172 research outputs found
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Modelling GTPase dynamics to understand rhoa-driven cancer cell invasion
Metastasis, initially driven by cells migrating and invading through the local environment, leads to most cancer-associated deaths. Cells can use a variety of modes to move in vitro, all of which depend on Rho GTPases at some level. While traditionally it was thought that Rac1 activity drives protrusive lamellipodia at the leading edge of a polarised cell while RhoA drives rear retraction, more recent work in 3D microenvironments has revealed a much more complicated picture of GTPase dynamics. In particular, RhoA activity can dominate the leading edge polymerisation of actin to form filopodial actinspike protrusions that drive more invasive cell migration. We recently described a potential mechanism to abrogate this pro-invasive localised leading edge Rac1 to RhoA switch via manipulation of a negative feedback loop that was revealed by adopting a logical modelling approach. Both challenging dogma and taking a formal, mathematical approach to understanding signalling involved in motility may be vital to harnessing harmful cell migration and preventing metastasis in future research
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A MAPK-Driven Feedback Loop Suppresses Rac Activity to Promote RhoA-Driven Cancer Cell Invasion
Data Availability: All relevant data are within the paper and its Supporting Information files.Supporting Information is available online at: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004909#sec024 .Cell migration in 3D microenvironments is fundamental to development, homeostasis and the pathobiology of diseases such as cancer. Rab-coupling protein (RCP) dependent co-trafficking of Ξ±5Ξ²1 and EGFR1 promotes cancer cell invasion into fibronectin (FN) containing extracellular matrix (ECM), by potentiating EGFR1 signalling at the front of invasive cells. This promotes a switch in RhoGTPase signalling to inhibit Rac1 and activate a RhoA-ROCK-Formin homology domain-containing 3 (FHOD3) pathway and generate filopodial actin-spike protrusions which drive invasion. To further understand the signalling network that drives RCP-driven invasive migration, we generated a Boolean logical model based on existing network pathways/models, where each node can be interrogated by computational simulation. The model predicted an unanticipated feedback loop, whereby Raf/MEK/ERK signalling maintains suppression of Rac1 by inhibiting the Rac-activating Sos1-Eps8-Abi1 complex, allowing RhoA activity to predominate in invasive protrusions. MEK inhibition was sufficient to promote lamellipodia formation and oppose filopodial actin-spike formation, and led to activation of Rac and inactivation of RhoA at the leading edge of cells moving in 3D matrix. Furthermore, MEK inhibition abrogated RCP/Ξ±5Ξ²1/EGFR1-driven invasive migration. However, upon knockdown of Eps8 (to suppress the Sos1-Abi1-Eps8 complex), MEK inhibition had no effect on RhoGTPase activity and did not oppose invasive migration, suggesting that MEK-ERK signalling suppresses the Rac-activating Sos1-Abi1-Eps8 complex to maintain RhoA activity and promote filopodial actin-spike formation and invasive migration. Our study highlights the predictive potential of mathematical modelling approaches, and demonstrates that a simple intervention (MEK-inhibition) could be of therapeutic benefit in preventing invasive migration and metastasis.JHRH is supported by a University of Manchester Presidentβs Doctoral Scholarship and BBSRC-DTP (BB/J014478/1). This work was supported by the Wellcome Trust (090453/Z/09/Z and 090453/Z/09/A to PTC)
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Graphene oxide activates canonical TGFΞ² signalling in a human chondrocyte cell line <i>via</i> increased plasma membrane tension
Data availability:
No new data. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.Footnote: Electronic supplementary information (ESI) available. See doi: https://doi.org/10.1039/d3nr06033k .Graphene Oxide (GO) has been shown to increase the expression of key cartilage genes and matrix components within 3D scaffolds. Understanding the mechanisms behind the chondroinductive ability of GO is critical for developing articular cartilage regeneration therapies but remains poorly understood. The objectives of this work were to elucidate the effects of GO on the key chondrogenic signalling pathway β TGFΞ² and identify the mechanism through which signal activation is achieved in human chondrocytes. Activation of canonical signalling was validated through GO-induced SMAD-2 phosphorylation and upregulation of known TGFΞ² response genes, while the use of a TGFΞ² signalling reporter assay allowed us to identify the onset of GO-induced signal activation which has not been previously reported. Importantly, we investigate the cellβmaterial interactions and molecular mechanisms behind these effects, establishing a novel link between GO, the plasma membrane and intracellular signalling. By leveraging fluorescent lifetime imaging (FLIM) and a membrane tension probe, we reveal GO-mediated increases in plasma membrane tension, in real-time for the first time. Furthermore, we report the activation of mechanosensory pathways which are known to be regulated by changes in plasma membrane tension and reveal the activation of endogenous latent TGFΞ² in the presence of GO, providing a mechanism for signal activation. The data presented here are critical to understanding the chondroinductive properties of GO and are important for the implementation of GO in regenerative medicine.The authors acknowledge financial support to LO from the UKRI Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training (CDT) in Advanced Biomedical Materials (EP/S022201/1). Additional support to SW from Medical Research Council (Grant MR/S002553/1). A. K. acknowledges the studentship from the UK Research and Innovation (UKRI) Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training programme (Graphene NOWNANO CDT; EP/L01548X/1). The authors acknowledge the Manchester Collaborative Centre for Inflammation Research (MCCIR) as the funding source for the FACSVerse. The ICN2 has been supported by the Severo Ochoa Centres of Excellence programme [SEV-2017-0706] and is currently supported by the Severo Ochoa Centres of Excellence programme, Grant CEX2021-001214-S, both funded by MCIN/AEI/10.13039.501100011033
Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion
The Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser435 by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser897 by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinomaβwhereas conditional knockout of another RCP cargo, Ξ±5 integrin, does not suppress pancreatic cancer metastasisβindicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo
Alcohol-induced retrograde facilitation renders witnesses of crime less suggestible to misinformation
RATIONALE: Research has shown that alcohol can have both detrimental and facilitating effects on memory: intoxication can lead to poor memory for information encoded after alcohol consumption (anterograde amnesia) and may improve memory for information encoded before consumption (retrograde facilitation). This study examined whether alcohol consumed after witnessing a crime can render individuals less vulnerable to misleading post-event information (misinformation). METHOD: Participants watched a simulated crime video. Thereafter, one third of participants expected and received alcohol (alcohol group), one third did not expect but received alcohol (reverse placebo), and one third did not expect nor receive alcohol (control). After alcohol consumption, participants were exposed to misinformation embedded in a written narrative about the crime. The following day, participants completed a cued-recall questionnaire about the event. RESULTS: Control participants were more likely to report misinformation compared to the alcohol and reverse placebo group. CONCLUSION: The findings suggest that we may oversimplify the effect alcohol has on suggestibility and that sometimes alcohol can have beneficial effects on eyewitness memory by protecting against misleading post-event information
Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation
Copyright Β© 2013 The American Society of Human Genetics. Published by Elsevier Inc.Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease
The Rho-Family GTPase Rac1 Regulates Integrin Localization in Drosophila Immunosurveillance Cells
BACKGROUND: When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila Ξ²-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of Ξ± and Ξ² subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. RESULTS: In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. SIGNIFICANCE: We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29Β°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes
Endosomal integrin signals for survival.
The mechanisms underlying integrin-dependent signalling are a topic of continued study. Endocytosed integrins are now shown to drive assembly of signalling complexes on the cytoplasmic face of endocytic membranes to promote cancer cell survival and increase metastatic capacity following cell detachment
Interplay between cell adhesion and growth factor receptors: from the plasma membrane to the endosomes
The emergence of multicellular animals could only take place once evolution had produced molecular mechanisms for cell adhesion and communication. Today, all metazoans express integrin-type adhesion receptors and receptors for growth factors. Integrins recognize extracellular matrix proteins and respective receptors on other cells and, following ligand binding, can activate the same cellular signaling pathways that are regulated by growth factor receptors. Recent reports have indicated that the two receptor systems also collaborate in many other ways. Here, we review the present information concerning the role of integrins as assisting growth factor receptors and the interplay between the receptors in cell signaling and in the orchestration of receptor recycling
Targeting Ξ±Ξ½Ξ²3 and Ξ±Ξ½Ξ²5 inhibits photon-induced hypermigration of malignant glioma cells
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