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Your search keyword '"François Sipieter"' showing total 9 results
Start Over Author "François Sipieter" Topic 0302 clinical medicine
9 results on '"François Sipieter"'

1. Les nesprines, des protéines de l’enveloppe nucléaire, contrôlent mécaniquement différentes transitions épithélio-mésenchymateuses

Author

Louis Laurent, François Sipieter, Nicolas Borghi, Université de Lille, VIB-UGent Center for Inflammation Research [Gand, Belgique] (IRC), VIB [Belgium], Universiteit Gent = Ghent University [Belgium] (UGENT), Institut Jacques Monod UMR 7592 Université Paris Diderot and CNRS, 75205 Paris cedex 13, Universiteit Gent = Ghent University (UGENT), Institut Jacques Monod (IJM (UMR_7592)), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
0303 health sciences, 03 medical and health sciences, 0302 clinical medicine, [SDV]Life Sciences [q-bio], General Medicine, 030217 neurology & neurosurgery, General Biochemistry, Genetics and Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

International audience; No abstract available

Published
2021

2. Simultaneous readout of multiple FRET pairs using photochromism

Author

Peter Dedecker, Christophe Stove, François Sipieter, Thijs Roebroek, Wim Vandenberg, Siewert Hugelier, and Jin Zhang

Subjects
0301 basic medicine, Fluorescence-lifetime imaging microscopy, Resonant inductive coupling, Extracellular signal-regulated kinases, General Physics and Astronomy, Biosensing Techniques, Fluorescence imaging, 0302 clinical medicine, Chlorocebus aethiops, Fluorescence Resonance Energy Transfer, Absorption (electromagnetic radiation), 0303 health sciences, Multidisciplinary, Imaging and sensing, Multidisciplinary Sciences, Chemistry, 030220 oncology & carcinogenesis, COS Cells, Optoelectronics, Science & Technology - Other Topics, Algorithms, Materials science, Science, Time-Lapse Imaging, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Photochromism, Animals, Humans, Fluorescent Dyes, 030304 developmental biology, Science & Technology, business.industry, Extramural, General Chemistry, Fluorescent proteins, Cyclic AMP-Dependent Protein Kinases, Acceptor, Luminescent Proteins, HEK293 Cells, Wide-field fluorescence microscopy, 030104 developmental biology, Förster resonance energy transfer, Microscopy, Fluorescence, Biophysics, business, Biosensor, 030217 neurology & neurosurgery, HeLa Cells
Abstract

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent protein kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for extracellular signal-regulated kinase (ERK) activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells., Performing multiple FRET measurements at once can be challenging. Here the authors report a method to discriminate between overlapping FRET pairs, even if the fluorophores display almost identical absorption and emission spectra, based on the photochromism of the donor fluorophores.

Published
2021

3. Nesprins are mechanotransducers that discriminate epithelial–mesenchymal transition programs

Author

Edgar R. Gomes, Pietro Salvatore Carollo, Cécile Sykes, Bruno Cadot, Théophile Déjardin, François Sipieter, Cynthia Seiler, Damien Cuvelier, Patricia M. Davidson, Nicolas Borghi, Institut Jacques Monod (IJM (UMR_7592)), Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Physico-Chimie Curie [Institut Curie] (PCC), Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Myologie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre de recherche en Myologie – U974 SU-INSERM, Repositório da Universidade de Lisboa, and Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPC)

Subjects
Epithelial-Mesenchymal Transition, [SDV]Life Sciences [q-bio], LINC complex, Cell, Biophysics, Biology, Mechanotransduction, Cellular, Article, 03 medical and health sciences, 0302 clinical medicine, Cell Signaling, medicine, Epithelial–mesenchymal transition, Cytoskeleton, Integral membrane protein, beta Catenin, 030304 developmental biology, Cell Nucleus, 0303 health sciences, Nesprin, Migration, Motility, Nuclear Proteins, Cell Biology, Transmembrane protein, 3. Good health, Cell biology, medicine.anatomical_structure, Signal transduction, 030217 neurology & neurosurgery
Abstract

© 2020 Déjardin et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/)., LINC complexes are transmembrane protein assemblies that physically connect the nucleoskeleton and cytoskeleton through the nuclear envelope. Dysfunctions of LINC complexes are associated with pathologies such as cancer and muscular disorders. The mechanical roles of LINC complexes are poorly understood. To address this, we used genetically encoded FRET biosensors of molecular tension in a nesprin protein of the LINC complex of fibroblastic and epithelial cells in culture. We exposed cells to mechanical, genetic, and pharmacological perturbations, mimicking a range of physiological and pathological situations. We show that nesprin experiences tension generated by the cytoskeleton and acts as a mechanical sensor of cell packing. Moreover, nesprin discriminates between inductions of partial and complete epithelial-mesenchymal transitions. We identify the implicated mechanisms, which involve α-catenin capture at the nuclear envelope by nesprin upon its relaxation, thereby regulating β-catenin transcription. Our data thus implicate LINC complex proteins as mechanotransducers that fine-tune β-catenin signaling in a manner dependent on the epithelial-mesenchymal transition program., This material is based on work supported by the Centre national de la recherche scientifique (CNRS), Agence nationale de la recherche (ANR; grants ANR-13-JSV5-0007 and ANR-14-CE09-0006), France BioImaging (ANR-10-INBS-04), la Ligue contre le Cancer (REMX17751 to P.M. Davidson), and the Fondation ARC pour la Recherche sur le Cancer (PDF20161205227 to P.M. Davidson). P.S. Carollo has received funding from the European Union’s Horizon 2020 Framework Programme for Research and Innovation (Marie Skłodowska-Curie grant agreement 665850-INSPIRE) and acknowledges the Ecole Doctorale Frontières de l'Innovation en Recherche et Éducation (FIRE) Programme Bettencourt. E.R. Gomes was supported by a European Research Council consolidator grant (617676).

Published
2020

4. Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling

Author

Livio Trusolino, Francesco Sassi, Sander Mertens, Benjamin Cappe, Ingrid Verlaan-Klink, Ravian L. van Ineveld, Bas Ponsioen, Sylvia F. Boj, Simone Kersten, Julian R. Buissant des Amorie, Dimitrios Laskaris, Rob G. J. Vries, Franck B. Riquet, Andrea Bertotti, Jasmin B. Post, François Sipieter, Johannes L. Bos, Peter Vandenabeele, Hugo J. Snippert, Holger Rehmann, University Medical Center [Utrecht], Department of Biomedical Molecular Biology [Ghent], Universiteit Gent = Ghent University [Belgium] (UGENT), Institut Jacques Monod (IJM (UMR_7592)), and Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
MAPK/ERK pathway, endocrine system diseases, cell-to-cell heterogeneity, pan-HER inhibition, [SDV]Life Sciences [q-bio], Mitogen-activated protein kinase kinase, medicine.disease_cause, 0302 clinical medicine, Epidermal growth factor receptor, oncogenic signaling, EGFR inhibitors, 0303 health sciences, Tumor, biology, Kinase, Chemistry, 3. Good health, Cell biology, ErbB Receptors, Gene Expression Regulation, Neoplastic, Organoids, 030220 oncology & carcinogenesis, KRAS, Signal transduction, Single-Cell Analysis, Colorectal Neoplasms, patient-derived organoids, Proto-Oncogene Proteins B-raf, MAP Kinase Signaling System, EGFR, FRET biosensors, Article, Cell Line, Proto-Oncogene Proteins p21(ras), 03 medical and health sciences, Cell Line, Tumor, medicine, Humans, Protein kinase A, neoplasms, Protein Kinase Inhibitors, 030304 developmental biology, Mitogen-Activated Protein Kinase Kinases, Neoplastic, Cell Biology, ERK oscillations, Colorectal cancer, digestive system diseases, Gene Expression Regulation, Mutation, biology.protein
Abstract

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC. Ponsioen et al. use a FRET‐based ERK biosensor EKAREN5 in patient‐derived organoids to show that EGFR activity amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways.

Published
2020

5. Characteristic ERK1/2 signaling dynamics distinguishes necroptosis from apoptosis

Author

Peter Vandenabeele, Wim Declercq, Jolien Bridelance, Laurent Héliot, Pierre Vincent, Franck B. Riquet, François Sipieter, Aymeric Leray, Elke De Schutter, Benjamin Cappe, Guy Van Camp, Paco Hulpiau, Universiteit Gent = Ghent University [Belgium] (UGENT), VIB-UGent Center for Inflammation Research [Gand, Belgique] (IRC), VIB [Belgium], Université de Lille, Laboratoire Interdisciplinaire Carnot de Bourgogne [Dijon] (LICB), Université de Bourgogne (UB)-Université de Technologie de Belfort-Montbeliard (UTBM)-Centre National de la Recherche Scientifique (CNRS), University of Antwerp (UA), VIB Center for Inflammation Research [Ghent, Belgium], Antwerp University Hospital [Edegem] (UZA), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Adaptation Biologique et Vieillissement = Biological Adaptation and Ageing (B2A), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Universiteit Gent = Ghent University (UGENT), Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB), Université de Technologie de Belfort-Montbeliard (UTBM)-Université de Bourgogne (UB)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Riquet, Franck, Université de Bourgogne (UB), and Leray, Aymeric

Subjects
Cell biology, Programmed cell death, Science, [SDV]Life Sciences [q-bio], Necroptosis, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, PROTEIN, MECHANISMS, ESCRT, ACTIVATION, 03 medical and health sciences, 0302 clinical medicine, INFLAMMATION, Gene expression, Medicine and Health Sciences, KINASE, Biology, 030304 developmental biology, 0303 health sciences, Multidisciplinary, IDENTIFICATION, Chemistry, NECROSIS, Dynamics (mechanics), Biology and Life Sciences, Erk1 2 signaling, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV] Life Sciences [q-bio], Biological sciences, Biomolecular engineering, CELL-DEATH, Apoptosis, Cell culture, 030220 oncology & carcinogenesis, Tumor necrosis factor alpha, BIOSENSORS, Human medicine
Abstract

International audience; ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular re- sponses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensi- tizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and nec- roptosis. We also decrypted a temporally shifted amplitude- and frequency-modu- lated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

Published
2021

6. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

Author

Julien Roul, Catherine Chapuis, Marc Tramier, François Sipieter, Sergi Padilla-Parra, Claire Demeautis, Franck B. Riquet, Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Universiteit Gent = Ghent University [Belgium] (UGENT), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Region Bretagne, IBiSA, Vandenabeele's group research funding: Fonds Wetenschappelijk Onderzoek [FWO G.0875.11], Vandenabeele's group research funding: Methusalem grant (Bijzonder Onderzoeksfonds) [BOF09/01M00709], Vlaams Instituut voor Biotechnologie (VIB), Belgian grants (Interuniversity Attraction Poles) [IAP 7/32], Ghent Researchers On Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium, Foundation against Cancer [2012-188], Agence Nationale pour la Recherche (ANR) [ANR-11-BSV5-0023, ANR-13-BSV2-0016-02], [FWO G.0973.11], Ligue Contre le Cancer Comite d'Ille et Vilaine, Comite du Maine et Loire et Comite de la Sarthe, Nuffield Department of Medicine Leadership Fellowship from University of Oxford, [FWO G.0172.12], Welcome Trust [090532/Z/09/X], [FWO G.0A45.12 N], Rennes Metropole, [FWO G.0787.13 N], Lille 1 University, [FWO G.0C31.14 N], CNRS, UR1, Centre National de la Recherche Scientifique (CNRS), Ghent University, Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Universiteit Gent = Ghent University (UGENT), ANR-11-BSV5-0023,KinBioFRET,Biosenseurs FRET pour la dynamique spatio-temporelle d'activités kinase par FLIM rapide et anisotropie de fluorescence sous microscope(2011), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Ghent University [Belgium] (UGENT), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
0301 basic medicine, resonance energy-transfer, ERK ACTIVITY, Mitogen-Activated Protein Kinase 3, CYCLIC-AMP, PROTEIN, activated protein-kinase, Biosensing Techniques, RESONANCE ENERGY-TRANSFER, growth-factor receptor, 0302 clinical medicine, erk activity, Medicine and Health Sciences, Cyclic AMP, Fluorescence Resonance Energy Transfer, cyclic-amp, Cellular localization, ComputingMilieux_MISCELLANEOUS, Mitogen-Activated Protein Kinase 1, Multidisciplinary, biology, Chemistry, Kinase, Optical Imaging, FLUORESCENT, Crosstalk (biology), Mitogen-activated protein kinase, map kinase, Signal Transduction, Cytological Techniques, Article, 03 medical and health sciences, Humans, fluorescent protein, SIGNAL-REGULATED KINASE, Kinase activity, Protein kinase A, large stokes shift, a activity, Fluorescent Dyes, [SDV.GEN]Life Sciences [q-bio]/Genetics, signal-regulated kinase, Biology and Life Sciences, PROTEIN-KINASE, MAP KINASE, Cyclic AMP-Dependent Protein Kinases, ACTIVATED, 030104 developmental biology, Förster resonance energy transfer, A ACTIVITY, biology.protein, Biophysics, GROWTH-FACTOR RECEPTOR, LARGE STOKES SHIFT, 030217 neurology & neurosurgery, HeLa Cells
Abstract

Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.

Published
2017

7. From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells

Author

Jean-François Bodart, Corentin Spriet, Franck B. Riquet, Pierre Vincent, Dave Trinel, Laurent Héliot, Aymeric Leray, Pauline Vandame, François Sipieter, Intégration cellulaire des Signaux Neuromodulateurs (ICSN), Adaptation Biologique et Vieillissement = Biological Adaptation and Ageing (B2A), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Morris, MC, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0303 health sciences, Cellular functions, Context (language use), Computational biology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, 03 medical and health sciences, 0302 clinical medicine, Förster resonance energy transfer, Biophysics, Kinase activity, 030217 neurology & neurosurgery, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

Biological processes are intrinsically dynamic. Although traditional methods provide valuable insights for the understanding of many biological phenomena, the possibility of measuring, quantifying, and localizing proteins within a cell, a tissue, and even an embryo has revolutionized our train of thoughts and has encouraged scientists to develop molecular tools for the assessment of protein or protein complex dynamics within their physiological context. These ongoing efforts rest on the emergence of biophotonic techniques and the continuous improvement of fluorescent probes, allowing precise and reliable measurements of dynamic cellular functions. The march of the “ in vivo biochemistry” has begun, already yielding breathtaking results.

Published
2013

8. Single Wavelength Excitation Dual Color Flim for Multiplexing Genetically Encoded FRET Biosensors

Author

François Sipieter, Franck B. Riquet, Sergi Padilla-Parra, Julien Roul, Catherine Chapuis, Claire Demeautis, Marc Tramier, Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )

Subjects
0303 health sciences, Conformational change, Chemistry, [SDV]Life Sciences [q-bio], Biophysics, macromolecular substances, Fluorescence, Acceptor, 03 medical and health sciences, symbols.namesake, 0302 clinical medicine, Förster resonance energy transfer, Nuclear magnetic resonance, Stokes shift, symbols, Multiplex, Biosensor, 030217 neurology & neurosurgery, Cellular localization, 030304 developmental biology
Abstract

Genetically encoded Forster Resonance Energy Transfert (FRET) biosensors are powerful tools for monitoring spatiotemporal biochemical activities in living samples. By labeling a probe protein with a pair of fluorescent proteins, FRET measurement allows to follow a conformational change of the probe sensor to a specific activity. A very exciting challenge is to follow two FRET biosensors at the same time in the same sample and in the same cellular compartment. But the multiplex approach suffers from two limitations: (i) a spectral bleed-through of the first acceptor in the second donor emission band that depends on the concentration of the two biosensors and (ii) the multiple excitation wavelengths which necessitates sequential acquisition that is not adequate to follow fast signal changes in highly dynamic biochemical activities.Here, we report a method alleviating from both limitations. Taking advantage of the long stoke shift of LSSmOrange, we have used 440 nm single excitation wavelength of the two donor mTFP1 and LSSmOrange and a dual color FLIM to simultaneously measure two genetically encoded FRET biosensors. Moreover, thanks to the non-fluorescent acceptor sREACh for mTFP1 and of red-shifted mKate2 for LSSmOrange, we were able to neglect any spectral bleed-trough. With a dual spectral FLIM system we were able to detect fluorescence lifetime images of mTFP1 and LSSmOrange simultaneously and in the same cellular localization. We validated our approach by applying this methodology to simultaneously determine ERK and PKA activation in the same Hela cell using EKAR2G and AKAR4 biosensors respectively modified with mTFP1/sREACh and LSSmOrange/mKate2 pairs. By activating PKA with forskolin, the ERK pathway is not activated as expected. But surprisingly, by activating ERK with EGF, PKA is also activated denoting a cross talk between these two signaling pathway in Hela cell line.

Published
2016

9. When Separation Strengthens Ties

Author

Helena Canever, François Sipieter, Nicolas Borghi, Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Borghi, Nicolas

Subjects
0303 health sciences, Tight junction, [SDV]Life Sciences [q-bio], Epithelial Cells, Cell Biology, Compartmentalization (psychology), Biology, Models, Biological, Phase Transition, Cell biology, [SDV] Life Sciences [q-bio], 03 medical and health sciences, 0302 clinical medicine, Cytoplasm, Animals, Humans, Zonula Occludens Proteins, Epithelial barrier function, Zebrafish, 030217 neurology & neurosurgery, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

Phase separation underlies functional compartmentalization in living systems. Two recent studies (Beutel et al. and Schwayer et al.) show that zonula occludens (ZO) proteins of tight junctions (TJs) condense into compartments within the cytoplasm that display liquid properties. This ability to condense predicts normal TJ assembly and epithelial barrier function which are essential for vertebrate embryogenesis.

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