PI4P-mediated solid-like Merlin condensates orchestrate Hippo pathway regulation.

Despite recent studies implicating liquid-like biomolecular condensates in diverse cellular processes, many biomolecular condensates exist in a solid-like state, and their function and regulation are less understood. We show that the tumor suppressor Merlin, an upstream regulator of the Hippo pathway, localizes to both cell junctions and medial apical cortex in Drosophila epithelia, with the latter forming solid-like condensates that activate Hippo signaling. Merlin condensation required phosphatidylinositol-4-phosphate (PI4P)-mediated plasma membrane targeting and was antagonistically controlled by Pez and cytoskeletal tension through plasma membrane PI4P regulation. The solid-like material properties of Merlin condensates are essential for physiological function and protect the condensates against external perturbations. Collectively, these findings uncover an essential role for solid-like condensates in normal physiology and reveal regulatory mechanisms for their formation and disassembly.

High-affinity biomolecular interactions are modulated by low-affinity binders.

The strength of molecular interactions is characterized by their dissociation constants (KD). Only high-affinity interactions (KD ≤ 10-8 M) are extensively investigated and support binary on/off switches. However, such analyses have discounted the presence of low-affinity binders (KD > 10-5 M) in the cellular environment. We assess the potential influence of low-affinity binders on high-affinity interactions. By employing Gillespie stochastic simulations and continuous methods, we demonstrate that the presence of low-affinity binders can alter the kinetics and the steady state of high-affinity interactions. We refer to this effect as 'herd regulation' and have evaluated its possible impact in two different contexts including sex determination in Drosophila melanogaster and in signalling systems that employ molecular thresholds. We have also suggested experiments to validate herd regulation in vitro. We speculate that low-affinity binders are prevalent in biological contexts where the outcomes depend on molecular thresholds impacting homoeostatic regulation.

Dysfunction of Mitochondrial Dynamics Induces Endocytosis Defect and Cell Damage in Drosophila Nephrocytes.

Mitochondria are crucial for cellular ATP production. They are highly dynamic organelles, whose morphology and function are controlled through mitochondrial fusion and fission. The specific roles of mitochondria in podocytes, the highly specialized cells of the kidney glomerulus, remain less understood. Given the significant structural, functional, and molecular similarities between mammalian podocytes and Drosophila nephrocytes, we employed fly nephrocytes to explore the roles of mitochondria in cellular function. Our study revealed that alterations in the Pink1-Park (mammalian PINK1-PRKN) pathway can disrupt mitochondrial dynamics in Drosophila nephrocytes. This disruption led to either fragmented or enlarged mitochondria, both of which impaired mitochondrial function. The mitochondrial dysfunction subsequently triggered defective intracellular endocytosis, protein aggregation, and cellular damage. These findings underscore the critical roles of mitochondria in nephrocyte functionality.

Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.

RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.

Analysis of Mitophagy Reporters in Drosophila.

Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.

Endocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi responses.

RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.

Kismet/CHD7/CHD8 and Amyloid Precursor Protein-like Regulate Synaptic Levels of Rab11 at the Drosophila Neuromuscular Junction.

The transmembrane protein ß-amyloid precursor protein (APP) is central to the pathophysiology of Alzheimer's disease (AD). The ß-amyloid hypothesis posits that aberrant processing of APP forms neurotoxic ß-amyloid aggregates, which lead to the cognitive impairments observed in AD. Although numerous additional factors contribute to AD, there is a need to better understand the synaptic function of APP. We have found that Drosophila APP-like (APPL) has both shared and non-shared roles at the synapse with Kismet (Kis), a chromatin helicase binding domain (CHD) protein. Kis is the homolog of CHD7 and CHD8, both of which are implicated in neurodevelopmental disorders including CHARGE Syndrome and autism spectrum disorders, respectively. Loss of function mutations in kis and animals expressing human APP and BACE in their central nervous system show reductions in the glutamate receptor subunit, GluRIIC, the GTPase Rab11, and the bone morphogenetic protein (BMP), pMad, at the Drosophila larval neuromuscular junction (NMJ). Similarly, processes like endocytosis, larval locomotion, and neurotransmission are deficient in these animals. Our pharmacological and epistasis experiments indicate that there is a functional relationship between Kis and APPL, but Kis does not regulate appl expression at the larval NMJ. Instead, Kis likely influences the synaptic localization of APPL, possibly by promoting rab11 transcription. These data identify a potential mechanistic connection between chromatin remodeling proteins and aberrant synaptic function in AD.

OTUD6 deubiquitination of RPS7/eS7 on the free 40 S ribosome regulates global protein translation and stress.

Ribosomes are regulated by evolutionarily conserved ubiquitination/deubiquitination events. We uncover the role of the deubiquitinase OTUD6 in regulating global protein translation through deubiquitination of the RPS7/eS7 subunit on the free 40 S ribosome in vivo in Drosophila. Coimmunoprecipitation and enrichment of monoubiquitinated proteins from catalytically inactive OTUD6 flies reveal RPS7 as the ribosomal substrate. The 40 S protein RACK1 and E3 ligases CNOT4 and RNF10 function upstream of OTUD6 to regulate alkylation stress. OTUD6 interacts with RPS7 specifically on the free 40 S, and not on 43 S/48 S initiation complexes or the translating ribosome. Global protein translation levels are bidirectionally regulated by OTUD6 protein abundance. OTUD6 protein abundance is physiologically regulated in aging and in response to translational and alkylation stress. Thus, OTUD6 may promote translation initiation, the rate limiting step in protein translation, by titering the amount of 40 S ribosome that recycles.

EVs move messes, not messages, at the synapse.

Extracellular vesicles are known for intercellular signaling roles but can also serve to simply dispose of unwanted cargoes. In this issue, Bostelman and Broihier discuss new work from Rodal and colleagues (https://doi.org/10.1083/jcb.202405025) that refutes prior work by showing that extracellular vesicles at Drosophila neuromuscular junctions are not required for signaling and instead likely serve a proteostasis role.

A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

Evolutionary adaptation of an HP1-protein chromodomain integrates chromatin and DNA sequence signals.

Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino's chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino's chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.

Multi-labeling Live Imaging to Quantify Gene Expression Dynamics During Drosophila Embryonic Development.

Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules. In this protocol, we describe the successful implementation of a combination of MS2/MCP and PP7/PCP systems in living Drosophila embryos to further our understanding of transcriptional dynamics during development. Our technique can be extended to visualize transcriptional activities of multiple genes or alleles simultaneously, characterize allele-specific expression of a target gene, and quantitatively analyze RNA polymerase II activity in a single-cell resolution.

Deficiency of ValRS-m Causes Male Infertility in Drosophila melanogaster.

Drosophila spermatogenesis involves the renewal of germline stem cells, meiosis of spermatocytes, and morphological transformation of spermatids into mature sperm. We previously demonstrated that Ocnus (ocn) plays an essential role in spermatogenesis. The ValRS-m (Valyl-tRNA synthetase, mitochondrial) gene was down-regulated in ocn RNAi testes. Here, we found that ValRS-m-knockdown induced complete sterility in male flies. The depletion of ValRS-m blocked mitochondrial behavior and ATP synthesis, thus inhibiting the transition from spermatogonia to spermatocytes, and eventually, inducing the accumulation of spermatogonia during spermatogenesis. To understand the intrinsic reason for this, we further conducted transcriptome-sequencing analysis for control and ValRS-m-knockdown testes. The differentially expressed genes (DEGs) between these two groups were selected with a fold change of ≥2 or ≤1/2. Compared with the control group, 4725 genes were down-regulated (dDEGs) and 2985 genes were up-regulated (uDEGs) in the ValRS-m RNAi group. The dDEGs were mainly concentrated in the glycolytic pathway and pyruvate metabolic pathway, and the uDEGs were primarily related to ribosomal biogenesis. A total of 28 DEGs associated with mitochondria and 6 meiosis-related genes were verified to be suppressed when ValRS-m was deficient. Overall, these results suggest that ValRS-m plays a wide and vital role in mitochondrial behavior and spermatogonia differentiation in Drosophila.

Translational regulation enhances distinction of cell types in the nervous system.

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

Murine glial protrusion transcripts predict localized Drosophila glial mRNAs involved in plasticity.

The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia are both known to contain long cytoplasmic processes, while localized transcripts have only been studied extensively in neurons, not glia, especially in intact nervous systems. Here, we predict 1,740 localized Drosophila glial transcripts by extrapolating from our meta-analysis of seven existing studies characterizing the localized transcriptomes and translatomes of synaptically associated mammalian glia. We demonstrate that the localization of mRNAs in mammalian glial projections strongly predicts the localization of their high-confidence Drosophila homologs in larval motor neuron-associated glial projections and are highly statistically enriched for genes associated with neurological diseases. We further show that some of these localized glial transcripts are specifically required in glia for structural plasticity at the nearby neuromuscular junction synapses. We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important in disease.

Storage protein SfSP8 mediates larval starvation tolerance of Spodoptera frugiperda.

BACKGROUND: Energy homeostasis is vital for insects to survive food shortages. This study investigated the starvation tolerance of Spodoptera frugiperda, which invaded China in 2019, focusing on its storage protein family, crucial for energy balance. 10 storage protein family members were identified and their expression patterns at different development stages and under different starvation stress were analyzed. METHODS AND RESULTS: We used qPCR to evaluate the expression levels of storage protein family members under various larval instars and starvation conditions. We discovered that, among above 10 members, only 2 storage proteins, SfSP8 and SfSP7 showed significant upregulation in response to starvation stress. Notably, SfSP8 upregulated markedly after 24 h of fasting, whereas SfSP7 exhibited a delayed response, with significant upregulation observed only after 72 h of starvation. Then we significantly reduced the starvation tolerance of larvae through RNAi-mediated knockdown of SfSP8 and also altered the starvation response of SfSP7 from a late to an early activation pattern. Finally, we constructed transgenic Drosophila melanogaster with heterologous overexpressing SfSP8 revealed that the starvation tolerance of the transgenic line was significantly stronger than that of wild-type lines. CONCLUSIONS: SfSP8 was the core storage protein member that mediated the starvation tolerance of larvae of S. frugiperda. Our study on the novel function of storage proteins in mediating larval starvation tolerance of S. frugiperda is conducive to understanding the strong colonization of this terrible invasive pest.

The Activin Branch Ligand Daw Regulates the Drosophila melanogaster Immune Response and Lipid Metabolism against the Heterorhabditis bacteriophora Serine Carboxypeptidase.

Despite impressive advances in the broad field of innate immunity, our understanding of the molecules and signaling pathways that control the host immune response to nematode infection remains incomplete. We have shown recently that Transforming Growth Factor-ß (TGF-ß) signaling in the fruit fly Drosophila melanogaster is activated by nematode infection and certain TGF-ß superfamily members regulate the D. melanogaster anti-nematode immune response. Here, we investigate the effect of an entomopathogenic nematode infection factor on host TGF-ß pathway regulation and immune function. We find that Heterorhabditis bacteriophora serine carboxypeptidase activates the Activin branch in D. melanogaster adults and the immune deficiency pathway in Activin-deficient flies, it affects hemocyte numbers and survival in flies deficient for Activin signaling, and causes increased intestinal steatosis in Activin-deficient flies. Thus, insights into the D. melanogaster signaling pathways and metabolic processes interacting with H. bacteriophora pathogenicity factors will be applicable to entomopathogenic nematode infection of important agricultural insect pests and vectors of disease.

Peroxisomal cholesterol metabolism regulates yap-signaling, which maintains intestinal epithelial barrier function and is altered in Crohn's disease.

Intestinal epithelial cells line the luminal surface to establish the intestinal barrier, where the cells play essential roles in the digestion of food, absorption of nutrients and water, protection from microbial infections, and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling, which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells, indeed, is linked to gastrointestinal disorders such as irritable bowel syndrome, inflammatory bowel disease, and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and, therefore, homeostasis. Here, we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol, which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover, analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn's Disease revealed that the dysregulation of peroxisome number, excessive cellular cholesterol, and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn's Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis.

Airway specific deregulation of asthma-related serpins impairs tracheal architecture and oxygenation in D. melanogaster.

Serine proteases are important regulators of airway epithelial homeostasis. Altered serum or cellular levels of two serpins, Scca1 and Spink5, have been described for airway diseases but their function beyond antiproteolytic activity is insufficiently understood. To close this gap, we generated fly lines with overexpression or knockdown for each gene in the airways. Overexpression of both fly homologues of Scca1 and Spink5 induced the growth of additional airway branches, with more variable results for the respective knockdowns. Dysregulation of Scca1 resulted in a general delay in fruit fly development, with increases in larval and pupal mortality following overexpression of this gene. In addition, the morphological changes in the airways were concomitant with lower tolerance to hypoxia. In conclusion, the observed structural changes of the airways evidently had a strong impact on the airway function in our model as they manifested in a lower physical fitness of the animals. We assume that this is due to insufficient tissue oxygenation. Future work will be directed at the identification of key molecular regulators following the airway-specific dysregulation of Scca1 and Spink5 expression.

Molecular mechanisms of proteoglycan-mediated semaphorin signaling in axon guidance.

The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.