The Hob proteins are novel and conserved lipid-binding proteins at ER-PM contact sites.

Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.

Polycomb group genes are required for neuronal pruning in Drosophila.

BACKGROUND: Pruning that selectively eliminates unnecessary or incorrect neurites is required for proper wiring of the mature nervous system. During Drosophila metamorphosis, dendritic arbourization sensory neurons (ddaCs) and mushroom body (MB) γ neurons can selectively prune their larval dendrites and/or axons in response to the steroid hormone ecdysone. An ecdysone-induced transcriptional cascade plays a key role in initiating neuronal pruning. However, how downstream components of ecdysone signalling are induced remains not entirely understood. RESULTS: Here, we identify that Scm, a component of Polycomb group (PcG) complexes, is required for dendrite pruning of ddaC neurons. We show that two PcG complexes, PRC1 and PRC2, are important for dendrite pruning. Interestingly, depletion of PRC1 strongly enhances ectopic expression of Abdominal B (Abd-B) and Sex combs reduced, whereas loss of PRC2 causes mild upregulation of Ultrabithorax and Abdominal A in ddaC neurons. Among these Hox genes, overexpression of Abd-B causes the most severe pruning defects, suggesting its dominant effect. Knockdown of the core PRC1 component Polyhomeotic (Ph) or Abd-B overexpression selectively downregulates Mical expression, thereby inhibiting ecdysone signalling. Finally, Ph is also required for axon pruning and Abd-B silencing in MB γ neurons, indicating a conserved function of PRC1 in two types of pruning. CONCLUSIONS: This study demonstrates important roles of PcG and Hox genes in regulating ecdysone signalling and neuronal pruning in Drosophila. Moreover, our findings suggest a non-canonical and PRC2-independent role of PRC1 in Hox gene silencing during neuronal pruning.

A novel function for Rab1 and Rab11 during secretory granule maturation.

Regulated exocytosis is an essential process whereby specific cargo proteins are secreted in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi network (TGN); after budding from the TGN, granules undergo modifications, including an increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here, we leverage the Drosophila larval salivary glands as a model to characterize a novel role for Rab GTPases during granule maturation. We find that secretory granules increase in size ∼300-fold between biogenesis and release, and loss of Rab1 or Rab11 reduces granule size. Surprisingly, we find that Rab1 and Rab11 localize to secretory granule membranes. Rab11 associates with granule membranes throughout maturation, and Rab11 recruits Rab1. In turn, Rab1 associates specifically with immature granules and drives granule growth. In addition to roles in granule growth, both Rab1 and Rab11 appear to have additional functions during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new role for Rab GTPases in secretory granule maturation.

The bridge-like lipid transfer protein (BLTP) gene group: introducing new nomenclature based on structural homology indicating shared function.

The HUGO Gene Nomenclature Committee assigns unique symbols and names to human genes. The use of approved nomenclature enables effective communication between researchers, and there are multiple examples of how the usage of unapproved alias symbols can lead to confusion. We discuss here a recent nomenclature update (May 2022) for a set of genes that encode proteins with a shared repeating ß-groove domain. Some of the proteins encoded by genes in this group have already been shown to function as lipid transporters. By working with researchers in the field, we have been able to introduce a new root symbol (BLTP, which stands for "bridge-like lipid transfer protein") for this domain-based gene group. This new nomenclature not only reflects the shared domain in these proteins, but also takes into consideration the mounting evidence of a shared lipid transport function.

Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover.

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. Here we show that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) and CryAB. NUAK biochemically and genetically interacts with Drosophila Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. We further demonstrate that Fil is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, our results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases.

Hobbit regulates intracellular trafficking to drive insulin-dependent growth during Drosophila development.

All animals must coordinate growth rate and timing of maturation to reach the appropriate final size. Here, we describe hobbit, a novel and conserved gene identified in a forward genetic screen for Drosophila animals with small body size. hobbit is highly conserved throughout eukaryotes, but its function remains unknown. We demonstrate that hobbit mutant animals have systemic growth defects because they fail to secrete insulin. Other regulated secretion events also fail in hobbit mutant animals, including mucin-like 'glue' protein secretion from the larval salivary glands. hobbit mutant salivary glands produce glue-containing secretory granules that are reduced in size. Importantly, secretory granules in hobbit mutant cells lack essential membrane fusion machinery required for exocytosis, including Synaptotagmin 1 and the SNARE SNAP-24. These membrane fusion proteins instead accumulate inside enlarged late endosomes. Surprisingly, however, the Hobbit protein localizes to the endoplasmic reticulum. Our results suggest that Hobbit regulates a novel step in intracellular trafficking of membrane fusion proteins. Our studies also suggest that genetic control of body size, as a measure of insulin secretion, is a sensitive functional readout of the secretory machinery.

Eyeless/Pax6 initiates eye formation non-autonomously from the peripodial epithelium.

The transcription factor Pax6 is considered the master control gene for eye formation because (1) it is present within the genomes and retina/lens of all animals with a visual system; (2) severe retinal defects accompany its loss; (3) Pax6 genes have the ability to substitute for one another across the animal kingdom; and (4) Pax6 genes are capable of inducing ectopic eye/lens in flies and mammals. Many roles of Pax6 were first elucidated in Drosophila through studies of the gene eyeless (ey), which controls both growth of the entire eye-antennal imaginal disc and fate specification of the eye. We show that Ey also plays a surprising role within cells of the peripodial epithelium to control pattern formation. It regulates the expression of decapentaplegic (dpp), which is required for initiation of the morphogenetic furrow in the eye itself. Loss of Ey within the peripodial epithelium leads to the loss of dpp expression within the eye, failure of the furrow to initiate, and abrogation of retinal development. These findings reveal an unexpected mechanism for how Pax6 controls eye development in Drosophila.

Allocation of distinct organ fates from a precursor field requires a shift in expression and function of gene regulatory networks.

A common occurrence in metazoan development is the rise of multiple tissues/organs from a single uniform precursor field. One example is the anterior forebrain of vertebrates, which produces the eyes, hypothalamus, diencephalon, and telencephalon. Another instance is the Drosophila wing disc, which generates the adult wing blade, the hinge, and the thorax. Gene regulatory networks (GRNs) that are comprised of signaling pathways and batteries of transcription factors parcel the undifferentiated field into discrete territories. This simple model is challenged by two observations. First, many GRN members that are thought to control the fate of one organ are actually expressed throughout the entire precursor field at earlier points in development. Second, each GRN can simultaneously promote one of the possible fates choices while repressing the other alternatives. It is therefore unclear how GRNs function to allocate tissue fates if their members are uniformly expressed and competing with each other within the same populations of cells. We address this paradigm by studying fate specification in the Drosophila eye-antennal disc. The disc, which begins its development as a homogeneous precursor field, produces a number of adult structures including the compound eyes, the ocelli, the antennae, the maxillary palps, and the surrounding head epidermis. Several selector genes that control the fates of the eye and antenna, respectively, are first expressed throughout the entire eye-antennal disc. We show that during early stages, these genes are tasked with promoting the growth of the entire field. Upon segregation to distinct territories within the disc, each GRN continues to promote growth while taking on the additional roles of promoting distinct primary fates and repressing alternate fates. The timing of both expression pattern restriction and expansion of functional duties is an elemental requirement for allocating fates within a single field.

Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements.

The eyes absent (eya) gene of the fruit fly, Drosophila melanogaster, is a member of an evolutionarily conserved gene regulatory network that controls eye formation in all seeing animals. The loss of eya leads to the complete elimination of the compound eye while forced expression of eya in non-retinal tissues is sufficient to induce ectopic eye formation. Within the developing retina eya is expressed in a dynamic pattern and is involved in tissue specification/determination, cell proliferation, apoptosis, and cell fate choice. In this report we explore the mechanisms by which eya expression is spatially and temporally governed in the developing eye. We demonstrate that multiple cis-regulatory elements function cooperatively to control eya transcription and that spacing between a pair of enhancer elements is important for maintaining correct gene expression. Lastly, we show that the loss of eya expression in sine oculis (so) mutants is the result of massive cell death and a progressive homeotic transformation of retinal progenitor cells into head epidermis.

A steroid-controlled global switch in sensitivity to apoptosis during Drosophila development.

Precise control over activation of the apoptotic machinery is critical for development, tissue homeostasis and disease. In Drosophila, the decision to trigger apoptosis--whether in response to developmental cues or to DNA damage--converges on transcription of inhibitor of apoptosis protein (IAP) antagonists reaper, hid and grim. Here we describe a parallel process that regulates the sensitivity to, rather than the execution of, apoptosis. This process establishes developmental windows that are permissive or restrictive for triggering apoptosis, where the status of cells determines their capacity to die. We characterize one switch in the sensitivity to apoptotic triggers, from restrictive to permissive, that occurs during third-instar larval (L3) development. Early L3 animals are highly resistant to induction of apoptosis by expression of IAP-antagonists, DNA-damaging agents and even knockdown of the IAP diap1. This resistance to apoptosis, however, is lost in wandering L3 animals after acquiring a heightened sensitivity to apoptotic triggers. This switch in sensitivity to death activators is mediated by a change in mechanisms available for activating endogenous caspases, from an apoptosome-independent to an apoptosome-dependent pathway. This switch in apoptotic pathways is regulated in a cell-autonomous manner by the steroid hormone ecdysone, through changes in expression of critical pro-, but not anti-, apoptotic genes. This steroid-controlled switch defines a novel, physiologically-regulated, mechanism for controlling sensitivity to apoptosis and provides new insights into the control of apoptosis during development.

INO80-dependent regression of ecdysone-induced transcriptional responses regulates developmental timing in Drosophila.

Sequential pulses of the steroid hormone ecdysone regulate the major developmental transitions in Drosophila, and the duration of each developmental stage is determined by the length of time between ecdysone pulses. Ecdysone regulates biological responses by directly initiating target gene transcription. In turn, these transcriptional responses are known to be self-limiting, with mechanisms in place to ensure regression of hormone-dependent transcription. However, the biological significance of these transcriptional repression mechanisms remains unclear. Here we show that the chromatin remodeling protein INO80 facilitates transcriptional repression of ecdysone-regulated genes during prepupal development. In ino80 mutant animals, inefficient repression of transcriptional responses to the late larval ecdysone pulse delays the onset of the subsequent prepupal ecdysone pulse, resulting in a significantly longer prepupal stage. Conversely, increased expression of ino80 is sufficient to shorten the prepupal stage by increasing the rate of transcriptional repression. Furthermore, we demonstrate that enhancing the rate of regression of the mid-prepupal competence factor ßFTZ-F1 is sufficient to determine the timing of head eversion and thus the duration of prepupal development. Although ino80 is conserved from yeast to humans, this study represents the first characterization of a bona fide ino80 mutation in any metazoan, raising the possibility that the functions of ino80 in transcriptional repression and developmental timing are evolutionarily conserved.

Translational control by the DEAD Box RNA helicase belle regulates ecdysone-triggered transcriptional cascades.

Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarily conserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

Down-regulation of inhibitor of apoptosis levels provides competence for steroid-triggered cell death.

A pulse of the steroid hormone ecdysone triggers the destruction of larval salivary glands during Drosophila metamorphosis through a transcriptional cascade that converges on reaper (rpr) and head involution defective (hid) induction, resulting in caspase activation and cell death. We identify the CREB binding protein (CBP) transcriptional cofactor as essential for salivary gland cell death. We show that CBP acts 1 d before the onset of metamorphosis in apparent response to a mid-third instar ecdysone pulse, when CBP is necessary and sufficient for down-regulation of the Drosophila inhibitor of apoptosis 1 (DIAP1). It is only after DIAP1 levels are reduced that salivary glands become competent to die through rpr/hid-mediated cell death. Before this time, high levels of DIAP1 block salivary gland cell death, even in the presence of ectopic rpr expression. This study shows that naturally occurring changes in inhibitor of apoptosis levels can be critical for regulating cell death during development. It also provides a molecular mechanism for the acquisition of competence in steroid signaling pathways.

A novel superfamily of bridge-like lipid transfer proteins.

Lipid transfer proteins mediate nonvesicular transport of lipids at membrane contact sites to regulate the lipid composition of organelle membranes. Recently, a new type of bridge-like lipid transfer protein has emerged; these proteins contain a long hydrophobic groove and can mediate bulk transport of lipids between organelles. Here, we review recent insights into the structure of these proteins and identify a repeating modular unit that we propose to name the repeating ß-groove (RBG) domain. This new structural understanding conceptually unifies all the RBG domain-containing lipid transfer proteins as members of an RBG protein superfamily. We also examine the biological functions of these lipid transporters in normal physiology and disease and speculate on the evolutionary origins of RBG proteins in bacteria.

The Hob proteins: Putative, novel lipid transfer proteins at ER-PM contact sites.

Nonvesicular transfer of lipids at membrane contact sites (MCS) has recently emerged as a critical process for cellular function. Lipid transfer proteins (LTPs) mediate this unique transport mechanism, and although several LTPs are known, the cellular complement of these proteins continues to expand. Our recent work has revealed the highly conserved but poorly characterized Hobbit/Hob proteins as novel, putative LTPs at endoplasmic reticulum-plasma membrane (ER-PM) contact sites. Using both S. cerevisiae and D. melanogaster model systems, we demonstrated that the Hob proteins localize to ER-PM contact sites via an N-terminal ER membrane anchor and conserved C-terminal sequences. These conserved C-terminal sequences bind to phosphoinositides (PIPs), and the distribution of PIPs is disrupted in hobbit mutant cells. Recently released structural models of the Hob proteins exhibit remarkable similarity to other bona fide LTPs, like VPS13A and ATG2, that function at MCS. Hobbit is required for viability in Drosophila, suggesting that the Hob proteins are essential genes that may mediate lipid transfer at MCS.

Mistargeting of secretory cargo in retromer-deficient cells.

Intracellular trafficking is a basic and essential cellular function required for delivery of proteins to the appropriate subcellular destination; this process is especially demanding in professional secretory cells, which synthesize and secrete massive quantities of cargo proteins via regulated exocytosis. The Drosophila larval salivary glands are composed of professional secretory cells that synthesize and secrete mucin proteins at the onset of metamorphosis. Using the larval salivary glands as a model system, we have identified a role for the highly conserved retromer complex in trafficking of secretory granule membrane proteins. We demonstrate that retromer-dependent trafficking via endosomal tubules is induced at the onset of secretory granule biogenesis, and that recycling via endosomal tubules is required for delivery of essential secretory granule membrane proteins to nascent granules. Without retromer function, nascent granules do not contain the proper membrane proteins; as a result, cargo from these defective granules is mistargeted to Rab7-positive endosomes, where it progressively accumulates to generate dramatically enlarged endosomes. Retromer complex dysfunction is strongly associated with neurodegenerative diseases, including Alzheimer's disease, characterized by accumulation of amyloid ß (Aß). We show that ectopically expressed amyloid precursor protein (APP) undergoes regulated exocytosis in salivary glands and accumulates within enlarged endosomes in retromer-deficient cells. These results highlight recycling of secretory granule membrane proteins as a critical step during secretory granule maturation and provide new insights into our understanding of retromer complex function in secretory cells. These findings also suggest that missorting of secretory cargo, including APP, may contribute to the progressive nature of neurodegenerative disease.

Translational induction of ATF4 during integrated stress response requires noncanonical initiation factors eIF2D and DENR.

The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.

A genetic screen identifies new regulators of steroid-triggered programmed cell death in Drosophila.

The steroid hormone ecdysone triggers the rapid and massive destruction of larval tissues through transcriptional cascades that culminate in rpr and hid expression and caspase activation. Here we describe the use of genetic screens to further our understanding of this steroid-triggered programmed cell death response. Pupal lethal mutants were screened for specific defects in larval salivary gland destruction. A pilot screen using existing P-element collections resulted in the identification of mutations in known cell death regulators, E74 and hid, as well as multiple alleles in CBP (nejire) and dTrf2. A large-scale EMS mutagenesis screen on the third chromosome resulted in the recovery of 48 mutants. These include seven multiallelic complementation groups, at least five of which do not map to regions or genes previously associated with cell death. Five mutants display defects in the transcriptional induction of rpr and hid, and all display a penetrant block in caspase activation. Three were mapped to specific genes: CG5146, which encodes a protein of unknown function, Med24, which encodes a component of the RNA polymerase II mediator complex, and CG7998, which encodes a putative mitochondrial malate dehydrogenase. These genetic screens provide new directions for understanding the regulation of programmed cell death during development.

Reconsidering the Passive Diffusion Model of Steroid Hormone Cellular Entry.

Steroid hormones have long been thought to enter target cells via passive diffusion through the plasma membrane. Now, reporting in Developmental Cell, Okamoto et al. (2018) demonstrate that, at least for Drosophila, steroid hormones require a protein transporter for cellular entry.