A Microsphere Immunoassay for the Quantitative Detection of Antigens in Cell Culture Supernatant.

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ∼1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

Tip60-FOXO regulates JNK signaling mediated apoptosis in Drosophila.

The JNK signaling pathway plays crucial roles in various physiological processes, including cell proliferation, differentiation, migration, apoptosis, and stress response. Dysregulation of this pathway is closely linked to the onset and progression of numerous major diseases, such as developmental defects and tumors. Identifying and characterizing novel components of the JNK signaling pathway to enhance and refine its network hold significant scientific and clinical importance for the prevention and treatment of associated cancers. This study utilized the model organism Drosophila and employed multidisciplinary approaches encompassing genetics, developmental biology, biochemistry, and molecular biology to investigate the interplay between Tip60 and the JNK signaling pathway, and elucidated its regulatory mechanisms. Our findings suggest that loss of Tip60 acetyltransferase activity results in JNK signaling pathway activation and subsequent induction of JNK-dependent apoptosis. Genetic epistasis analysis reveals that Tip60 acts downstream of JNK, paralleling with the transcription factor FOXO. The biochemical results confirm that Tip60 can bind to FOXO and acetylate it. Introduction of human Tip60 into Drosophila effectively mitigates apoptosis induced by JNK signaling activation, underscoring conserved regulatory role of Tip60 in the JNK signaling pathway from Drosophila to humans. This study further enhances our understanding of the regulatory network of the JNK signaling pathway. By revealing the role and mechanism of Tip60 in JNK-dependent apoptosis, it unveils new insights and potential therapeutic avenues for preventing and treating associated cancers.

Topoisomerase I is an evolutionarily conserved key regulator for satellite DNA transcription.

RNA Polymerase (RNAP) II transcription on non-coding repetitive satellite DNAs plays an important role in chromosome segregation, but a little is known about the regulation of satellite transcription. We here show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite DNAs on human centromeres. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation. Interestingly, in response to DNA double-stranded breaks (DSBs), α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner, and these DSB-induced α-satellite RNAs form into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.

Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation.

Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.

The Endosomal Sorting Complex, ESCRT, has diverse roles in blood progenitor maintenance, lineage choice and immune response.

Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT). However, the roles of ESCRT in stem cell and progenitor maintenance are not resolved. Parsing signaling pathways in relation to the canonical role of ESCRT poses a challenge. The Drosophila hematopoietic organ, the larval lymph gland, provides a path to dissect the roles of cellular trafficking pathways such as ESCRT in blood development and maintenance. Drosophila has 13 core ESCRT components. Knockdown of individual ESCRTs showed that only Vps28 and Vp36 were required in all lymph gland progenitors. Using the well-conserved ESCRT-II complex as an example of the range of phenotypes seen upon ESCRT depletion, we show that ESCRTs have cell-autonomous as well as non-autonomous roles in progenitor maintenance and differentiation. ESCRT depletion also sensitized posterior lobe progenitors to respond to immunogenic wasp infestation. We also identify key heterotypic roles for ESCRT in position-dependent control of Notch activation to suppress crystal cell differentiation. Our study shows that the cargo sorting machinery determines the identity of progenitors and their adaptability to the dynamic microenvironment. These mechanisms for control of cell fate may tailor developmental diversity in multiple contexts.

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila central nervous system.

Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

Recurrent Duplication and Diversification of a Vital DNA Repair Gene Family Across Drosophila.

Maintaining genome integrity is vital for organismal survival and reproduction. Essential, broadly conserved DNA repair pathways actively preserve genome integrity. However, many DNA repair proteins evolve adaptively. Ecological forces like UV exposure are classically cited drivers of DNA repair evolution. Intrinsic forces like repetitive DNA, which also imperil genome integrity, have received less attention. We recently reported that a Drosophila melanogaster-specific DNA satellite array triggered species-specific, adaptive evolution of a DNA repair protein called Spartan/MH. The Spartan family of proteases cleave hazardous, covalent crosslinks that form between DNA and proteins ("DNA-protein crosslink repair"). Appreciating that DNA satellites are both ubiquitous and universally fast-evolving, we hypothesized that satellite DNA turnover spurs adaptive evolution of DNA-protein crosslink repair beyond a single gene and beyond the D. melanogaster lineage. This hypothesis predicts pervasive Spartan gene family diversification across Drosophila species. To study the evolutionary history of the Drosophila Spartan gene family, we conducted population genetic, molecular evolution, phylogenomic, and tissue-specific expression analyses. We uncovered widespread signals of positive selection across multiple Spartan family genes and across multiple evolutionary timescales. We also detected recurrent Spartan family gene duplication, divergence, and gene loss. Finally, we found that ovary-enriched parent genes consistently birthed functionally diverged, testis-enriched daughter genes. To account for Spartan family diversification, we introduce a novel mechanistic model of antagonistic coevolution that links DNA satellite evolution and adaptive regulation of Spartan protease activity. This framework promises to accelerate our understanding of how DNA repeats drive recurrent evolutionary innovation to preserve genome integrity.

An integrative sensor of body states: how the mushroom body modulates behavior depending on physiological context.

The brain constantly compares past and present experiences to predict the future, thereby enabling instantaneous and future behavioral adjustments. Integration of external information with the animal's current internal needs and behavioral state represents a key challenge of the nervous system. Recent advancements in dissecting the function of the Drosophila mushroom body (MB) at the single-cell level have uncovered its three-layered logic and parallel systems conveying positive and negative values during associative learning. This review explores a lesser-known role of the MB in detecting and integrating body states such as hunger, thirst, and sleep, ultimately modulating motivation and sensory-driven decisions based on the physiological state of the fly. State-dependent signals predominantly affect the activity of modulatory MB input neurons (dopaminergic, serotoninergic, and octopaminergic), but also induce plastic changes directly at the level of the MB intrinsic and output neurons. Thus, the MB emerges as a tightly regulated relay station in the insect brain, orchestrating neuroadaptations due to current internal and behavioral states leading to short- but also long-lasting changes in behavior. While these adaptations are crucial to ensure fitness and survival, recent findings also underscore how circuit motifs in the MB may reflect fundamental design principles that contribute to maladaptive behaviors such as addiction or depression-like symptoms.

Modeling neurodegenerative and neurodevelopmental disorders in the Drosophila mushroom body.

The common fruit fly Drosophila melanogaster provides a powerful platform to investigate the genetic, molecular, cellular, and neural circuit mechanisms of behavior. Research in this model system has shed light on multiple aspects of brain physiology and behavior, from fundamental neuronal function to complex behaviors. A major anatomical region that modulates complex behaviors is the mushroom body (MB). The MB integrates multimodal sensory information and is involved in behaviors ranging from sensory processing/responses to learning and memory. Many genes that underlie brain disorders are conserved, from flies to humans, and studies in Drosophila have contributed significantly to our understanding of the mechanisms of brain disorders. Genetic mutations that mimic human diseases-such as Fragile X syndrome, neurofibromatosis type 1, Parkinson's disease, and Alzheimer's disease-affect MB structure and function, altering behavior. Studies dissecting the effects of disease-causing mutations in the MB have identified key pathological mechanisms, and the development of a complete connectome promises to add a comprehensive anatomical framework for disease modeling. Here, we review Drosophila models of human neurodevelopmental and neurodegenerative disorders via the effects of their underlying mutations on MB structure, function, and the resulting behavioral alterations.

Skewing information flow through pre- and postsynaptic plasticity in the mushroom bodies of Drosophila.

Animal brains need to store information to construct a representation of their environment. Knowledge of what happened in the past allows both vertebrates and invertebrates to predict future outcomes by recalling previous experience. Although invertebrate and vertebrate brains share common principles at the molecular, cellular, and circuit-architectural levels, there are also obvious differences as exemplified by the use of acetylcholine versus glutamate as the considered main excitatory neurotransmitters in the respective central nervous systems. Nonetheless, across central nervous systems, synaptic plasticity is thought to be a main substrate for memory storage. Therefore, how brain circuits and synaptic contacts change following learning is of fundamental interest for understanding brain computations tied to behavior in any animal. Recent progress has been made in understanding such plastic changes following olfactory associative learning in the mushroom bodies (MBs) of Drosophila A current framework of memory-guided behavioral selection is based on the MB skew model, in which antagonistic synaptic pathways are selectively changed in strength. Here, we review insights into plasticity at dedicated Drosophila MB output pathways and update what is known about the plasticity of both pre- and postsynaptic compartments of Drosophila MB neurons.

Diverse memory paradigms in Drosophila reveal diverse neural mechanisms.

In this review, we aggregated the different types of learning and memory paradigms developed in adult Drosophila and attempted to assess the similarities and differences in the neural mechanisms supporting diverse types of memory. The simplest association memory assays are conditioning paradigms (olfactory, visual, and gustatory). A great deal of work has been done on these memories, revealing hundreds of genes and neural circuits supporting this memory. Variations of conditioning assays (reversal learning, trace conditioning, latent inhibition, and extinction) also reveal interesting memory mechanisms, whereas mechanisms supporting spatial memory (thermal maze, orientation memory, and heat box) and the conditioned suppression of innate behaviors (phototaxis, negative geotaxis, anemotaxis, and locomotion) remain largely unexplored. In recent years, there has been an increased interest in multisensory and multicomponent memories (context-dependent and cross-modal memory) and higher-order memory (sensory preconditioning and second-order conditioning). Some of this work has revealed how the intricate mushroom body (MB) neural circuitry can support more complex memories. Finally, the most complex memories are arguably those involving social memory: courtship conditioning and social learning (mate-copying and egg-laying behaviors). Currently, very little is known about the mechanisms supporting social memories. Overall, the MBs are important for association memories of multiple sensory modalities and multisensory integration, whereas the central complex is important for place, orientation, and navigation memories. Interestingly, several different types of memory appear to use similar or variants of the olfactory conditioning neural circuitry, which are repurposed in different ways.

Glial metabolism versatility regulates mushroom body-driven behavioral output in Drosophila.

Providing metabolic support to neurons is now recognized as a major function of glial cells that is conserved from invertebrates to vertebrates. However, research in this field has focused for more than two decades on the relevance of lactate and glial glycolysis for neuronal energy metabolism, while overlooking many other facets of glial metabolism and their impact on neuronal physiology, circuit activity, and behavior. Here, we review recent work that has unveiled new features of glial metabolism, especially in Drosophila, in the modulation of behavioral traits involving the mushroom bodies (MBs). These recent findings reveal that spatially and biochemically distinct modes of glucose-derived neuronal fueling are implemented within the MB in a memory type-specific manner. In addition, cortex glia are endowed with several antioxidant functions, whereas astrocytes can serve as pro-oxidant agents that are beneficial to redox signaling underlying long-term memory. Finally, glial fatty acid oxidation seems to play a dual fail-safe role: first, as a mode of energy production upon glucose shortage, and, second, as a factor underlying the clearance of excessive oxidative load during sleep. Altogether, these integrated studies performed in Drosophila indicate that glial metabolism has a deterministic role on behavior.

Drosophila learning and memory centers and the actions of drugs of abuse.

Drug addiction and the circuitry for learning and memory are intimately intertwined. Drugs of abuse create strong, inappropriate, and lasting memories that contribute to many of their destructive properties, such as continued use despite negative consequences and exceptionally high rates of relapse. Studies in Drosophila melanogaster are helping us understand how drugs of abuse, especially alcohol, create memories at the level of individual neurons and in the circuits where they function. Drosophila is a premier organism for identifying the mechanisms of learning and memory. Drosophila also respond to drugs of abuse in ways that remarkably parallel humans and rodent models. An emerging consensus is that, for alcohol, the mushroom bodies participate in the circuits that control acute drug sensitivity, not explicitly associative forms of plasticity such as tolerance, and classical associative memories of their rewarding and aversive properties. Moreover, it is becoming clear that drugs of abuse use the mushroom body circuitry differently from other behaviors, potentially providing a basis for their addictive properties.

Cognitive limits of larval Drosophila: testing for conditioned inhibition, sensory preconditioning, and second-order conditioning.

Drosophila larvae are an established model system for studying the mechanisms of innate and simple forms of learned behavior. They have about 10 times fewer neurons than adult flies, and it was the low total number of their neurons that allowed for an electron microscopic reconstruction of their brain at synaptic resolution. Regarding the mushroom body, a central brain structure for many forms of associative learning in insects, it turned out that more than half of the classes of synaptic connection had previously escaped attention. Understanding the function of these circuit motifs, subsequently confirmed in adult flies, is an important current research topic. In this context, we test larval Drosophila for their cognitive abilities in three tasks that are characteristically more complex than those previously studied. Our data provide evidence for (i) conditioned inhibition, as has previously been reported for adult flies and honeybees. Unlike what is described for adult flies and honeybees, however, our data do not provide evidence for (ii) sensory preconditioning or (iii) second-order conditioning in Drosophila larvae. We discuss the methodological features of our experiments as well as four specific aspects of the organization of the larval brain that may explain why these two forms of learning are observed in adult flies and honeybees, but not in larval Drosophila.

Roles of feedback and feed-forward networks of dopamine subsystems: insights from Drosophila studies.

Across animal species, dopamine-operated memory systems comprise anatomically segregated, functionally diverse subsystems. Although individual subsystems could operate independently to support distinct types of memory, the logical interplay between subsystems is expected to enable more complex memory processing by allowing existing memory to influence future learning. Recent comprehensive ultrastructural analysis of the Drosophila mushroom body revealed intricate networks interconnecting the dopamine subsystems-the mushroom body compartments. Here, we review the functions of some of these connections that are beginning to be understood. Memory consolidation is mediated by two different forms of network: A recurrent feedback loop within a compartment maintains sustained dopamine activity required for consolidation, whereas feed-forward connections across compartments allow short-term memory formation in one compartment to open the gate for long-term memory formation in another compartment. Extinction and reversal of aversive memory rely on a similar feed-forward circuit motif that signals omission of punishment as a reward, which triggers plasticity that counteracts the original aversive memory trace. Finally, indirect feed-forward connections from a long-term memory compartment to short-term memory compartments mediate higher-order conditioning. Collectively, these emerging studies indicate that feedback control and hierarchical connectivity allow the dopamine subsystems to work cooperatively to support diverse and complex forms of learning.

Presynaptic regulators in memory formation.

The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we investigate the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the Drosophila mushroom body Kenyon cells. Our findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.

Future avenues in Drosophila mushroom body research.

How does the brain translate sensory information into complex behaviors? With relatively small neuronal numbers, readable behavioral outputs, and an unparalleled genetic toolkit, the Drosophila mushroom body (MB) offers an excellent model to address this question in the context of associative learning and memory. Recent technological breakthroughs, such as the freshly completed full-brain connectome, multiomics approaches, CRISPR-mediated gene editing, and machine learning techniques, led to major advancements in our understanding of the MB circuit at the molecular, structural, physiological, and functional levels. Despite significant progress in individual MB areas, the field still faces the fundamental challenge of resolving how these different levels combine and interact to ultimately control the behavior of an individual fly. In this review, we discuss various aspects of MB research, with a focus on the current knowledge gaps, and an outlook on the future methodological developments required to reach an overall view of the neurobiological basis of learning and memory.

Sensory encoding and memory in the mushroom body: signals, noise, and variability.

To survive in changing environments, animals need to learn to associate specific sensory stimuli with positive or negative valence. How do they form stimulus-specific memories to distinguish between positively/negatively associated stimuli and other irrelevant stimuli? Solving this task is one of the functions of the mushroom body, the associative memory center in insect brains. Here we summarize recent work on sensory encoding and memory in the Drosophila mushroom body, highlighting general principles such as pattern separation, sparse coding, noise and variability, coincidence detection, and spatially localized neuromodulation, and placing the mushroom body in comparative perspective with mammalian memory systems.