The Intrinsically Disordered N Terminus in Atg12 from Yeast Is Necessary for the Functional Structure of the Protein.

The Atg12 protein in yeast is an indispensable polypeptide in the highly conserved ubiquitin-like conjugation system operating in the macroautophagy/autophagy pathway. Atg12 is covalently conjugated to Atg5 through the action of Atg7 and Atg10; the Atg12-Atg5 conjugate binds Atg16 to form an E3 ligase that functions in a separate conjugation pathway involving Atg8. Atg12 is comprised of a ubiquitin-like (UBL) domain preceded at the N terminus by an intrinsically disordered protein region (IDPR), a domain that comprises a major portion of the protein but remains elusive in its conformation and function. Here, we show that the IDPR in unconjugated Atg12 is positioned in proximity to the UBL domain, a configuration that is important for the functional structure of the protein. A major deletion in the IDPR disrupts intactness of the UBL domain at the unconjugated C terminus, and a mutation in the predicted α0 helix in the IDPR prevents Atg12 from binding to Atg7 and Atg10, which ultimately affects the protein function in the ubiquitin-like conjugation cascade. These findings provide evidence that the IDPR is an indispensable part of the Atg12 protein from yeast.

DNA damage checkpoint activation affects peptidoglycan synthesis and late divisome components in Bacillus subtilis.

During normal DNA replication, all cells encounter damage to their genetic material. As a result, organisms have developed response pathways that provide time for the cell to complete DNA repair before cell division occurs. In Bacillus subtilis, it is well established that the SOS-induced cell division inhibitor YneA blocks cell division after genotoxic stress; however, it remains unclear how YneA enforces the checkpoint. Here, we identify mutations that disrupt YneA activity and mutations that are refractory to the YneA-induced checkpoint. We find that YneA C-terminal truncation mutants and point mutants in or near the LysM peptidoglycan binding domain render YneA incapable of checkpoint enforcement. In addition, we develop a genetic method which isolated mutations in the ftsW gene that completely bypassed checkpoint enforcement while also finding that YneA interacts with late divisome components FtsL, Pbp2b, and Pbp1. Characterization of an FtsW variant resulted in considerably shorter cells during the DNA damage response indicative of hyperactive initiation of cell division and bypass of the YneA-enforced DNA damage checkpoint. With our results, we present a model where YneA inhibits septal cell wall synthesis by binding peptidoglycan and interfering with interaction between late arriving divisome components causing DNA damage checkpoint activation.

The world's first (and probably last) autophagy video game.

Macroautophagy/autophagy is the process by which portions of the cytoplasm are sequestered within a transient compartment and delivered to the degradative organelle of the cell, the vacuole or lysosome. Autophagy is a fundamental cytoprotective mechanism, and defects in this process are associated with many diseases. For example, the inability to degrade certain cargo such as mitochondria may lead to neurodegenerative disorders such as Parkinson disease. Autophagic cargo can be many different things including organelles, but also proteins and protein aggregates, nucleic acids, and lipids. Much of our understanding of autophagy comes from studies in baker's yeast, Saccharomyces cerevisiae. In that organism, autophagy begins at the phagophore assembly site (PAS), which nucleates the initial sequestering compartment, referred to as a phagophore. With the help of autophagy-related (Atg) proteins and lipid addition, the phagophore membrane expands to enclose damaged or superfluous cytoplasmic components, eventually closing into a completed double-membrane vesicle called the autophagosome. The autophagosome is delivered to the degradative organelle where it fuses, releasing the encapsulated cargo into the interior of the organelle where it is broken down into macromolecular building blocks. The resulting building blocks are released back into the cytosol for reuse. Video games are modern expressions of art incorporating illustration, animation, and mechanistic design. While often underappreciated as a scientific art form, video games can beautifully express scientific topics in a way that is both intuitive and engaging, especially to a younger audience.

Bruchpiloting synaptic connections: Egfr in autophagy-mediated neuronal circuit development.

Macroautophagy/autophagy is involved in many aspects of human development including the formation of neuronal circuits. A recent study from Dutta et al. found that the recruitment of Egfr (Epidermal growth factor receptor) to synapses suppresses autophagic degradation of presynaptic proteins, a requirement for proper neuronal circuit development. The findings suggest that Egfr inactivation during a distinct critical interval in late development results in increased levels of autophagy in the brain and decreased neuronal circuit development. Furthermore, the presence of brp (bruchpilot) in the synapse is critical for proper neuronal functioning over this same period. Dutta and colleagues found that increased autophagy due to Egfr inactivation results in decreased brp levels and, therefore, reduced neuronal connectivity. Through live cell imaging, it was determined that only the synaptic branches that accumulate both Egfr and brp are stabilized, allowing for the persistence of active zones, further supporting the importance of both Egfr and brp in the brain. While Dutta and colleagues collected these data based on studies conducted on Drosophila brains, the findings provide great insight as to how these different proteins may be implicated in human neurology.

Dimerization-dependent membrane tethering by Atg23 is essential for yeast autophagy.

Eukaryotes maintain cellular health through the engulfment and subsequent degradation of intracellular cargo using macroautophagy. The function of Atg23, despite being critical to the efficiency of this process, is unclear due to a lack of biochemical investigations and an absence of any structural information. In this study, we use a combination of in vitro and in vivo methods to show that Atg23 exists primarily as a homodimer, a conformation facilitated by a putative amphipathic helix. We utilize small-angle X-ray scattering to monitor the overall shape of Atg23, revealing that it contains an extended rod-like structure spanning approximately 320 Å. We also demonstrate that Atg23 interacts with membranes directly, primarily through electrostatic interactions, and that these interactions lead to vesicle tethering. Finally, mutation of the hydrophobic face of the putative amphipathic helix completely precludes dimer formation, leading to severely impaired subcellular localization, vesicle tethering, Atg9 binding, and autophagic efficiency.

Atg23 is a vesicle-tethering protein.

Small 30-nm vesicles containing the integral membrane protein Atg9 provide the initial membrane source for autophagy in yeast. Atg23 is an Atg9 binding protein that is required for Atg9 vesicle trafficking but whose exact function is unknown. In our recent paper, we explored the function of Atg23 using an approach combining cellular biology and biochemistry on purified protein. We determined that Atg23 is an elongated dimer spanning 320 Å in length. We also demonstrated that Atg23 is a membrane-binding and -tethering protein. Furthermore, we identified a series of amino acids residing in a putative coiled-coil region that when mutated prevent Atg23 dimer formation resulting in a stable Atg23 monomer. Last, we demonstrated that when monomeric Atg23 is expressed in yeast lacking Atg23, this leads to a loss of Atg23 puncta, a reduction in Atg9 puncta, a reduction in nonselective autophagy and a complete block in the cytoplasm-to-vacuole targeting (Cvt) pathway.

Post-transcriptional regulation of ATG1 is a critical node that modulates autophagy during distinct nutrient stresses.

Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches.

The expanding role of Atg8.

It would be quite convenient if every protein had one distinct function, one distinct role in just a single cellular process. In the field of macroautophagy/autophagy, however, we are increasingly finding that this is not the case; several autophagy proteins have two or more roles within the process of autophagy and many even "moonlight" as functional members of entirely different cellular processes. This is perhaps best exemplified by the Atg8-family proteins. These dynamic proteins have already been reported to serve several functions both within autophagy (membrane tethering, membrane fusion, binding to cargo receptors, binding to autophagy machinery) and beyond (LC3-associated phagocytosis, formation of EDEMosomes, immune signaling) but as Maruyama and colleagues suggest in their recent report, this list of functions may not yet be complete.

Structure of human ATG9A: how holey art thou?

Several studies have provided insight into the unique intracellular localization, dynamic trafficking and diverse repertoire of binding partners of Atg9/ATG9, but structural details of the protein have remained elusive. Guardia and colleagues now report the structure of human ATG9A to a resolution of 2.9 Å, revealing, among other features, an elaborate system of tunnels permeating the ATG9A protein complex.

Applications of machine learning in behavioral ecology: Quantifying avian incubation behavior and nest conditions in relation to environmental temperature.

In the age of machine learning, building programs that take advantage of the speed and specificity of algorithm development can greatly aid efforts to quantify and interpret changes in animal behavior in response to abiotic environmental factors, like temperature. For both endotherms and ectotherms, temperature can affect everything from daily energy budgets to nesting behaviors. For instance, in birds environmental temperature plays a key role in shaping parental incubation behavior and temperatures experienced by embryos. Recent research indicates that temperatures experienced by embryos affect viability and are important in shaping fitness-related traits in young birds, sparking renewed interest in relationships among environmental factors, parental incubation behavior, and incubation temperature. Incubation behavior of birds can be monitored non-invasively by placing thermal probes into the nest and analyzing temperature fluctuations that occur as parents attend and leave the nest (on- and off-bouts, respectively). When other measures of temperature (e.g., ambient air or operative temperature) are collected simultaneously with incubation temperature it is possible to compare shifts in behavior with environmental changes. To improve analysis of incubation behavior using these large thermal data sets we developed a program, NestIQ, that uses machine learning to guide parameter optimization allowing it to track the behavior of diverse species. NestIQ's algorithm was tested using six species incubating in lab or field scenarios, that exhibit unique incubation patterns. This stand-alone and open source software is operated through a graphical user interface (i.e., no user programming is required) that provides important behavioral and thermal output statistics. Further, measures of environmental temperature can be imported alongside nest temperature into the program, which then reports various attributes of environmental temperature during shifts in parental behavior. This program will improve the ability of avian ecologists to interpret a critical parental care behavior that can be used across diverse incubation scenarios and species. Although specifically designed for quantifying avian incubation, NestIQ has the potential for broader applications, including basking and nesting behaviors of non-avian reptiles in relation to ambient temperature.

Watch What You (Self-) Eat: Autophagic Mechanisms that Modulate Metabolism.

Autophagy is an evolutionarily conserved lysosome- or vacuole-dependent catabolic pathway in eukaryotes. Autophagy functions basally for cellular quality control and is induced to act as an alternative source of basic metabolites during nutrient deprivation. These functions of autophagy are intimately connected to the regulation of metabolism, and the metabolic status of the cell in turn controls the nature and extent of autophagic induction. Here, we highlight the co-regulation of autophagy and metabolism with a special focus on selective autophagy that, along with bulk autophagy, plays a central role in regulating and rewiring metabolic circuits. We outline the metabolic signals that activate these pathways, the mechanisms involved, and the downstream effects and implications while recognizing yet unanswered questions. We also discuss the role of autophagy in the development and maintenance of adipose tissue, an emerging player in systemic metabolic homeostasis, and describe what is currently known about the complex relationship between autophagy and cancer.