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Cellular and Molecular Life Sciences :... Jun 2024Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a...
Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca-binding sites in Scramb1 are essential for its activity raising the possibility that Ca signaling may control the assembly of SDs by impacting on Scramb1 activity.
Topics: Animals; Podocytes; Drosophila Proteins; Phospholipid Transfer Proteins; Membrane Proteins; Drosophila melanogaster; Membrane Microdomains; Intercellular Junctions
PubMed: 38878170
DOI: 10.1007/s00018-024-05287-z -
Nature Communications Jun 2024Genome organization is thought to underlie cell type specific gene expression, yet how it is regulated in progenitors to produce cellular diversity is unknown. In...
Genome organization is thought to underlie cell type specific gene expression, yet how it is regulated in progenitors to produce cellular diversity is unknown. In Drosophila, a developmentally-timed genome reorganization in neural progenitors terminates competence to produce early-born neurons. These events require downregulation of Distal antenna (Dan), part of the conserved pipsqueak DNA-binding superfamily. Here we find that Dan forms liquid-like condensates with high protein mobility, and whose size and subnuclear distribution are balanced with its DNA-binding. Further, we identify a LARKS domain, a structural motif associated with condensate-forming proteins. Deleting just 13 amino acids from LARKS abrogates Dan's ability to retain the early-born neural fate gene, hunchback, in the neuroblast nuclear interior and maintain competence in vivo. Conversely, domain-swapping with LARKS from known phase-separating proteins rescues Dan's effects on competence. Together, we provide in vivo evidence for condensate formation and the regulation of progenitor nuclear architecture underlying neuronal diversification.
Topics: Animals; Cell Nucleus; DNA-Binding Proteins; Drosophila melanogaster; Drosophila Proteins; Gene Expression Regulation, Developmental; Neural Stem Cells; Neurons; Protein Domains; Transcription Factors
PubMed: 38877037
DOI: 10.1038/s41467-024-49326-6 -
Genetics Jun 2024To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for...
To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound is regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, while the wound does not close in the absence of calcium influx, we find that reduced calcium influx can still initiate repair processes, albeit leading to severe repair phenotypes. Thus, our results suggest that, in addition to initiating repair events, the quantity of calcium influx is important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair.
PubMed: 38874345
DOI: 10.1093/genetics/iyae101 -
Frontiers in Neuroscience 2024The sensitivity of the eye at night would lead to complete saturation of the eye during the day. Therefore, the sensitivity of the eye must be down-regulated during the...
The sensitivity of the eye at night would lead to complete saturation of the eye during the day. Therefore, the sensitivity of the eye must be down-regulated during the day to maintain visual acuity. In the Drosophila eye, the opening of TRP and TRPL channels leads to an influx of Ca that triggers down-regulation of further responses to light, including the movement of the TRPL channel and Gα proteins out of signaling complexes found in actin-mediated microvillar extensions of the photoreceptor cells (the rhabdomere). The eye also exhibits a light entrained-circadian rhythm, and we have recently observed that one component of this rhythm (BDBT) becomes undetectable by antibodies after exposure to light even though immunoblot analyses still detect it in the eye. BDBT is necessary for normal circadian rhythms, and in several circadian and visual mutants this eye-specific oscillation of detection is lost. Many phototransduction signaling proteins (e.g., Rhodopsin, TRP channels and Gα) also become undetectable shortly after light exposure, most likely due to a light-induced compaction of the rhabdomeric microvilli. The circadian protein BDBT might be involved in light-induced changes in the rhabdomere, and if so this could indicate that circadian clocks contribute to the daily adaptations of the eye to light. Likewise, circadian oscillations of clock proteins are observed in photoreceptors of the mammalian eye and produce a circadian oscillation in the ERG. Disruption of circadian rhythms in the eyes of mammals causes neurodegeneration in the eye, demonstrating the importance of the rhythms for normal eye function.
PubMed: 38872947
DOI: 10.3389/fnins.2024.1401721 -
MicroPublication Biology 2024Wnt signalling coordinates growth and cell fate decisions during development and mis-regulation of Wnt signalling in adults is associated with a range of conditions,...
Wnt signalling coordinates growth and cell fate decisions during development and mis-regulation of Wnt signalling in adults is associated with a range of conditions, including cancer and neurodegenerative diseases. Therefore, means of modulating Wnt proteins and/or cofactors could have significant therapeutic potential. As a first step towards enumerating the Wnt interactome, we devised an proximity labelling strategy to identify proteins that interact with Wingless (Wg), the main Wnt. We engineered the locus to express a functional TurboID-Wg fusion at endogenous levels and identified interactors by streptavidin pull-down from embryos, followed by mass spectrometry. Further analysis may in future extend the screen coverage and deliver functional validation of the newly identified interactors.
PubMed: 38872844
DOI: 10.17912/micropub.biology.001210 -
PLoS Genetics Jun 2024Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that...
Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.
Topics: RNA Splicing; Animals; Transcription, Genetic; Introns; Drosophila melanogaster; Y Chromosome; RNA, Messenger; Drosophila Proteins; Drosophila; Male; Humans
PubMed: 38870220
DOI: 10.1371/journal.pgen.1011241 -
Cell Reports Jun 2024Wnt/Wingless (Wg) signaling is critical in development and disease, including cancer. Canonical Wnt signaling is mediated by β-catenin/Armadillo (Arm in Drosophila)...
Wnt/Wingless (Wg) signaling is critical in development and disease, including cancer. Canonical Wnt signaling is mediated by β-catenin/Armadillo (Arm in Drosophila) transducing signals to the nucleus, with IFT-A/Kinesin 2 complexes promoting nuclear translocation of β-catenin/Arm. Here, we demonstrate that a conserved small N-terminal Arm/β-catenin peptide binds to IFT140, acting as a dominant interference tool to attenuate Wg/Wnt signaling in vivo. Arm expression antagonizes endogenous Wnt/Wg signaling, resulting in the reduction of its target expression. Arm inhibits Wg/Wnt signaling by interfering with nuclear translocation of endogenous Arm/β-catenin, and this can be modulated by levels of wild-type β-catenin or IFT140, with the Arm effect being enhanced or suppressed. Importantly, this mechanism is conserved in mammals with the equivalent β-catenin peptide blocking nuclear translocation and pathway activation, including in cancer cells. Our work indicates that Wnt signaling can be regulated by a defined N-terminal β-catenin peptide and thus might serve as an entry point for therapeutic applications to attenuate Wnt/β-catenin signaling.
Topics: beta Catenin; Animals; Drosophila Proteins; Wnt Signaling Pathway; Cell Nucleus; Humans; Armadillo Domain Proteins; Wnt1 Protein; Active Transport, Cell Nucleus; Drosophila melanogaster; Peptides; Protein Binding; Amino Acid Sequence; Transcription Factors
PubMed: 38870008
DOI: 10.1016/j.celrep.2024.114362 -
ELife Jun 2024Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. Here, we investigate the genetic basis underlying the embryonic onset of...
Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. Here, we investigate the genetic basis underlying the embryonic onset of movement in focusing on the role played by small non-coding RNAs (microRNAs, miRNAs). To this end, we first develop a quantitative behavioural pipeline capable of tracking embryonic movement in large populations of fly embryos, and using this system, discover that the miRNA plays a role in the emergence of movement. Through the combination of spectral analysis of embryonic motor patterns, cell sorting and RNA in situs, genetic reconstitution tests, and neural optical imaging we define that miR-2b-1 influences the emergence of embryonic movement by exerting actions in the developing nervous system. Furthermore, through the combination of bioinformatics coupled to genetic manipulation of miRNA expression and phenocopy tests we identify a previously uncharacterised (but evolutionarily conserved) chloride channel encoding gene - which we term () - as a genetic target that mechanistically links to the onset of movement. Cell-specific genetic reconstitution of expression in a null miRNA mutant background, followed by behavioural assays and target gene analyses, suggest that affects the emergence of movement through effects in sensory elements of the embryonic circuitry, rather than in the motor domain. Our work thus reports the first miRNA system capable of regulating embryonic movement, suggesting that other miRNAs are likely to play a role in this key developmental process in as well as in other species.
Topics: Animals; MicroRNAs; Drosophila melanogaster; Gene Expression Regulation, Developmental; Movement; Embryo, Nonmammalian; Drosophila; Drosophila Proteins
PubMed: 38869942
DOI: 10.7554/eLife.95209 -
ELife Jun 2024The generation of distinct cell fates during development depends on asymmetric cell division of progenitor cells. In the central and peripheral nervous system of...
The generation of distinct cell fates during development depends on asymmetric cell division of progenitor cells. In the central and peripheral nervous system of progenitor cells respectively called neuroblasts or sensory organ precursors use PAR polarity during mitosis to control cell fate determination in their daughter cells. How polarity and the cell cycle are coupled, and how the cell cycle machinery regulates PAR protein function and cell fate determination is poorly understood. Here, we generate an analog sensitive allele of CDK1 and reveal that its partial inhibition weakens but does not abolish apical polarity in embryonic and larval neuroblasts and leads to defects in polarisation of fate determinants. We describe a novel in vivo phosphorylation of Bazooka, the homolog of PAR-3, on Serine180, a consensus CDK phosphorylation site. In some tissular contexts, phosphorylation of Serine180 occurs in asymmetrically dividing cells but not in their symmetrically dividing neighbours. In neuroblasts, Serine180 phosphomutants disrupt the timing of basal polarisation. Serine180 phosphomutants also affect the specification and binary cell fate determination of sensory organ precursors as well as Baz localisation during their asymmetric cell divisions. Finally, we show that CDK1 phosphorylates Serine-S180 and an equivalent Serine on human PAR-3 in vitro.
Topics: Animals; Phosphorylation; Drosophila Proteins; CDC2 Protein Kinase; Cell Polarity; Drosophila melanogaster; Neural Stem Cells; Sense Organs; Intracellular Signaling Peptides and Proteins
PubMed: 38869055
DOI: 10.7554/eLife.97902 -
Genes & Development Jun 2024Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by...
Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of , , and mutant We found that the gene expression profiles of and larvae are markedly different than larvae despite similar reductions in H4K20me1. mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of , indicating that the developmental defects of animals are largely due to H4K20me1-independent effects on gene expression. Furthermore, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in and mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin and therefore that H4K20me1 does not play a large role in gene expression.
Topics: Animals; Drosophila melanogaster; Histones; Drosophila Proteins; Methylation; Lysine; Histone-Lysine N-Methyltransferase; Gene Expression Regulation, Developmental; Mutation; Transcriptome; Larva
PubMed: 38866557
DOI: 10.1101/gad.351698.124