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Acute Effects of Drugs on Caenorhabditis elegans Movement Reveal Complex Responses and Plasticity.

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Acute Effects of Drugs on Caenorhabditis elegans Movement Reveal Complex Responses and Plasticity.

G3 (Bethesda). 2018 Jul 30;:

Authors: Spensley M, Del Borrello S, Pajkic D, Fraser AG

Abstract
Many drugs act very rapidly; they can turn on or off their targets within minutes in a whole animal. What are the acute effects of drug treatment and how does an animal respond to these? We developed a simple assay to measure the acute effects of drugs on C. elegans movement and examined the effects of a range of compounds including neuroactive drugs, toxins, environmental stresses and novel compounds on worm movement over a time period of 3 hours. We find that many treatments show complex acute responses, where a phase of rapid paralysis is followed by one or more recovery phases. The recoveries are not the result of some generic stress response but are specific to the drug e.g. recovery from paralysis due to a neuroactive drug requires neurotransmitter pathways whereas recovery from a metabolic inhibitor requires metabolic changes. Finally, we also find that acute responses can vary greatly across development and that there is extensive and complex natural variation in acute responses. In summary, acute responses are sensitive probes of the ability of biological networks to respond to drug treatment and these responses can reveal the action of unexplored pathways.

PMID: 30061375 [PubMed - as supplied by publisher]



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mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β.

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mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β.

J Biol Chem. 2018 Jul 30;:

Authors: Bautista SJ, Boras I, Vissa A, Mecica N, Yip CM, Kim PK, Antonescu CN

Abstract
Glycogen synthase kinase 3β (GSK3β) phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3β substrates, such as c-myc and snail, are nuclear transcription factors, suggesting the possibility that GSK3β function is controlled through its nuclear localization. Here, using ARPE-19 and MDA-MB-231 human cell lines, we found that inhibition of mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3β from the cytosol to the nucleus and to a GSK3β-dependent reduction of the levels of both c-myc and snail. mTORC1 is known to be controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance, and we observed here that AMPK activation or amino acid deprivation promotes GSK3β nuclear localization in an mTORC1-dependent manner. GSK3β was detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through perturbation of RAB7, member RAS oncogene family 7 (Rab7) resulted in loss of GSK3β from lysosomes and in enhanced GSK3β nuclear localization as well as GSK3β-dependent reduction of c-myc levels. These findings indicate that the nuclear localization and function of GSK3β is suppressed by mTORC1 and suggest a link between metabolic conditions sensed by mTORC1 and GSK3β-dependent regulation of transcriptional networks controlling cellular biomass production.

PMID: 30061153 [PubMed - as supplied by publisher]



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Skin-derived precursor cells undergo substrate-dependent galvanotaxis that can be modified by neighbouring cells.

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Skin-derived precursor cells undergo substrate-dependent galvanotaxis that can be modified by neighbouring cells.

Stem Cell Res. 2018 Jul 19;31:95-101

Authors: Iwasa SN, Popovic MR, Morshead CM

Abstract
Many cell types respond to electric fields (EFs) through cell migration, a process termed galvanotaxis. The galvanotactic response is critical for development and wound healing. Here we investigate whether skin-derived precursor cells (SKPs), which have the potential to differentiate into mesodermal and peripheral neural cell types, undergo directed migration in the presence of an EF. We found that EF application promotes SKP migration towards the anode. The migratory response is substrate-dependent as SKPs undergo directed migration on laminin and Matrigel, but not collagen. The majority of SKPs express the undifferentiated cell markers nestin, fibronectin and Sox2, after both EF application and in sister cultures with no EF application, suggesting that EFs do not promote cell differentiation. Co-cultures of SKPs and brain-derived neural precursor cells (NPCs), a population of cells that undergo rapid, cathode-directed migration, reveal that in the presence of NPCs an increased percentage of SKPs undergo galvanotaxis, providing evidence that cells can provide cues to modify the galvanotactic response. We propose that a better understanding of SKP migration in the presence of EFs may provide insight into improved strategies for wound repair.

PMID: 30059907 [PubMed - as supplied by publisher]



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Integrating genetic and protein-protein interaction networks maps a functional wiring diagram of a cell.

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Integrating genetic and protein-protein interaction networks maps a functional wiring diagram of a cell.

Curr Opin Microbiol. 2018 Jul 27;45:170-179

Authors: VanderSluis B, Costanzo M, Billmann M, Ward HN, Myers CL, Andrews BJ, Boone C

Abstract
Systematic experimental approaches have led to construction of comprehensive genetic and protein-protein interaction networks for the budding yeast, Saccharomyces cerevisiae. Genetic interactions capture functional relationships between genes using phenotypic readouts, while protein-protein interactions identify physical connections between gene products. These complementary, and largely non-overlapping, networks provide a global view of the functional architecture of a cell, revealing general organizing principles, many of which appear to be evolutionarily conserved. Here, we focus on insights derived from the integration of large-scale genetic and protein-protein interaction networks, highlighting principles that apply to both unicellular and more complex systems, including human cells. Network integration reveals fundamental connections involving key functional modules of eukaryotic cells, defining a core network of cellular function, which could be elaborated to explore cell-type specificity in metazoans.

PMID: 30059827 [PubMed - as supplied by publisher]



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Melatonin receptors limit dopamine reuptake by regulating dopamine transporter cell-surface exposure.

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Melatonin receptors limit dopamine reuptake by regulating dopamine transporter cell-surface exposure.

Cell Mol Life Sci. 2018 Jul 24;:

Authors: Benleulmi-Chaachoua A, Hegron A, Le Boulch M, Karamitri A, Wierzbicka M, Wong V, Stagljar I, Delagrange P, Ahmad R, Jockers R

Abstract
Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT1 and MT2 receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT1 or MT2 receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT1 and MT2) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.

PMID: 30043140 [PubMed - as supplied by publisher]



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A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases.

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A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases.

Structure. 2018 Jun 26;:

Authors: Gorelik M, Manczyk N, Pavlenco A, Kurinov I, Sidhu SS, Sicheri F

Abstract
Skp1-Cul1-F-box (SCF) E3 ligases constitute the largest and best-characterized family of the multisubunit E3 ligases with important cellular functions and numerous disease links. The specificity of an SCF E3 ligase is established by one of the 69 human F-box proteins that are recruited to Cul1 through the Skp1 adaptor. We previously reported generation of ubiquitin variants (UbVs) targeting Fbw7 and Fbw11, which inhibit ligase activity by binding at the F-box-Skp1 interface to competitively displace Cul1. In the present study, we employed an optimized engineering strategy to generate specific binding UbVs against 17 additional Skp1-F-box complexes. We validated our design strategy and uncovered the structural basis of binding specificity by crystallographic analyses of representative UbVs bound to Skp1-Fbl10 and Skp1-Fbl11. Our study highlights the power of combining phage display with structure-based design to develop UbVs targeting specific protein surfaces.

PMID: 30033217 [PubMed - as supplied by publisher]



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Single-Cell RNA Sequencing: A New Window into Cell Scale Dynamics.

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Single-Cell RNA Sequencing: A New Window into Cell Scale Dynamics.

Biophys J. 2018 Jul 11;:

Authors: Dasgupta S, Bader GD, Goyal S

Abstract
Single-cell genomics has recently emerged as a powerful tool for observing multicellular systems at a much higher level of resolution and depth than previously possible. High-throughput single-cell RNA sequencing techniques are able to simultaneously quantify expression levels of several thousands of genes within individual cells for tens of thousands of cells within a complex tissue. This has led to development of novel computational methods to analyze this high-dimensional data, investigating longstanding and fundamental questions regarding the granularity of cell types, the definition of cell states, and transitions from one cell type to another along developmental trajectories. In this perspective, we outline this emerging field starting from the "input data" (e.g., quantifying transcription levels in single cells), which are analyzed to define "identities" (e.g., cell types, states, and key genes) and to build "interactions" using models that can infer relations and transitions between cells.

PMID: 30033145 [PubMed - as supplied by publisher]



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The shieldin complex mediates 53BP1-dependent DNA repair.

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The shieldin complex mediates 53BP1-dependent DNA repair.

Nature. 2018 Jul 18;:

Authors: Noordermeer SM, Adam S, Setiaputra D, Barazas M, Pettitt SJ, Ling AK, Olivieri M, Álvarez-Quilón A, Moatti N, Zimmermann M, Annunziato S, Krastev DB, Song F, Brandsma I, Frankum J, Brough R, Sherker A, Landry S, Szilard RK, Munro MM, McEwan A, Goullet de Rugy T, Lin ZY, Hart T, Moffat J, Gingras AC, Martin A, van Attikum H, Jonkers J, Lord CJ, Rottenberg S, Durocher D

Abstract
53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

PMID: 30022168 [PubMed - as supplied by publisher]



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Two protein/protein interaction assays in one go.

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Two protein/protein interaction assays in one go.

Mol Syst Biol. 2018 Jul 18;14(7):e8485

Authors: Taipale M

PMID: 30021847 [PubMed - in process]



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New and Prospective Roles for lncRNAs in Organelle Formation and Function.

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New and Prospective Roles for lncRNAs in Organelle Formation and Function.

Trends Genet. 2018 Jul 14;:

Authors: Krause HM

Abstract
The observation that long noncoding RNAs (lncRNAs) represent the majority of transcripts in humans has led to a rapid increase in interest and study. Most of this interest has focused on their roles in the nucleus. However, increasing evidence is beginning to reveal even more functions outside the nucleus, and even outside cells. Many of these roles are mediated by newly discovered properties, including the ability of lncRNAs to interact with lipids, membranes, and disordered protein domains, and to form differentially soluble RNA-protein sub-organelles. This review explores the possibilities enabled by these new properties and abilities, such as likely roles in exosome formation and function.

PMID: 30017312 [PubMed - as supplied by publisher]



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