Macroautophagy is essential to cell survival during starvation and proceeds by the growth of a double-membraned phagophore, which engulfs cytosol and other substrates. The synthesis and recognition ...of the lipid phosphatidylinositol 3-phosphate, PI(3)P, is essential for autophagy. The key autophagic PI(3)P sensors, which are conserved from yeast to humans, belong to the PROPPIN family. Here we report the crystal structure of the yeast PROPPIN Hsv2. The structure consists of a seven-bladed β-propeller and, unexpectedly, contains two pseudo-equivalent PI(3)P binding sites on blades 5 and 6. These two sites both contribute to membrane binding in vitro and are collectively required for full autophagic function in yeast. These sites function in concert with membrane binding by a hydrophobic loop in blade 6, explaining the specificity of the PROPPINs for membrane-bound PI(3)P. These observations thus provide a structural and mechanistic framework for one of the conserved central molecular recognition events in autophagy.
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► Crystal structure of the yeast PROPPIN Hsv2 ► Two sites in propeller blades 5 and 6 bind to phosphatidylinositol 3-phosphate ► Aromatic residues of a loop in propeller blade 6 help bind the membrane ► Both PI(3)P binding sites in the aromatic loop residues are important in autophagy
Macroautophagy is a bulk clearance mechanism in which the double-membraned phagophore grows and engulfs cytosolic material. In yeast, the phagophore nucleates from a cluster of 20–30 nm diameter ...Atg9-containing vesicles located at a multiprotein assembly known as the preautophagosomal structure (PAS). The crystal structure of a 2:2:2 complex of the earliest acting PAS proteins, Atg17, Atg29, and Atg31, was solved at 3.05 Å resolution. Atg17 is crescent shaped with a 10 nm radius of curvature. Dimerization of the Atg17-Atg31-Atg29 complex is critical for both PAS formation and autophagy, and each dimer contains two separate and complete crescents. Upon induction of autophagy, Atg17-Atg31-Atg29 assembles with Atg1 and Atg13, which in turn initiates the formation of the phagophore. The C-terminal EAT domain of Atg1 was shown to sense membrane curvature, dimerize, and tether lipid vesicles. These data suggest a structural mechanism for the organization of Atg9 vesicles into the early phagophore.
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► Crystal structure of the yeast Atg17-Atg31-Atg29 autophagy initiation complex ► Atg17 is crescent shaped and functions as a dimer of two crescents ► C-terminal EAT domain of Atg1 binds and tethers highly curved vesicles ► Atg1 EAT domain binds Atg17 via Atg13, leading to a model for the PAS
The crystal structure of the Atg17-Atg31-Atg29 autophagy initiation complex reveals a dimer that adopts two crescent scaffolds. Atg1 associates with this initiation complex and also tethers highly curved vesicles, organizing them into the early phagophore.
In the Brazilian Cerrado, the Red-and-green Macaw (Ara chloropterus) populations are facing an accelerated rate of habitat loss. Despite this, their feeding areas and primary food sources remain ...poorly understood. In this study, I assessed the relationship between the diet of the Red-and-green Macaw and available food resources in a habitat mosaic from the fragmented Cerrado in Mato Grosso do Sul State, Brazil. Red-and-green Macaws fed on 20 native and five exotic species, mainly in dry habitats (Cerrado, dry forest, and an urban area along the Maracaju Cliffs) during the dry season, while year-round foraging in the riparian vegetation (Aquidauana River and streams). Then, the number of feeding macaws paralleled variations in food abundance and diversity, besides the number of food species. On the other hand, by using a wide variety of abundant foods, macaws' diet breadth presented high values throughout the year. The seasonal consumption of large-seeded fruits across the habitat mosaic displayed a gradient ranging from the urban area to dry habitats, along which macaws ate from exotic to Cerrado species. In this respect, Terminalia catappa seeds and Mangifera indica fruit pulp were important for Red-and-green Macaws in the urban area during the wet season, while Caryocar brasiliense seeds comprised the same in the Cerrado. At this site, both Dipteryx alata and Buchenavia tomentosa seeds composed much of the Red-and-green Macaws' diet during the dry season. Between those habitats, in the watercourse vegetation, macaws frequently foraged on palm fruits across seasons. Therefore, throughout the year, the abundance and variety of food resources strongly influenced the number of foraging Red-and-green Macaws across the habitat mosaic. Understanding the effect of varying seed availability on the spatial and temporal abundance patterns of Red-and-green Macaws, which primarily feed on large-seeded species, is central to developing effective conservation strategies. Due to the accelerated habitat loss, the Maracaju Cliffs emerge as crucial for the Red-and-green Macaw among the Cerrado remnants of Mato Grosso do Sul.
The edge effect has impacts on seed and seedling survival due to modifications in biotic and abiotic factors. Often, large-seeded tree species lost seed vectors in the forest edge due to the rarity ...or absence of large frugivores at this habitat type. In this study, I compared the seedling abundance and distribution of the palm Syagrus flexuosa between edges and interiors of three large Cerrado remnants. In every remnant, the number of seedlings around parent palms in the edge was smaller than around palm individuals located in the Cerrado interior. Moreover, the distribution of seedlings around parent palms differed between edges and interiors. In the edges, most seedlings were found under parent crowns, while in the interiors, the contrary occurred. The high concentration of seedlings under parent palms suggests a decrease of seed dispersal at the edges. Because S. flexuosa is a widely distributed palm that serves as an important resource for several animals along Cerrado habitats, changes on the regeneration process of this palm due to edge effects can further impact frugivore populations. Therefore, the decline of seedling establishment along forest edges implies changes in the Cerrado regeneration dynamics, which may compromise the persistence of ecological processes and animal communities.
Alzheimer's disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (Aβ) in the brain has historically been ...associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that Aβ is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e., microglia), which culminates in neuronal damage and cognitive decline. To test this hypothesis, many in vitro systems have been established to study Aβ-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale.
We used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic Aβ or LPS and INFγ.
We found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model that best resembles primary microglia. Surprisingly, synthetic Aβ does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of Aβ formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INFγ activate microglia and induce transcriptional changes that resemble many, but not all, aspects of the transcriptomic profiles of disease associated microglia (DAM) present in the AD brain.
These results suggest that synthetic Aβ treatment of innate immune cell cultures does not recapitulate transcriptional profiles observed in microglia from AD brains. In contrast, treating IMGL with LPS and INFγ induces transcriptional changes similar to those observed in microglia detected in AD brains.
Autophagy-related 13 (Atg13) is a key early-acting factor in autophagy and the major locus for nutrient-dependent regulation of autophagy by Tor. The 2.3-Å resolution crystal structure of the ...N-terminal domain of Atg13 reveals a previously unidentified HORMA (Hop1p, Rev1p and Mad2) domain similar to that of the spindle checkpoint protein Mad2. Mad2 has two different stable conformations, O-Mad2 and C-Mad2, and the Atg13 HORMA structure corresponds to the C-Mad2 state. The Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PI) 3-kinase subunit Atg14 but is not required for Atg1 interaction or Atg13 recruitment to the preautophagosomal structure. The Atg13 HORMA structure reveals a pair of conserved Arg residues that constitute a putative phosphate sensor. One of the Arg residues is in the region corresponding to the “safety belt” conformational switch of Mad2, suggesting conformational regulation of phosphate binding. These two Arg residues are essential for autophagy, suggesting that the Atg13 HORMA domain could function as a phosphoregulated conformational switch.
Palms are an important component of Neotropical communities as they are often diverse and abundant. In some areas, palms occur in high density and act as limiting factor in tree recruitment by ...limiting tree seedling and sapling abundance. In this study, I evaluated the intensity of seed mortality caused by insects in Attalea geraensis, in a large area of preserved Cerrado (Serra do Cabral, MG, Brazil) during wet season when both A. geraensis fruits and bruchid beetles were abundant. I collected a total of 63 infructescences which had from 3 fruits and 7 seeds to 82 fruits and 251 seeds. Endocarps had from 1 to 6 seeds. Seed mortality per infructescence due to beetles (Pachymerus cardo) was intense, and increases positively and disproportionally according to seed number per infructescence. Besides that, average proportions of seeds preyed upon by insects were consistently high (> 0.83), irrespective of seed number per endocarp. Positive density-dependent seed mortality caused by specialized natural enemies has been assumed to promote species rarity, an important feature of species coexistence in Neotropical forests. Then, the intense seed mortality documented in this study suggests that seed predators may contribute to the richness and diversity of plant species in the Cerrado, the richest and most endangered savanna in the world.
Highlights • The phagophore grows from a few Atg9-positive vesicles of ∼20 nm diameter. • Atg17, Atg29, and Atg31 form a structure that potentially scaffolds vesicles. • Atg1 and Atg13 join Atg17 at ...the PAS in starvation. • The phagophore grows and closes, becoming the autophagosome.
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•Atg16 in the budding yeast S. cerevisiae is an intrinsically disordered polypeptide.•The C terminal amphipathic helix (at amino acid residues 113–131) anchors Atg16 on membranes.•The ...coiled-coil domain of Atg16 (at amino acid residues 64–99) mediates homodimerization in vivo.•Both membrane binding and homodimerization are required for Atg16 function in autophagy.
Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.
Selective autophagy is the capture of specific cytosolic contents in double-membrane vesicles that subsequently fuse with the vacuole or lysosome, thereby delivering cargo for degradation. Selective ...autophagy receptors (SARs) mark the cargo for degradation and, in yeast, recruit Atg11, the scaffolding protein for selective autophagy initiation. The mitochondrial protein Atg32 is the yeast SAR that mediates mitophagy, the selective autophagic capture of mitochondria. Atg11–Atg32 interactions concentrate Atg32 into puncta that are thought to represent sites of mitophagy initiation. However, it is unclear how Atg11 concentrates Atg32 to generate mitophagy initiation sites. We show here that the coiled coil 3 (CC3) domain of Atg11 is required for concentrating Atg32 into puncta. We determined the structure of the majority of the CC3, demonstrating that the CC3 forms a parallel homodimer whose dimer interface is formed by a small number of hydrophobic residues. We further show that the CC3 interface is not required for Atg11 dimerization but is required for shaping Atg32 into functional mitophagy initiation sites and for delivery of mitochondria to the vacuole. Our findings suggest that Atg11 self-interactions help concentrate SARs as a necessary precondition for cargo capture.
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•The outer mitochondrial membrane protein Atg32 is clustered to start mitophagy.•The third coiled coil domain of Atg11 is required for Atg32 clustering.•The 2.03 Å crystal structure of Atg11699–800 was determined.•Mutation of the dimer interface of Atg11699–800 blocks Atg32 clustering.•Atg32 puncta must be shaped by Atg11 for mitophagy.
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