Nitric oxide gas acts as a short-range signaling molecule in vast array of important physiological processes, many which include major changes gene expression. How these genomic responses are induced, however, is poorly understood. Here, using genetic and chemical manipulations, we show that nitric produced the Drosophila prothoracic gland, where it via nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with heterodimer partner, hormone 3 (DHR3)....

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes early embryos, NPCs reside not only within NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether how they contribute to density NE. Here, we show that insert into NE ER feeds rapid expansion in Drosophila blastoderm embryos. We...

Abstract During morphogenesis, remodelling of cell shape requires the expansion or contraction plasma membrane domains. Here we identify a mechanism underlying restructuring apical surface during epithelial morphogenesis in Drosophila . We show that retraction villous protrusions and subsequent flattening is an endocytosis-driven morphogenetic process. Quantitation endogenously tagged GFP::Rab5 dynamics reveals massive increase endocytosis correlates with changes morphology. This accompanied...

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where when nuclear regulated by small chemical ligands and/or protein partners, we have used `ligand sensor'system to visualize spatial activity patterns for each the 18 Drosophila live developing animals. Transgenic lines were established express ligand binding domain receptor fused DNA-binding yeast GAL4. When combined with...

Report31 October 2019Open Access Transparent process Optogenetic inhibition of Delta reveals digital Notch signalling output during tissue differentiation Ranjith Viswanathan Developmental Biology Unit, European Molecular Laboratory, Heidelberg, GermanyCollaboration for a joint PhD degree between EMBL and Heidelberg University, Faculty Biosciences Search more papers by this author Aleksandar Necakov Germany Department Biological Science, Brock St. Catharines, ON, Canada Mateusz Trylinski...

Abstract The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of intracellular Domain (NICD). Using combination immunohistochemistry, RNA situ , Optogenetics and super-resolution live imaging transcription human cells, we show N1ICD can form condensates positively facilitate target expression. We determined undergoes Phase Separation Coupled Percolation (PSCP) transcriptional...

INTRODUCTIONFluorescent in situ hybridization (FISH) is commonly used to analyze the three-dimensional distribution of RNAs intact embryos and tissues. Tyramide signal amplification (TSA) significantly increases sensitivity resolution FISH probe signals. This protocol includes optimized TSA-FISH procedures for Drosophila embryos, ovaries, larval Instructions are given preparation RNA probes, collection fixation tissues, TSA-mediated detection including options high-throughput processing...

Retinoic acid (RA) is the biologically active metabolite of vitamin A and has become a well-established factor that induces neurite outgrowth regeneration in both vertebrates invertebrates. However, underlying regulatory mechanisms may mediate RA-induced sprouting remain unclear. In past decade, microRNAs have emerged as important regulators nervous system development regeneration, been shown to contribute processes such sprouting. few studies demonstrated role miRNAs By miRNA sequencing...

Nucleus raphéobscurus (NRo) modulates hypoglossal (XII) nerve motor output in the vitro transverse brain stem slice of neonatal rats (1–5 days old); chemical ablation NRo and its focal CO 2 acidification modulated bursting rhythm XII nerves. We microinjected a 4.5 mM solution kainic acid into to disrupt cellular activity observed that was temporarily abolished ( n = 10). also -acidified (pH 6.00 ± 0.01) artificial cerebrospinal fluid (aCSF) 6), pre-Bötzinger complex (PBC) as well control...

The discovery of fluorescence two centuries ago ushered in, what is today, an illuminating field science rooted in the rational design photochromic molecules for task-specific bio-, material-, and medical-driven applications. Today, this includes applications bioimaging diagnosis, photodynamic therapy regimes, addition to photovoltaic devices solar cells, among a vast multitude other usages. In furthering indispensable area daily life modern-day scientific research, we report herein...

Drosophila melanogaster has been used as a model system to identify and characterize genetic contributions development, homeostasis, investigate the molecular determinants of numerous human diseases. While there exist many differences at genetic, structural, level, signalling components cellular machineries are conserved between humans. For this reason, can extensively model, study pathologies. The extensive resources available make powerful one. Over years, sophisticated rapidly expanding...

Integrative top-down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review methodology to ensure deepest possible quantitative proteome analyses. Here, we establish optimized general protocol extracts improve reduction proteoforms and, thus, resolution in 2DE. Dithiothreitol (DTT), tributylphosphine (TBP), 2-hydroxyethyldisulfide (HED),...

Abstract Background Ubiquitylation (ubi) of the intracellular domain Notch ligand Delta (Dl) by E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) on lysines (Ks) is thought to be essential for its signalling activity. Nevertheless, we have previously shown that DlK2R-HA, a Dl variant where all Ks in (ICD) are replaced structurally similar arginine (R), still possess weak activity if over-expressed. This suggests ubi not absolutely required signalling. However, it known whether residual...

Abstract Spatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development multicellular organisms. The signal dynamics transferring information vivo is incompletely understood. Here we employ genome engineering Drosophila melanogaster to generate functional optogenetic allele Notch ligand Delta (opto-Delta), which replaces both copies endogenous wild type locus. Using clonal analysis, show that activation blocks through cis-inhibition...

With advancements in multicomponent molecular biological tools, the need for versatile, rapid and cost-effective cloning that enables successful combinatorial assembly of DNA plasmids interest is becoming increasingly important. Unfortunately, current platforms fall short regarding affordability, ease and, above all, ability to iteratively remove individual cassettes at will. Herein we construct, implement make available a broad set vectors, called PlayBack allow expression several different...

Abstract Notch signaling is a critical regulator of multiple developmental processes through its ability to control gene expression, and thereby influence cell fate specification proliferation, direct cell-cell communication. Although signalling has been implicated in myogenesis during late embryogenesis, the role early mesoderm development largely unexplored. Endocytosis ligand Delta receptor extracellular domain, step pathway activation, extensively observed ventral Drosophila embryo,...

<title>Abstract</title> Cells and organisms adjust their growth based on the availability of cholesterol, which is essential for cellular functions. However, mechanisms by cells sense cholesterol levels translate these into signals are not fully understood. We report that rapidly activates master growth-regulatory TOR pathway in <italic>Drosophila</italic>tissues. identify nuclear receptor HR3, an ortholog mammalian RORα, as factor cholesterol-induced activation. demonstrate HR3 binds...

Retinoic acid (RA) is the biologically active metabolite of vitamin A and has become a well-established factor that induces neurite outgrowth regeneration in both vertebrates invertebrates. However, underlying regulatory mechanisms may mediate RA-induced sprouting remain unclear. In past decade, microRNAs have emerged as important regulators nervous system development regeneration, been shown to contribute processes such sprouting. few studies demonstrated role miRNAs By miRNA-Sequencing...

Insulin resistance (IR) is associated with reductions in neuronal proteins often observed Alzheimer's disease (AD), however, the mechanisms through which IR promotes neurodegeneration/AD pathogenesis are poorly understood. Metformin (MET), a potent activator of metabolic regulator AMPK used to treat but its effectiveness for AD unclear. We have previously shown that chronic activation impairs neurite growth and protein synthesis SH-SY5Y neurons, was not explored. Therefore, we examined...

Retinoic acid, the active metabolite of Vitamin A, is important for appropriate development nervous system (e.g., neurite outgrowth) as well cognition memory formation) in adult brain. We have shown that many effects retinoids are conserved CNS mollusc, Lymnaea stagnalis. RXRs predominantly nuclear receptors, but RXR (LymRXR) exhibits a non-nuclear distribution CNS, where it also implicated non-genomic retinoid functions. As such, we developed Drosophila organ culture-based to examine...

Neuronal growth and synaptic function are dependent on precise protein production turnover at the synapse. AMPK-activated kinase (AMPK) represents a metabolic node involved in energy sensing regulating homeostasis. However, there is ambiguity surrounding role of AMPK neuronal health. This study examined effect chronic activation markers growth. Retinoic-acid-differentiated SH-SY5Y human neuroblastoma cells were treated with A-769662 (100 nM) or Compound C (30 for 1, 3, 5 days before AMPK,...

The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression

Abstract Notch signaling is a critical regulator of multiple developmental processes through its ability to control gene expression, and thereby influence cell fate specification proliferation direct cell-cell communication. Although signalling has been implicated in myogenesis during late embryogenesis, the role early mesoderm development largely unexplored. Endocytosis ligand receptor extracellular domain observed mesectoderm therefore indicative activity. Here we present evidence that...