Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors inDrosophila

Journal of Neuroscience, Journal Year: 2024, Volume and Issue: 44(33), P. e2377232024 - e2377232024

Published: June 27, 2024

To visualize the cellular and subcellular localization of neuromodulatory G-protein–coupled receptors in Drosophila , we implement a molecular strategy recently used to add epitope tags ionotropic at their endogenous loci. Leveraging evolutionary conservation identify sites more likely permit insertion tag, generated constitutive conditional tagged alleles for 5-HT1A 5-HT2A 5-HT2B Oct β 1R 2R two isoforms OAMB mGluR . The allow restricted expression receptor specific cell types, an option not available any previous reagents label these proteins. We show patterns female brains that localize mushroom bodies (MBs) central complex, respectively, as predicted by roles sleep. By contrast, unexpected enrichment Octβ1R complex nerve terminals lobular columnar cells visual system suggest new hypotheses about functions sites. Using additional allele serotonin transporter, marker serotonergic tracts, demonstrate diverse spatial relationships between postsynaptic 5-HT presynaptic neurons, consistent with importance both synaptic volume transmission. Finally, use it localizes distinct within MBs Kenyon autoreceptor.

Language: Английский

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RiboTag RNA Sequencing Identifies Local Translation of HSP70 in Astrocyte Endfeet After Cerebral Ischemia

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(1), P. 309 - 309

Published: Jan. 1, 2025

Brain ischemia causes disruption in cerebral blood flow and blood–brain barrier integrity, which are normally maintained by astrocyte endfeet. Emerging evidence points to dysregulation of the translatome during ischemia, but its effects on endfoot unknown. In this study, we aimed investigate early a rodent reperfusion model stroke. To do so, immunoprecipitated astrocyte-specific tagged ribosomes (RiboTag IP) from mechanically isolated brain microvessels. mice subjected middle artery occlusion contralateral controls, sequenced ribosome-bound RNAs perivascular endfeet identified 205 genes that were differentially expressed after ischemia. The main biological processes associated with these included proteostasis, inflammation, cell cycle/death, metabolism. Transcription factors whose targets enriched amongst upregulated translating HSF1, master regulator heat shock response. most highly HSF1-dependent Hspa1a Hspa1b, encode inducible HSP70. Using qPCR, Western blot, immunohistochemistry, confirmed HSP70 is This coincided an increase ubiquitination across proteome suggests induces proteostasis These findings suggest robust response proteotoxic stress Modulating may be strategy preserve function BBB integrity ischemic

Language: Английский

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Dynamic local mRNA localization and translation occurs during the postnatal molecular maturation of perivascular astrocytic processes

Glia, Journal Year: 2024, Volume and Issue: 72(4), P. 777 - 793

Published: Jan. 8, 2024

Abstract Astrocytes are highly ramified and send out perivascular processes (PvAPs) that entirely sheathe the brain's blood vessels. PvAPs equipped with an enriched molecular repertoire sustains astrocytic regulatory functions at vascular interface. In mouse, PvAP development starts after birth is essentially complete by postnatal day (P) 15. Progressive maturation also occurs over this period, acquisition of proteins in PvAPs. The mechanisms controlling have not been extensively characterized. We reported previously mRNAs distributed unequally mature locally translated. Since dynamic mRNA localization local translation influence cell's polarity, we hypothesized they might sustain Here, used a combination biology imaging approaches to demonstrate accompanied transport polysomal into PvAPs, rough endoplasmic reticulum (RER) network Golgi cisternae, translation. By focusing on genes selectively or specifically expressed astrocytes, characterized developmental profile mRNAs, from P5 P60. found some polarized progressively towards Lastly, expression developing perturbed mouse model megalencephalic leukoencephalopathy subcortical cysts. Our results indicate

Language: Английский

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Spatial and Temporal Single-Cell Profiling of RNA Compartmentalization in Neurons with Nanotweezers

ACS Nano, Journal Year: 2025, Volume and Issue: unknown

Published: May 6, 2025

Emerging techniques for mapping mRNAs within the subcellular compartments of live cells hold great promise advancing our understanding spatial distribution transcripts and enabling study single-cell dynamics in health disease. This is particularly critical polarized cells, such as neurons, where mRNA compartmentalization essential regulating gene expression, defects these localization mechanisms are linked to numerous neurological disorders. However, many analysis require a compromise between precision, live-cell measurements, nondestructive access single their native microenvironment. To overcome challenges, we employ technology that have recently developed, nanotweezer, which features nanoscale footprint (∼100 nm), avoids cytoplasmic fluid aspiration, enables rapid RNA isolation from living with minimal invasiveness. Using this tool, investigate soma dendrites hippocampal neurons at different stages neuronal development. By combining precise targeting sequential sampling, track changes abundance dendritic spine regions same neuron, both before after stimulation. minimally invasive approach time-resolved, expression profiling cell. could provide insights into advance biological processes complex diseases.

Language: Английский

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Revisiting the development of cerebellar inhibitory interneurons in the light of single-cell genetic analyses

Histochemistry and Cell Biology, Journal Year: 2023, Volume and Issue: 161(1), P. 5 - 27

Published: Nov. 8, 2023

The present review aims to provide a short update of our understanding the inhibitory interneurons cerebellum. While these cells constitute but minority all cerebellar neurons, their functional significance is increasingly being recognized. For one, cortex are now known clearly more diverse group than traditional grouping as stellate, basket, and Golgi suggests, this diversity substantiated by single-cell genetic data. past decade or so has also provided important information about in nuclei. Significantly, developmental studies have revealed that specification formation fundamentally differ from, say, cortical interneurons, define mode diversification critically dependent on spatiotemporally patterned external signals. Last, not least, years, dysfunction could be linked with clinically defined deficits. I hope review, however fragmentary, may stimulate interest help focus research towards

Language: Английский

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Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors inDrosophila

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 27, 2023

To visualize the cellular and subcellular localization of neuromodulatory G-protein coupled receptors (GPCRs) in

Language: Английский

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In vivo timelapse imaging and analysis of Golgi satellite organelle distribution and movement in the neural progenitor cells of the brain

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Feb. 21, 2024

Abstract The dividing stem cells of the developing brain are radial glial neural progenitor (NPCs), multifunctional that proliferate to generate all brain, but also act as scaffolds for their migrating neuron progeny, guideposts pathfinding growing axons and regulators synaptic activity. These remarkable perform these very different activities while remaining in contact with inner outer surface ever-growing brain. NPCs synthesize proteins locally support compartmentalized protein expression required specialized functions, it is not clear how necessary processing normally occurs Golgi apparatus achieved at locations far from cell body. satellites, motile organelles members maturation machinery, control glycosylation polarized like neurons. To investigate whether rely on we expressed a fluorescent reporter label satellites intact brains Xenopus laevis tadpoles. Quantitative analysis vivo timelapse images revealed dynamic, distribute throughout cell, suggesting have local proteostasis diverse functions.

Language: Английский

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Transcriptomic Evidence Reveals the Dysfunctional Mechanism of Synaptic Plasticity Control in ASD

Genes, Journal Year: 2024, Volume and Issue: 16(1), P. 11 - 11

Published: Dec. 25, 2024

A prominent endophenotype in Autism Spectrum Disorder (ASD) is the synaptic plasticity dysfunction, yet molecular mechanism remains elusive. As a prototype, we investigate postsynaptic signal transduction network glutamatergic neurons and integrate single-cell nucleus transcriptomics data from Prefrontal Cortex (PFC) to unveil malfunction of translation control. We devise an innovative highly dependable pipeline transform our acquired into mRNA Signaling-Regulatory Network (mSiReN) analyze it at RNA level. employ Cell-Specific Inference via Integer Value Programming Causal Reasoning (CS-NIVaCaR) identify core modules Probabilistic Contextualization for Regulatory Networks (CS-ProComReN) quantitatively reveal activated sub-pathways involving MAPK1, MKNK1, RPS6KA5, MTOR across different cell types ASD. The results indicate that specific pivotal molecules, such as EIF4EBP1 EIF4E, lacking Differential Expression (DE) characteristics responsible protein with long-term potentiation (LTP) or depression (LTD), are dysregulated. further uncover distinct activation patterns causally linked EIF4EBP1-EIF4E module excitatory inhibitory neurons. Importantly, work introduces methodology leveraging extensive parse network, transforming mSiReN, mapping back These algorithms can serve potent tools systems biology other omics regulatory networks. Furthermore, biomarkers within sub-pathways, revealed by identifying convergent dysregulation, illuminate potential diagnostic prognostic factors

Language: Английский

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Dynamic local mRNA distribution and translation influence the postnatal molecular maturation of perivascular astrocytic processes

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: July 28, 2023

Abstract Astrocytes (the main glial cells in the brain) are highly ramified and send out perivascular processes (PvAPs) that entirely sheathe brain’s blood vessels. PvAPs equipped with an enriched molecular repertoire sustains astrocytic regulatory functions at vascular interface. In mouse, PvAP development starts after birth is essentially complete by postnatal day (P) 15. Progressive maturation also occurs over this period, acquisition of proteins PvAPs. The mechanisms controlling have not been extensively characterized. We reported previously mRNAs distributed unequally mature locally translated. Since dynamic mRNA distribution local translation influence cell’s polarity, we hypothesized they might sustain Here, used a combination biology imaging approaches to demonstrate accompanied transport polysomal into PvAPs, rough endoplasmic reticulum (RER) network Golgi cisternae, translation. By focusing on genes selectively or specifically expressed astrocytes, characterized developmental profile mRNAs, from P5 P60. Furthermore, found perturbed mouse model megalencephalic leukoencephalopathy subcortical cysts. Lastly, some polarized progressively towards Our results indicate Summary statement Local operates during astrocyte contribute their maturation.

Language: Английский

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Convergent Transcriptomic Evidence Reveals the Dysfunctional Quantitative Mechanism of Synaptic Plasticity Control in ASD

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 26, 2023

Abstract A prominent endophenotype in Autism Spectrum Disorder (ASD) is synaptic plasticity dysfunction, yet the molecular mechanism remains elusive. As a prototype, we investigated postsynaptic signal transduction network glutamatergic neurons and integrated transcriptomics to unveil malfunction of translation control. We devised an innovative highly dependable pipeline transform our acquired into mRNA Signaling-Regulatory Network (mSiReN) analyze it at RNA level. employed Cell-Specific Inference via Integer Value Programming Causal Reasoning (CS-NIVaCaR) identify core modules Probabilistic Contextualization for Regulatory Networks (CS-ProComReN) quantitatively reveal activated sub-pathways involving MAPK1, MKNK1, RPS6KA5, MTOR across different cell types ASD. The results indicate that specific pivotal molecules, such as EIF4EBP1 EIF4E, lacking Differential Expression (DE) characteristics responsible protein with long-term potentiation (LTP) or depression (LTD), are dysregulated. further uncovered distinct activation patterns causally linked EIF4EBP1-EIF4E module excitatory inhibitory neurons. Importantly, work has introduced methodology leveraging extensive data parse network, transforming mSiReN, mapping back These algorithms can serve potent tools systems biology other omics regulatory networks. Furthermore, biomarkers within sub-pathways, revealed by identifying convergent dysregulation, illuminate potential diagnostic prognostic factors

Language: Английский

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