Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest

Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.     

The small GTPases Rab9A and Rab23 function at distinct steps in autophagy during Group A Streptococcus infection

Autophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in immunity. Here we identified the small GTPases Rab9A and Rab23 as novel autophagy regulators during Group A streptococcus (GAS) infection. Rab9A was recruited to GAS-containing autophagosome-like vacuoles (GcAVs) after autophagosomal maturation and its activity was required for GcAV enlargement and eventual lysosomal fusion. GcAV enlargement appeared to be related to homotypic fusion of GcAVs with Rab9A. Rab23 was recruited to GAS-capturing forming autophagosomes. Knockdown of Rab23 expression decreased both LC3- and Atg5-positive GAS formation and caused the accumulation of LC3-positive structures that did not associate with intracellular GAS. It was suggested, therefore, that Rab23 is required for GcAV formation and is involved in GAS targeting of autophagic vacuoles. Furthermore, knockdown of Rab9A or Rab23 expression impaired the degradation of intracellular GAS. Therefore, our data reveal that the Rab9A and Rab23 GTPases play crucial roles in autophagy of GAS. However, neither Rab9A nor Rab23 were localized to starvation-induced autophagosomes. Not only Rab9A but also Rab23 was dispensable for starvation-induced autophagosome formation. These findings demonstrate that specific Rab proteins function at distinct steps during autophagy in response to GAS infection.     

TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain-containing Rab guanosine triphosphatase-activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)-positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.     

Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites

Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double‐membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long‐standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant‐negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)‐II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.     

Autophagy Induced by Calcium Phosphate Precipitates Involves Endoplasmic Reticulum Membranes in Autophagosome Biogenesis

Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.     

Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24

Rab GTPases comprises a large family of proteins, with more than 50 gene products localized in distinct subcellular compartments. Rab24 is a member of this family whose function is not presently known. In order to elucidate the role of this protein we have generated a GFP-tagged Rab24 and studied the distribution of this chimera by fluorescence microscopy. GFP-Rab24 showed a perinuclear reticular localization that often encircled the nucleus. This reticular pattern partially overlapped with ER markers, cis-Golgi, and the ER-Golgi intermediate compartment. Surprisingly, when GFP-Rab24-transfected cells were starved to induce autophagy the distribution of the protein changed dramatically. GFP-Rab24 localized in large dots, cup-shaped structures and ring-shaped vesicles. Some of these vesicles were labeled with monodansylcadaverine , a specific autophagosome marker. In the presence of vinblastine, an agent that induces the formation of very large autophagic vesicles, GFP-Rab24 accumulated in the large vacuoles that were also labeled by monodansylcadaverine. Furthermore, Rab24 colocalized with LC3, a mammalian homolog of the yeast protein Apg8/Aut7, an essential gene for autophagy. This is the first report indicating that Rab24 localizes on autophagosomes, suggesting that this Rab protein is involved in the autophagic pathway.     

The Atg17-Atg31-Atg29 complex and Atg11 regulate autophagosome-vacuole fusion

The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles fuse with the vacuole/lysosome. Genetic studies in yeasts have characterized more than 40 autophagy-related (Atg) proteins required for autophagy, and the majority of these proteins play roles in autophagosome formation. The fusion of autophagosomes with the vacuole is mediated by the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, these factors are not autophagosome-vacuole fusion specific. We recently showed that 2 autophagy scaffold proteins, the Atg17-Atg31-Atg29 complex and Atg11, regulate autophagosome-vacuole fusion by recruiting the vacuolar SNARE Vam7 to the phagophore assembly site (PAS), where an autophagosome forms in yeast.     

Small but mighty: Atg8s and Rabs in membrane dynamics during autophagy

Autophagy is a degradative pathway during which autophagosomes are formed that enwrap cytosolic material destined for turnover within the lytic compartment. Autophagosome biogenesis requires controlled lipid and membrane rearrangements to allow the formation of an autophagosomal seed and its subsequent elongation into a fully closed and fusion-competent double membrane vesicle. Different membrane remodeling events are required, which are orchestrated by the distinct autophagy machinery. An important player among these autophagy proteins is the small lipid-modifier Atg8. Atg8 proteins facilitate various aspects of autophagosome formation and serve as a binding platform for autophagy factors. Also Rab GTPases have been implicated in autophagosome biogenesis. As Atg8 proteins interact with several Rab GTPase regulators, they provide a possible link between autophagy progression and Rab GTPase activity. Here, we review central aspects in membrane dynamics during autophagosome biogenesis with a focus on Atg8 proteins and selected Rab GTPases.     

RNA-Dependent Protein Kinase Is Essential for 2-Methoxyestradiol-Induced Autophagy in Osteosarcoma Cells

Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgical resection and adjunctive chemotherapy are the only widely available options of treatment for this disease. Anti-tumor compound 2-Methoxyestradiol (2-ME) triggers cell death through the induction of apoptosis in osteosarcoma cells, but not in normal osteoblasts. In this report, we have investigated whether autophagy plays a role in 2-ME actions on osteosarcoma cells. Transmission electron microscopy imaging shows that 2-ME treatment leads to the accumulation of autophagosomes in human osteosarcoma cells. 2-ME induces the conversion of the microtubule-associated protein LC3-I to LC3-II, a biochemical marker of autophagy that is correlated with the formation of autophagosomes. Conversion to LC3-II is accompanied by protein degradation in 2-ME-treated cells. 2-ME does not induce autophagosome formation in normal primary human osteoblasts. In addition, 2-ME-dependent autophagosome formation in osteosarcoma cells requires ATG7 expression. Furthermore, 2-ME does not induce accumulation of autophagosomes in osteosarcoma cells that express dominant negative mutant RNA-dependent protein kinase (PKR) and are resistant to anti-proliferative and anti-tumor effects of 2-ME. Taken together, our study shows that 2-ME treatment induces PKR-dependent autophagy in osteosarcoma cells, and that autophagy could play an important role in 2-ME-mediated anti-tumor actions and in the control of osteosarcoma.     

WHAMM links actin assembly via the Arp2/3 complex to autophagy

Macroautophagy (hereafter autophagy) is the process by which cytosolic material destined for degradation is enclosed inside a double-membrane cisterna known as the autophagosome and processed for secretion and/or recycling. This process requires a large collection of proteins that converge on certain sites of the ER membrane to generate the autophagosome membrane. Recently, it was shown that actin accumulates around autophagosome precursors and could play a role in this process, but the mechanism and role of actin polymerization in autophagy were unknown. Here, we discuss our recent finding that the nucleation-promoting factor (NPF) WHAMM recruits and activates the Arp2/3 complex for actin assembly at sites of autophagosome formation on the ER. Using high-resolution, live-cell imaging, we showed that WHAMM forms dynamic puncta on the ER that comigrate with several autophagy markers, and propels the spiral movement of these puncta by an Arp2/3 complex-dependent actin comet tail mechanism. In starved cells, WHAMM accumulates at the interface between neighboring autophagosomes, whose number and size increases with WHAMM expression. Conversely, knocking down WHAMM, inhibiting the Arp2/3 complex or interfering with actin polymerization reduces the size and number of autophagosomes. These findings establish a link between Arp2/3 complex-mediated actin assembly and autophagy.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Phosphorylation of the exchange factor DENND3 by ULK in response to starvation activates Rab12 and induces autophagy

Unc-51-like kinases (ULKs) are the most upstream kinases in the initiation of autophagy, yet the molecular mechanisms underlying their function are poorly understood. We report a new role for ULK in the induction of autophagy. ULK-mediated phosphorylation of the guanine nucleotide exchange factor DENND3 at serines 554 and 572 upregulates its GEF activity toward the small GTPase Rab12. Through binding to LC3 and associating with LC3-positive autophagosomes, active Rab12 facilitates autophagosome trafficking, thus establishing a crucial role for the ULK/DENND3/Rab12 axis in starvation-induced autophagy.     

Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes

Nutrient deprivation of eukaryotic cells provokes a variety of stress responses, including autophagy. Autophagy is carried out by autophagosomes which sequester cytosolic components and organelles for degradation after fusion with protease-containing endosomes. To determine the role of microtubules in autophagy, we used nocodazole and vinblastine to disrupt microtubules and independently measured formation and fusion of autophagsosomes in primary rat hepatocytes. By measuring the translocation of GFP-LC3, an autophagosomal marker, to autophagosomes and the lipidation of GFP-LC3, we quantified the rate and magnitude of autophagosome formation. Starvation increased both the rate of autophagosome formation over the basal level and the total number of autophagosomes per cell. Maximal autophagosome formation required an intact microtubule network. Fusion of autophagosomes with endosomes, assayed by acquisition of protease-inhibitor sensitivity as well as overlap with LysoTracker Red-positive endosomes, required intact microtubules. Live-cell imaging demonstrated that autophagosomes were motile structures, and their movement also required microtubules. Interestingly, vinblastine stimulated autophagosome formation more than twofold before any discernable change in the microtubule network was observed. Stimulation of autophagosome formation by vinblastine was independent of nutrients and mTOR activity but was inhibited by depletion of the Autophagy proteins Atg5 and Atg6, known to be required for autophagy.     

Regulation of macroautophagy by mTOR and Beclin 1 complexes

Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations, including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in regulating autophagosome formation.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Rab11 facilitates cross-talk between autophagy and endosomal pathway through regulation of Hook localization

During autophagy, double-membrane autophagosomes deliver sequestered cytoplasmic content to late endosomes and lysosomes for degradation. The molecular mechanism of autophagosome maturation is still poorly characterized. The small GTPase Rab11 regulates endosomal traffic and is thought to function at the level of recycling endosomes. We show that loss of Rab11 leads to accumulation of autophagosomes and late endosomes in Drosophila melanogaster. Rab11 translocates from recycling endosomes to autophagosomes in response to autophagy induction and physically interacts with Hook, a negative regulator of endosome maturation. Hook anchors endosomes to microtubules, and we show that Rab11 facilitates the fusion of endosomes and autophagosomes by removing Hook from mature late endosomes and inhibiting its homodimerization. Thus induction of autophagy appears to promote autophagic flux by increased convergence with the endosomal pathway.     

Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes

Autophagy acts as a host-defense system against pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we identified Rab30 as a novel regulator of GAS-containing autophagosome-like vacuoles (GcAVs). We found that Rab30, a Golgi-resident Rab, was recruited to GcAVs in response to autophagy induction by GAS infection in epithelial cells. Rab30 recruitment was dependent upon its GTPase activity. In addition, the knockdown of Rab30 expression significantly reduced GcAV formation efficiency and impaired intracellular GAS degradation. Rab30 normally functions to maintain the structural integrity of the Golgi complex, but GcAV formation occurred even when the Golgi apparatus was disrupted. Although Rab30 also colocalized with a starvation-induced autophagosome, Rab30 was not required for autophagosome formation during starvation. These results suggest that Rab30 mediates autophagy against GAS independently of its normal cellular role in the structural maintenance of the Golgi apparatus, and autophagosome biogenesis during bacterial infection involves specific Rab GTPases.     

LAMTOR2/LAMTOR1 complex is required for TAX1BP1‐mediated xenophagy

Xenophagy, also known as antibacterial autophagy, plays a role in host defence against invading pathogens such as Group A Streptococcus (GAS) and Salmonella. In xenophagy, autophagy receptors are used in the recognition of invading pathogens and in autophagosome maturation and autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. In this study, we identified LAMTOR2 and LAMTOR1, also named p14 and p18, respectively, as previously unrecognised xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion. LAMTOR1 was localized to bacterium‐containing endosomes, and LAMTOR2 was recruited to bacterium‐containing damaged endosomes in a LAMTOR1‐dependent manner. LAMTOR2 was dispensable for the formation of autophagosomes targeting damaged membrane debris surrounding cytosolic bacteria, but it was critical for autolysosome formation, and LAMTOR2 interacted with the autophagy receptors NBR1, TAX1BP1, and p62 and was necessary for TAX1BP1 recruitment to pathogen‐containing autophagosomes. Notably, knockout of TAX1BP1 caused a reduction in autolysosome formation and subsequent bacterial degradation. Collectively, our findings demonstrated that the LAMTOR1/2 complex is required for recruiting TAX1BP1 to autophagosomes and thereby facilitating autolysosome formation during bacterial infection.     

Starvation-induced Hyperacetylation of Tubulin Is Required for the Stimulation of Autophagy by Nutrient Deprivation

The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.     

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.