19 results on '"Bousgouni V"'
Search Results
2. Robust optical autofocus system utilizing neural networks trained for extended range and time-course and automated multiwell plate imaging including single molecule localization microscopy
- Author
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Lightley, J., primary, Görlitz, F., additional, Kumar, S., additional, Kalita, R., additional, Kolbeinsson, A., additional, Garcia, E., additional, Alexandrov, Y, additional, Bousgouni, V., additional, Wysoczanski, R., additional, Barnes, P., additional, Donnelly, L., additional, Bakal, C., additional, Dunsby, C., additional, Neil, M.A.A., additional, Flaxman, S., additional, and French, P.M.W., additional
- Published
- 2021
- Full Text
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3. Robust optical autofocus system utilizing neural networks applied to automated multiwell plate STORM microscopy.
- Author
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Lightley, J., Görlitz, F., Kumar, S., Kalita, R., Kolbeinsson, A., Garcia, E., Alexandrov, Y., Bousgouni, V., Wysoczanski, R., Barnes, P., Donelly, L.., Bakal, C., Dunsby, C., Neil, M. A. A., Flaxman, S., and French, P. M. W.
- Published
- 2021
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4. The thiazolidinedione pioglitazone increases cholesterol biosynthetic gene expression in primary cortical neurons by a PPARgamma-independent mechanism.
- Author
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Cocks G, Wilde JI, Graham SJ, Bousgouni V, Virley D, Lovestone S, and Richardson J
- Published
- 2010
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5. Robust optical autofocus system utilizing neural networks applied to automated multiwell plate STORM microscopy
- Author
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Beaurepaire, Emmanuel, Ben-Yakar, Adela, Park, YongKeun, Lightley, J., Görlitz, F., Kumar, S., Kalita, R., Kolbeinsson, A., Garcia, E., Alexandrov, Y., Bousgouni, V., Wysoczanski, R., Barnes, P., Donelly, L.., Bakal, C., Dunsby, C., Neil, M. A. A., Flaxman, S., and French, P. M. W.
- Published
- 2021
- Full Text
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6. Biocompatibility characterisation of CMOS-based Lab-on-Chip electrochemical sensors for in vitro cancer cell culture applications.
- Author
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Beykou M, Bousgouni V, Moser N, Georgiou P, and Bakal C
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- Humans, Silicon Compounds chemistry, Cell Culture Techniques instrumentation, Electrochemical Techniques instrumentation, Electrochemical Techniques methods, Neoplasms, Biocompatible Materials chemistry, Biocompatible Materials pharmacology, Cell Line, Tumor, Biosensing Techniques instrumentation, Lab-On-A-Chip Devices, Cell Adhesion, Semiconductors, Cell Proliferation
- Abstract
Lab-on-Chip electrochemical sensors, such as Ion-Sensitive Field-Effect Transistors (ISFETs), are being developed for use in point-of-care diagnostics, such as pH detection of tumour microenvironments, due to their integration with standard Complementary Metal Oxide Semiconductor (CMOS) technology. With this approach, the passivation of the CMOS process is used as a sensing layer to minimise post-processing, and Silicon Nitride (Si
3 N4 ) is the most common material at the microchip surface. ISFETs have the potential to be used for cell-based assays however, there is a poor understanding of the biocompatibility of microchip surfaces. Here, we quantitatively evaluated cell adhesion, morphogenesis, proliferation and mechano-responsiveness of both normal and cancer cells cultured on a Si3 N4 , sensor surface. We demonstrate that both normal and cancer cell adhesion decreased on Si3 N4 . Activation of the mechano-responsive transcription regulators, YAP/TAZ, are significantly decreased in cancer cells on Si3 N4 in comparison to standard cell culture plastic, whilst proliferation marker, Ki67, expression markedly increased. Non-tumorigenic cells on chip showed less sensitivity to culture on Si3 N4 than cancer cells. Treatment with extracellular matrix components increased cell adhesion in normal and cancer cell cultures, surpassing the adhesiveness of plastic alone. Moreover, poly-l-ornithine and laminin treatment restored YAP/TAZ levels in both non-tumorigenic and cancer cells to levels comparable to those observed on plastic. Thus, engineering the electrochemical sensor surface with treatments will provide a more physiologically relevant environment for future cell-based assay development on chip., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2024
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7. Insights from multi-omic modeling of neurodegeneration in xeroderma pigmentosum using an induced pluripotent stem cell system.
- Author
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Badja C, Momen S, Koh GCC, Boushaki S, Roumeliotis TI, Kozik Z, Jones I, Bousgouni V, Dias JML, Krokidis MG, Young J, Chen H, Yang M, Docquier F, Memari Y, Valcarcel-Zimenez L, Gupta K, Kong LR, Fawcett H, Robert F, Zhao S, Degasperi A, Kumar Y, Davies H, Harris R, Frezza C, Chatgilialoglu C, Sarkany R, Lehmann A, Bakal C, Choudhary J, Fassihi H, and Nik-Zainal S
- Subjects
- Humans, Neurons metabolism, Neurons pathology, Oxidative Stress, Endoplasmic Reticulum Stress, Proteasome Endopeptidase Complex metabolism, Cell Differentiation, DNA Damage, Models, Biological, Multiomics, Xeroderma Pigmentosum pathology, Xeroderma Pigmentosum metabolism, Xeroderma Pigmentosum genetics, Induced Pluripotent Stem Cells metabolism
- Abstract
Xeroderma pigmentosum (XP) is caused by defective nucleotide excision repair of DNA damage. This results in hypersensitivity to ultraviolet light and increased skin cancer risk, as sunlight-induced photoproducts remain unrepaired. However, many XP patients also display early-onset neurodegeneration, which leads to premature death. The mechanism of neurodegeneration is unknown. Here, we investigate XP neurodegeneration using pluripotent stem cells derived from XP patients and healthy relatives, performing functional multi-omics on samples during neuronal differentiation. We show substantially increased levels of 5',8-cyclopurine and 8-oxopurine in XP neuronal DNA secondary to marked oxidative stress. Furthermore, we find that the endoplasmic reticulum stress response is upregulated and reversal of the mutant genotype is associated with phenotypic rescue. Critically, XP neurons exhibit inappropriate downregulation of the protein clearance ubiquitin-proteasome system (UPS). Chemical enhancement of UPS activity in XP neuronal models improves phenotypes, albeit inadequately. Although more work is required, this study presents insights with intervention potential., Competing Interests: Declaration of interests S.N.-Z. holds patents on mutational signature-based clinical algorithms not relevant to the research presented in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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8. Environmentally dependent and independent control of 3D cell shape.
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Dent LG, Curry N, Sparks H, Bousgouni V, Maioli V, Kumar S, Munro I, Butera F, Jones I, Arias-Garcia M, Rowe-Brown L, Dunsby C, and Bakal C
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- Humans, Cell Line, Tumor, Microtubules metabolism, Myosin Type II metabolism, Rho Guanine Nucleotide Exchange Factors metabolism, Rho Guanine Nucleotide Exchange Factors genetics, Melanoma pathology, Melanoma metabolism, Guanine Nucleotide Exchange Factors metabolism, Guanine Nucleotide Exchange Factors genetics, Cell Shape
- Abstract
How cancer cells determine their shape in response to three-dimensional (3D) geometric and mechanical cues is unclear. We develop an approach to quantify the 3D cell shape of over 60,000 melanoma cells in collagen hydrogels using high-throughput stage-scanning oblique plane microscopy (ssOPM). We identify stereotypic and environmentally dependent changes in shape and protrusivity depending on whether a cell is proximal to a flat and rigid surface or is embedded in a soft environment. Environmental sensitivity metrics calculated for small molecules and gene knockdowns identify interactions between the environment and cellular factors that are important for morphogenesis. We show that the Rho guanine nucleotide exchange factor (RhoGEF) TIAM2 contributes to shape determination in environmentally independent ways but that non-muscle myosin II, microtubules, and the RhoGEF FARP1 regulate shape in ways dependent on the microenvironment. Thus, changes in cancer cell shape in response to 3D geometric and mechanical cues are modulated in both an environmentally dependent and independent fashion., Competing Interests: Declaration of interests C.D. has a licensed granted patent on the optical arrangement for oblique plane microscopy (OPM) under patent nos. US 8582203 B2 and EP 2316048 B1., (Copyright © 2024. Published by Elsevier Inc.)
- Published
- 2024
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9. Actin nucleators safeguard replication forks by limiting nascent strand degradation.
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Nieminuszczy J, Martin PR, Broderick R, Krwawicz J, Kanellou A, Mocanu C, Bousgouni V, Smith C, Wen KK, Woodward BL, Bakal C, Shackley F, Aguilera A, Stewart GS, Vyas YM, and Niedzwiedz W
- Subjects
- Replication Protein A genetics, Replication Protein A metabolism, DNA, Single-Stranded genetics, Molecular Chaperones genetics, Actins genetics, DNA Replication
- Abstract
Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here, we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e. Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that β-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing β-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing β-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2023
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10. Quantitative imaging of single-cell phenotypes in cancer cells cultured on hydrogel surfaces.
- Author
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Bousgouni V and Bakal C
- Subjects
- High-Throughput Screening Assays methods, RNA, Small Interfering genetics, Diagnostic Imaging, Phenotype, Hydrogels, Neoplasms genetics
- Abstract
Small interfering RNA (siRNA) screening approaches used with quantitative single-cell analysis can uncover the roles of genes in cell morphogenesis. Here, we present a high-throughput automated phenotypic screening technique to quantify a single cell shape in cancer cells cultured on top of soft 3D hydrogels. We describe reverse transfection of cells with siRNAs and seeding of these cells on top of collagen, followed by image analysis to quantify morphology of a single cell and population levels in low-elasticity matrices. For complete details on the use and execution of this protocol, please refer to Bousgouni et al. (2022).
1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
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11. Robust deep learning optical autofocus system applied to automated multiwell plate single molecule localization microscopy.
- Author
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Lightley J, Görlitz F, Kumar S, Kalita R, Kolbeinsson A, Garcia E, Alexandrov Y, Bousgouni V, Wysoczanski R, Barnes P, Donnelly L, Bakal C, Dunsby C, Neil MAA, Flaxman S, and French PMW
- Subjects
- Single Molecule Imaging, Machine Learning, Microscopy methods, Deep Learning
- Abstract
We presenta robust, long-range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, for example due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition. To address the impact of (thermal or mechanical) fluctuations over time in the optical autofocus system itself, we utilize a convolutional neural network (CNN) that is trained over multiple days to account for such fluctuations. To address the trade-off between axial precision and range of the autofocus, we implement orthogonal optical readouts with separate CNN training data, thereby achieving an accuracy well within the 600 nm depth of field of our 1.3 numerical aperture objective lens over a defocus range of up to approximately +/-100 μm. We characterize the performance of this autofocus system and demonstrate its application to automated multiwell plate single molecule localization microscopy., (© 2021 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)
- Published
- 2022
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12. ARHGEF9 regulates melanoma morphogenesis in environments with diverse geometry and elasticity by promoting filopodial-driven adhesion.
- Author
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Bousgouni V, Inge O, Robertson D, Jones I, Clatworthy I, and Bakal C
- Abstract
Rho GTP Exchange Factors (RhoGEFs) and Rho GTPase Activating Proteins (RhoGAPs) are large families of molecules that regulate shape determination in all eukaryotes. In pathologies such as melanoma, RhoGEF and RhoGAP activity underpins the ability of cells to invade tissues of varying elasticity. To identify RhoGEFs and RhoGAPs that regulate melanoma cell shape on soft and/or stiff materials, we performed genetic screens, in tandem with single-cell quantitative morphological analysis. We show that ARHGEF9/Collybistin (Cb) is essential for cell shape determination on both soft and stiff materials, and in cells embedded in 3D soft hydrogel. ARHGEF9 is required for melanoma cells to invade 3D matrices. Depletion of ARHGEF9 results in loss of tension at focal adhesions decreased cell-wide contractility, and the inability to stabilize protrusions. Taken together we show that ARHGEF9 promotes the formation of actin-rich filopodia, which serves to establish and stabilize adhesions and determine melanoma cell shape., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)
- Published
- 2022
- Full Text
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13. A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division.
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Stojic L, Lun ATL, Mascalchi P, Ernst C, Redmond AM, Mangei J, Barr AR, Bousgouni V, Bakal C, Marioni JC, Odom DT, and Gergely F
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- HeLa Cells, High-Throughput Screening Assays, Humans, Mitosis genetics, Mitosis physiology, Proteins genetics, RNA Interference physiology, Cell Division genetics, Cell Division physiology, RNA, Long Noncoding genetics
- Abstract
Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.
- Published
- 2020
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14. WDR5, BRCA1, and BARD1 Co-regulate the DNA Damage Response and Modulate the Mesenchymal-to-Epithelial Transition during Early Reprogramming.
- Author
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Peñalosa-Ruiz G, Bousgouni V, Gerlach JP, Waarlo S, van de Ven JV, Veenstra TE, Silva JCR, van Heeringen SJ, Bakal C, Mulder KW, and Veenstra GJC
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- Animals, BRCA1 Protein metabolism, Chromatin genetics, Chromatin metabolism, DNA Repair, Gene Expression Profiling, Humans, Intracellular Signaling Peptides and Proteins metabolism, Mice, Phenotype, Signal Transduction, Transcription Factors genetics, Transcription Factors metabolism, Tumor Suppressor Proteins metabolism, Ubiquitin-Protein Ligases metabolism, BRCA1 Protein genetics, Cellular Reprogramming genetics, DNA Damage, Epithelial-Mesenchymal Transition genetics, Intracellular Signaling Peptides and Proteins genetics, Tumor Suppressor Proteins genetics, Ubiquitin-Protein Ligases genetics
- Abstract
Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features. This revealed five morphological phenotypes in early reprogramming, including one displaying large round colonies exhibiting an early block of reprogramming. Using RNA sequencing, we identified transcriptional changes associated with these phenotypes. Furthermore, double knockdown epistasis experiments revealed that BRCA1, BARD1, and WDR5 functionally interact and are required for the DNA damage response. In addition, the mesenchymal-to-epithelial transition is affected in Brca1, Bard1, and Wdr5 knockdowns. Our data provide a resource of chromatin-associated factors in early reprogramming and underline colony morphology as an important high-dimensional readout for reprogramming quality., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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15. RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer.
- Author
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Pascual-Vargas P, Cooper S, Sero J, Bousgouni V, Arias-Garcia M, and Bakal C
- Subjects
- Female, Humans, RNA Interference, GTPase-Activating Proteins, Triple Negative Breast Neoplasms enzymology, Triple Negative Breast Neoplasms pathology
- Abstract
In order to metastasise, triple negative breast cancer (TNBC) must make dynamic changes in cell shape. The shape of all eukaryotic cells is regulated by Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which activate Rho-family GTPases in response to mechanical and informational cues. In contrast, Rho GTPase-activating proteins (RhoGAPs) inhibit Rho GTPases. However, which RhoGEFs and RhoGAPS couple TNBC cell shape to changes in their environment is very poorly understood. Moreover, whether the activity of particular RhoGEFs and RhoGAPs become dysregulated as cells evolve the ability to metastasise is not clear. Towards the ultimate goal of identifying RhoGEFs and RhoGAPs that are essential for TNBC metastasis, we performed an RNAi screen to isolate RhoGEFs and RhoGAPs that contribute to the morphogenesis of the highly metastatic TNBC cell line LM2, and its less-metastatic parental cell line MDA-MB-231. For ~6 million cells from each cell line, we measured 127 different features following the depletion of 142 genes. Using a linear classifier scheme we also describe the morphological heterogeneity of each gene-depleted population.
- Published
- 2017
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16. Apolar and polar transitions drive the conversion between amoeboid and mesenchymal shapes in melanoma cells.
- Author
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Cooper S, Sadok A, Bousgouni V, and Bakal C
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- Animals, Cattle, Cell Line, Tumor, Cell Movement physiology, Cell Shape physiology, Collagen metabolism, Humans, Mesoderm metabolism, Neoplasm Invasiveness, Polar Bodies metabolism, Signal Transduction, Structure-Activity Relationship, rho GTP-Binding Proteins metabolism, Melanoma pathology
- Abstract
Melanoma cells can adopt two functionally distinct forms, amoeboid and mesenchymal, which facilitates their ability to invade and colonize diverse environments during the metastatic process. Using quantitative imaging of single living tumor cells invading three-dimensional collagen matrices, in tandem with unsupervised computational analysis, we found that melanoma cells can switch between amoeboid and mesenchymal forms via two different routes in shape space--an apolar and polar route. We show that whereas particular Rho-family GTPases are required for the morphogenesis of amoeboid and mesenchymal forms, others are required for transitions via the apolar or polar route and not amoeboid or mesenchymal morphogenesis per se. Altering the transition rates between particular routes by depleting Rho-family GTPases can change the morphological heterogeneity of cell populations. The apolar and polar routes may have evolved in order to facilitate conversion between amoeboid and mesenchymal forms, as cells are either searching for, or attracted to, particular migratory cues, respectively., (© 2015 Cooper et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
- Published
- 2015
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17. Signaling networks converge on TORC1-SREBP activity to promote endoplasmic reticulum homeostasis.
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Sanchez-Alvarez M, Finger F, Arias-Garcia Mdel M, Bousgouni V, Pascual-Vargas P, and Bakal C
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- Animals, Cell Line, Cell Proliferation, Drosophila melanogaster, Endoplasmic Reticulum Stress, Endoribonucleases metabolism, Fatty Acids, Unsaturated physiology, G1 Phase Cell Cycle Checkpoints, Gene Knockdown Techniques, Lipid Metabolism, RNA Interference, Unfolded Protein Response, Drosophila Proteins metabolism, Endoplasmic Reticulum physiology, Homeostasis, Signal Transduction, Sterol Regulatory Element Binding Proteins metabolism, Transcription Factors metabolism
- Abstract
The function and capacity of the endoplasmic reticulum (ER) is determined by multiple processes ranging from the local regulation of peptide translation, translocation, and folding, to global changes in lipid composition. ER homeostasis thus requires complex interactions amongst numerous cellular components. However, describing the networks that maintain ER function during changes in cell behavior and environmental fluctuations has, to date, proven difficult. Here we perform a systems-level analysis of ER homeostasis, and find that although signaling networks that regulate ER function have a largely modular architecture, the TORC1-SREBP signaling axis is a central node that integrates signals emanating from different sub-networks. TORC1-SREBP promotes ER homeostasis by regulating phospholipid biosynthesis and driving changes in ER morphology. In particular, our network model shows TORC1-SREBP serves to integrate signals promoting growth and G1-S progression in order to maintain ER function during cell proliferation.
- Published
- 2014
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18. Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity.
- Author
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Sailem H, Bousgouni V, Cooper S, and Bakal C
- Subjects
- Animals, Bayes Theorem, Cell Line, Cell Shape, Cluster Analysis, Cytoskeleton metabolism, Drosophila enzymology, Drosophila metabolism, Principal Component Analysis, RNA Interference, Signal Transduction, Software, rac GTP-Binding Proteins genetics, rho GTP-Binding Proteins genetics, rac GTP-Binding Proteins metabolism, rho GTP-Binding Proteins metabolism
- Abstract
One goal of cell biology is to understand how cells adopt different shapes in response to varying environmental and cellular conditions. Achieving a comprehensive understanding of the relationship between cell shape and environment requires a systems-level understanding of the signalling networks that respond to external cues and regulate the cytoskeleton. Classical biochemical and genetic approaches have identified thousands of individual components that contribute to cell shape, but it remains difficult to predict how cell shape is generated by the activity of these components using bottom-up approaches because of the complex nature of their interactions in space and time. Here, we describe the regulation of cellular shape by signalling systems using a top-down approach. We first exploit the shape diversity generated by systematic RNAi screening and comprehensively define the shape space a migratory cell explores. We suggest a simple Boolean model involving the activation of Rac and Rho GTPases in two compartments to explain the basis for all cell shapes in the dataset. Critically, we also generate a probabilistic graphical model to show how cells explore this space in a deterministic, rather than a stochastic, fashion. We validate the predictions made by our model using live-cell imaging. Our work explains how cross-talk between Rho and Rac can generate different cell shapes, and thus morphological heterogeneity, in genetically identical populations.
- Published
- 2014
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19. Differential RNAi screening provides insights into the rewiring of signalling networks during oxidative stress.
- Author
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Garcia MA, Alvarez MS, Sailem H, Bousgouni V, Sero J, and Bakal C
- Subjects
- Animals, Antioxidants metabolism, Cell Survival drug effects, Cells, Cultured, Drosophila Proteins genetics, Drosophila melanogaster drug effects, Drosophila melanogaster genetics, Endoribonucleases genetics, Gene Expression drug effects, Multigene Family, Oxidation-Reduction, Oxidative Stress, Protein Interaction Mapping, Proto-Oncogene Proteins c-jun genetics, RNA Interference, RNA, Small Interfering genetics, Signal Transduction drug effects, Stress, Physiological drug effects, Superoxides metabolism, Drosophila Proteins metabolism, Drosophila melanogaster metabolism, Endoribonucleases metabolism, Gene Regulatory Networks, Paraquat pharmacology, Proto-Oncogene Proteins c-jun metabolism
- Abstract
Reactive Oxygen Species (ROS) are a natural by-product of cellular growth and proliferation, and are required for fundamental processes such as protein-folding and signal transduction. However, ROS accumulation, and the onset of oxidative stress, can negatively impact cellular and genomic integrity. Signalling networks have evolved to respond to oxidative stress by engaging diverse enzymatic and non-enzymatic antioxidant mechanisms to restore redox homeostasis. The architecture of oxidative stress response networks during periods of normal growth, and how increased ROS levels dynamically reconfigure these networks are largely unknown. In order to gain insight into the structure of signalling networks that promote redox homeostasis we first performed genome-scale RNAi screens to identify novel suppressors of superoxide accumulation. We then infer relationships between redox regulators by hierarchical clustering of phenotypic signatures describing how gene inhibition affects superoxide levels, cellular viability, and morphology across different genetic backgrounds. Genes that cluster together are likely to act in the same signalling pathway/complex and thus make "functional interactions". Moreover we also calculate differential phenotypic signatures describing the difference in cellular phenotypes following RNAi between untreated cells and cells submitted to oxidative stress. Using both phenotypic signatures and differential signatures we construct a network model of functional interactions that occur between components of the redox homeostasis network, and how such interactions become rewired in the presence of oxidative stress. This network model predicts a functional interaction between the transcription factor Jun and the IRE1 kinase, which we validate in an orthogonal assay. We thus demonstrate the ability of systems-biology approaches to identify novel signalling events.
- Published
- 2012
- Full Text
- View/download PDF
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