a hydrophobic residue in position X₃. This LIR-like motif was necessary for autophagosome maturation, along with the domain of CALCOCO2 responsible for its binding to MYO6. Eventually, mutation of this LIR-like motif also resulted in an increased Salmonella Typhimurium intracellular proliferation, whereas bacteria were still efficiently targeted within nondegradative autophagosomes. Interestingly, the absence of the autophagy receptor OPTN also led to the accumulation of nondegradative autophagosomes, suggesting that other autophagy receptors could share CALCOCO2 dual functions in xenophagy.Having autophagy receptors ensuring both targeting and degradation of pathogens could be an important evolutionary advantage against infections. Indeed, this mechanism could help to reduce the delay necessary for maturation, thus avoiding adaptation of the pathogen to its new environment (as proposed for Coxiella burnetti, Listeria monocytogenes, and Legionella pneumophila) or its escape from the autophagosome. Conversely, pathogens could avoid autophagy entrapment or autophagic degradation by targeting CALCOCO2 or any other autophagy receptors, which could play similar roles. For instance Chikungunya virus was reported to target CALCOCO2 in human cells leading to increased virus replication. Nevertheless, redundancy among autophagy receptors could also ensure a selective immune advantage against pathogens targeting any one of these receptors.Our results and those from others suggest for now that CALCOCO2 serves as a docking platform for MYO6-bound endosomes, thus facilitating autophagosome maturation (Fig. 1). How this action is coordinated with CALCOCO2 directing pathogens to the phagophore membranes remains unclear. During xenophagy against Salmonella Typhimurium, CALCOCO2 interaction first with LC3C is necessary to further recruit other ATG8 orthologs and ensure the final degradation of bacteria. Since the LIR-like motifs bind several ATG8s, whereas the CLIR motif only mediates binding to LC3C, it is possible that binding of CALCOCO2 to LC3C induces conformational changes and uncovers the LIR-like motif that can be then engaged with other ATG8 orthologs to trigger autophagosome maturation. Moreover, it is still unclear whether the action of CALCOCO2 in autophagosome maturation is coordinated with other partners, such as STX17/SYNTAXIN 17, which is recruited on the external membrane of autophagosomes and regulate fusion with lysosomes. Open in a separate windowFigure 1.Schematic model for the dual role of CALCOCO2 in xenophagy. CALCOCO2 targets bacteria to the phagophore through its LC3C binding site (CLIR motif), and, independently, regulates autophagosome maturation through its LC3A, LC3B, or GABARAPL2 binding site (LIR-like motif) and its MYO6 interacting region.Our findings reveal a new role for the autophagy receptor CALCOCO2 in autophagosome maturation, unravelling another function for CALCOCO2 in cell autonomous defense against pathogens: CALCOCO2 not only targets pathogens to phagophore membranes, but also regulates subsequent maturation of pathogen-containing autophagosomes, thus assuring efficient degradation of autophagy-targeted pathogens.     

Foot-and-Mouth Disease Virus Induces Autophagosomes during Cell Entry via a Class III Phosphatidylinositol 3-Kinase-Independent Pathway

Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, ∼50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication.