Publications by authors named "Grégory Giannone"

46 Publications

Single-Protein Tracking to Study Protein Interactions During Integrin-Based Migration.

Methods Mol Biol 2021 ;2217:85-113

Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France.

Cell migration is a complex biophysical process which involves the coordination of molecular assemblies including integrin-dependent adhesions, signaling networks and force-generating cytoskeletal structures incorporating both actin polymerization and myosin activity. During the last decades, proteomic studies have generated impressive protein-protein interaction maps, although the subcellular location, duration, strength, sequence, and nature of these interactions are still concealed. In this chapter we describe how recent developments in superresolution microscopy (SRM) and single-protein tracking (SPT) start to unravel protein interactions and actions in subcellular molecular assemblies driving cell migration.
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http://dx.doi.org/10.1007/978-1-0716-0962-0_8DOI Listing
March 2021

Cell stretching is amplified by active actin remodelling to deform and recruit proteins in mechanosensitive structures.

Nat Cell Biol 2020 08 27;22(8):1011-1023. Epub 2020 Jul 27.

Interdisciplinary Institute for Neuroscience, UMR 5297, Université de Bordeaux, Bordeaux, France.

Detection and conversion of mechanical forces into biochemical signals controls cell functions during physiological and pathological processes. Mechanosensing is based on protein deformations and reorganizations, yet the molecular mechanisms are still unclear. Using a cell-stretching device compatible with super-resolution microscopy and single-protein tracking, we explored the nanoscale deformations and reorganizations of individual proteins inside mechanosensitive structures. We achieved super-resolution microscopy after live stretching on intermediate filaments, microtubules and integrin adhesions. Simultaneous single-protein tracking and stretching showed that while integrins followed the elastic deformation of the substrate, actin filaments and talin also displayed lagged and transient inelastic responses associated with active acto-myosin remodelling and talin deformations. Capturing acute reorganizations of single molecules during stretching showed that force-dependent vinculin recruitment is delayed and depends on the maturation of integrin adhesions. Thus, cells respond to external forces by amplifying transiently and locally cytoskeleton displacements, enabling protein deformation and recruitment in mechanosensitive structures.
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http://dx.doi.org/10.1038/s41556-020-0548-2DOI Listing
August 2020

A super-resolution platform for correlative live single-molecule imaging and STED microscopy.

Nat Methods 2019 12 21;16(12):1263-1268. Epub 2019 Oct 21.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.

Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.
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http://dx.doi.org/10.1038/s41592-019-0611-8DOI Listing
December 2019

Actin dynamics in cell migration.

Essays Biochem 2019 10;63(5):483-495

Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Spielmannstrasse 7, Braunschweig 38106, Germany.

Cell migration is an essential process, both in unicellular organisms such as amoeba and as individual or collective motility in highly developed multicellular organisms like mammals. It is controlled by a variety of activities combining protrusive and contractile forces, normally generated by actin filaments. Here, we summarize actin filament assembly and turnover processes, and how respective biochemical activities translate into different protrusion types engaged in migration. These actin-based plasma membrane protrusions include actin-related protein 2/3 complex-dependent structures such as lamellipodia and membrane ruffles, filopodia as well as plasma membrane blebs. We also address observed antagonisms between these protrusion types, and propose a model - also inspired by previous literature - in which a complex balance between specific Rho GTPase signaling pathways dictates the protrusion mechanism employed by cells. Furthermore, we revisit published work regarding the fascinating antagonism between Rac and Rho GTPases, and how this intricate signaling network can define cell behavior and modes of migration. Finally, we discuss how the assembly of actin filament networks can feed back onto their regulators, as exemplified for the lamellipodial factor WAVE regulatory complex, tightly controlling accumulation of this complex at specific subcellular locations as well as its turnover.
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http://dx.doi.org/10.1042/EBC20190015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823167PMC
October 2019

Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion.

Curr Biol 2019 09 15;29(17):2852-2866.e5. Epub 2019 Aug 15.

Interdisciplinary Institute for Neuroscience, Université de Bordeaux, UMR 5297, 33000 Bordeaux, France; Interdisciplinary Institute for Neuroscience, CNRS, UMR 5297, 33000 Bordeaux, France. Electronic address:

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion.
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http://dx.doi.org/10.1016/j.cub.2019.07.035DOI Listing
September 2019

A tessellation-based colocalization analysis approach for single-molecule localization microscopy.

Nat Commun 2019 05 30;10(1):2379. Epub 2019 May 30.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, 33076, France.

Multicolor single-molecule localization microscopy (λSMLM) is a powerful technique to reveal the relative nanoscale organization and potential colocalization between different molecular species. While several standard analysis methods exist for pixel-based images, λSMLM still lacks such a standard. Moreover, existing methods only work on 2D data and are usually sensitive to the relative molecular organization, a very important parameter to consider in quantitative SMLM. Here, we present an efficient, parameter-free colocalization analysis method for 2D and 3D λSMLM using tessellation analysis. We demonstrate that our method allows for the efficient computation of several popular colocalization estimators directly from molecular coordinates and illustrate its capability to analyze multicolor SMLM data in a robust and efficient manner.
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http://dx.doi.org/10.1038/s41467-019-10007-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542817PMC
May 2019

The inner life of integrin adhesion sites: From single molecules to functional macromolecular complexes.

Exp Cell Res 2019 06 31;379(2):235-244. Epub 2019 Mar 31.

Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR5297, 33000, Bordeaux, France; CNRS, Interdisciplinary Institute for Neuroscience, UMR5297, 33000, Bordeaux, France. Electronic address:

Cells are mechanical living machines that remodel their microenvironment by adhering and generating forces on the extracellular matrix (ECM) using integrin-dependent adhesion sites (IAS). In return, the biochemical and physical nature of the ECM determines cellular behavior and morphology during proliferation, differentiation and migration. IAS come in different shapes and forms. They have specific compositions, morphologies, mechanical and biochemical signaling activities, which serve different cellular functions. Proteomic studies showed that IAS are composed of a large repertoire of proteins that could be linked to different functional activities, including signaling, force-transmission and force-sensing. Thanks to recent technological advances in microscopy and protein engineering, it is now possible to localize single proteins in three dimensions inside IAS, determine their diffusive behaviors, orientations, and how much mechanical force is transmitted across individual components. Here, we review how researchers have used those tools to investigate how IAS components assemble and dynamically interact to produce diverse functions of adhesive structures.
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http://dx.doi.org/10.1016/j.yexcr.2019.03.036DOI Listing
June 2019

Using Single-Protein Tracking to Study Cell Migration.

Methods Mol Biol 2018 ;1749:291-311

Interdisciplinary Institute for Neuroscience, UMR 5297, University of Bordeaux, Bordeaux, France.

To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.
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http://dx.doi.org/10.1007/978-1-4939-7701-7_21DOI Listing
January 2019

Localization-based super-resolution imaging meets high-content screening.

Nat Methods 2017 Dec 30;14(12):1184-1190. Epub 2017 Oct 30.

Université de Bordeaux, Institut interdisciplinaire de Neurosciences, Bordeaux, France.

Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.
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http://dx.doi.org/10.1038/nmeth.4486DOI Listing
December 2017

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

Nat Protoc 2017 04 9;12(4):748-763. Epub 2017 Mar 9.

Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France.

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.
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http://dx.doi.org/10.1038/nprot.2017.010DOI Listing
April 2017

Organization and dynamics of the actin cytoskeleton during dendritic spine morphological remodeling.

Cell Mol Life Sci 2016 08 22;73(16):3053-73. Epub 2016 Apr 22.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, UMR 5297, 33000, Bordeaux, France.

In the central nervous system, most excitatory post-synapses are small subcellular structures called dendritic spines. Their structure and morphological remodeling are tightly coupled to changes in synaptic transmission. The F-actin cytoskeleton is the main driving force of dendritic spine remodeling and sustains synaptic plasticity. It is therefore essential to understand how changes in synaptic transmission can regulate the organization and dynamics of actin binding proteins (ABPs). In this review, we will provide a detailed description of the organization and dynamics of F-actin and ABPs in dendritic spines and will discuss the current models explaining how the actin cytoskeleton sustains both structural and functional synaptic plasticity.
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http://dx.doi.org/10.1007/s00018-016-2214-1DOI Listing
August 2016

Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

Cell 2016 Mar 25;165(1):100-110. Epub 2016 Feb 25.

Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.
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http://dx.doi.org/10.1016/j.cell.2016.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808403PMC
March 2016

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

Exp Cell Res 2016 04 10;343(1):28-34. Epub 2015 Nov 10.

Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France.

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.
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http://dx.doi.org/10.1016/j.yexcr.2015.11.004DOI Listing
April 2016

Super-resolution links vinculin localization to function in focal adhesions.

Nat Cell Biol 2015 Jul;17(7):845-7

Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France and CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France.

Integrin-based focal adhesions integrate biochemical and biomechanical signals from the extracellular matrix and the actin cytoskeleton. The combination of three-dimensional super-resolution imaging and loss- or gain-of-function protein mutants now links the nanoscale dynamic localization of proteins to their activation and function within focal adhesions.
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http://dx.doi.org/10.1038/ncb3196DOI Listing
July 2015

Single-molecule imaging in live cell using gold nanoparticles.

Methods Cell Biol 2015 7;125:13-27. Epub 2015 Jan 7.

Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d'Optique & CNRS, Talence, France.

Optimal single particle tracking experiments in live cells requires small and photostable probes, which do not modify the behavior of the molecule of interest. Current fluorescence-based microscopy of single molecules and nanoparticles is often limited by bleaching and blinking or by the probe size. As an alternative, we present in this chapter the synthesis of a small and highly specific gold nanoprobe whose detection is based on its absorption properties. We first present a protocol to synthesize 5-nm-diameter gold nanoparticles and functionalize them with a nanobody, a single-domain antibody from camelid, targeting the widespread green fluorescent protein (GFP)-tagged proteins with a high affinity. Then we describe how to detect and track these individual gold nanoparticles in live cell using photothermal imaging microscopy. The combination of a probe with small size, perfect photostability, high specificity, and versatility through the vast existing library of GFP-proteins, with a highly sensitive detection technique enables long-term tracking of proteins with minimal hindrance in confined and crowded environments such as intracellular space.
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http://dx.doi.org/10.1016/bs.mcb.2014.10.002DOI Listing
September 2015

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines.

Mol Biol Cell 2015 Mar 7;26(5):859-73. Epub 2015 Jan 7.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Unité Mixte de Recherche 5297, F-33000 Bordeaux, France Interdisciplinary Institute for Neuroscience, Centre Nationale de la Recherche Scientifique, Unité Mixte de Recherche 5297, F-33000 Bordeaux, France

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin-coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.
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http://dx.doi.org/10.1091/mbc.E14-06-1086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342023PMC
March 2015

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion.

EMBO J 2014 Dec 7;33(23):2745-64. Epub 2014 Oct 7.

Interdisciplinary Institute for Neuroscience, University Bordeaux UMR 5297, Bordeaux, France CNRS, Interdisciplinary Institute for Neuroscience UMR 5297, Bordeaux, France

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.
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http://dx.doi.org/10.15252/embj.201488837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282554PMC
December 2014

The cancer glycocalyx mechanically primes integrin-mediated growth and survival.

Nature 2014 Jul 25;511(7509):319-25. Epub 2014 Jun 25.

1] Department of Surgery and Center for Bioengineering and Tissue Regeneration, University of California, San Francisco, California 94143, USA [2] Bay Area Physical Sciences-Oncology Program, University of California, Berkeley, California 94720, USA [3] Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California 94143, USA [4] Departments of Anatomy and Bioengineering and Therapeutic Sciences and Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California, San Francisco, California 94143, USA.

Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.
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http://dx.doi.org/10.1038/nature13535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487551PMC
July 2014

Glutamate involvement in calcium-dependent migration of astrocytoma cells.

Cancer Cell Int 2014 19;14:42. Epub 2014 May 19.

Laboratoire de Biophotonique et Pharmacologie, CNRS, UMR 7213, Université de Strasbourg, Illkirch 67401, France.

Background: Astrocytoma are known to have altered glutamate machinery that results in the release of large amounts of glutamate into the extracellular space but the precise role of glutamate in favoring cancer processes has not yet been fully established. Several studies suggested that glutamate might provoke active killing of neurons thereby producing space for cancer cells to proliferate and migrate. Previously, we observed that calcium promotes disassembly of integrin-containing focal adhesions in astrocytoma, thus providing a link between calcium signaling and cell migration. The aim of this study was to determine how calcium signaling and glutamate transmission cooperate to promote enhanced astrocytoma migration.

Methods: The wound-healing model was used to assay migration of human U87MG astrocytoma cells and allowed to monitor calcium signaling during the migration process. The effect of glutamate on calcium signaling was evaluated together with the amount of glutamate released by astrocytoma during cell migration.

Results: We observed that glutamate stimulates motility in serum-starved cells, whereas in the presence of serum, inhibitors of glutamate receptors reduce migration. Migration speed was also reduced in presence of an intracellular calcium chelator. During migration, cells displayed spontaneous Ca(2+) transients. L-THA, an inhibitor of glutamate re-uptake increased the frequency of Ca(2+) oscillations in oscillating cells and induced Ca(2+) oscillations in quiescent cells. The frequency of migration-associated Ca(2+) oscillations was reduced by prior incubation with glutamate receptor antagonists or with an anti-β1 integrin antibody. Application of glutamate induced increases in internal free Ca(2+) concentration ([Ca(2+)]i). Finally we found that compounds known to increase [Ca(2+)]i in astrocytomas such as thapsigagin, ionomycin or the metabotropic glutamate receptor agonist t-ACPD, are able to induce glutamate release.

Conclusion: Our data demonstrate that glutamate increases migration speed in astrocytoma cells via enhancement of migration-associated Ca(2+) oscillations that in turn induce glutamate secretion via an autocrine mechanism. Thus, glutamate receptors are further validated as potential targets for astrocytoma cancer therapy.
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http://dx.doi.org/10.1186/1475-2867-14-42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032497PMC
May 2014

Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95.

J Neurosci 2013 Aug;33(32):13204-24

University of Bordeaux, Interdisciplinary Institute for Neuroscience, and Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5297, F-33000 Bordeaux, France.

The spatiotemporal organization of neurotransmitter receptors in postsynaptic membranes is a fundamental determinant of synaptic transmission and information processing by the brain. Using four independent super-resolution light imaging methods and EM of genetically tagged and endogenous receptors, we show that, in rat hippocampal neurons, AMPARs are often highly concentrated inside synapses into a few clusters of ∼70 nm that contain ∼20 receptors. AMPARs are stabilized reversibly in these nanodomains and diffuse freely outside them. Nanodomains are dynamic in their shape and position within synapses and can form or disappear within minutes, although they are mostly stable for up to 1 h. AMPAR nanodomains are often, but not systematically, colocalized with clusters of the scaffold protein PSD95, which are generally of larger size than AMPAR nanoclusters. PSD95 expression level regulates AMPAR nanodomain size and compactness in parallel to miniature EPSC amplitude. Monte Carlo simulations further indicate the impact of AMPAR concentration in clusters on the efficacy of synaptic transmission. The observation that AMPARs are highly concentrated in nanodomains, instead of diffusively distributed in the PSD as generally thought, has important consequences on our understanding of excitatory neurotransmission. Furthermore, our results indicate that glutamatergic synaptic transmission is controlled by the nanometer-scale regulation of the size of these highly concentrated nanodomains.
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http://dx.doi.org/10.1523/JNEUROSCI.2381-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619720PMC
August 2013

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells.

Sci Rep 2013 ;3:2387

LP2N, University of Bordeaux, UMR 5298, F-33405 Talence, France.

Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.
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http://dx.doi.org/10.1038/srep02387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737505PMC
February 2014

Neurexin-1β binding to neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1.

Cell Rep 2013 Jun 13;3(6):1996-2007. Epub 2013 Jun 13.

University Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France.

Adhesion between neurexin-1β (Nrx1β) and neuroligin-1 (Nlg1) induces early recruitment of the postsynaptic density protein 95 (PSD-95) scaffold; however, the associated signaling mechanisms are unknown. To dissociate the effects of ligand binding and receptor multimerization, we compared conditions in which Nlg1 in neurons was bound to Nrx1β or nonactivating HA antibodies. Time-lapse imaging, fluorescence recovery after photobleaching, and single-particle tracking demonstrated that in addition to aggregating Nlg1, Nrx1β binding stimulates the interaction between Nlg1 and PSD-95. Phosphotyrosine immunoblots and pull-down of gephyrin by Nlg1 peptides in vitro showed that Nlg1 can be phosphorylated at a unique tyrosine (Y782), preventing gephyrin binding. Expression of Nlg1 point mutants in neurons indicated that Y782 phosphorylation controls the preferential binding of Nlg1 to PSD-95 versus gephyrin, and accordingly the formation of inhibitory and excitatory synapses. We propose that ligand-induced changes in the Nlg1 phosphotyrosine level control the balance between excitatory and inhibitory scaffold assembly during synapse formation and stabilization.
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http://dx.doi.org/10.1016/j.celrep.2013.05.013DOI Listing
June 2013

A highly specific gold nanoprobe for live-cell single-molecule imaging.

Nano Lett 2013 Apr 6;13(4):1489-94. Epub 2013 Mar 6.

LP2N, Université de Bordeaux, Institut d'Optique & CNRS UMR 5298, Talence, France.

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Because of these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5 nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used green fluorescent proteins (GFPs) with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.
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http://dx.doi.org/10.1021/nl304561gDOI Listing
April 2013

High-content super-resolution imaging of live cell by uPAINT.

Methods Mol Biol 2013 ;950:95-110

Interdisciplinary Institute for Neuroscience, Université de Bordeaux, Bordeaux, France.

In this chapter, we present the uPAINT method (Universal Point Accumulation Imaging in Nanoscale Topography), a simple single-molecule super-resolution method which can be implemented on any wide field fluorescence microscope operating in oblique illumination. The key feature of uPAINT lies in recording high numbers of single molecules at the surface of a cell by constantly labeling while imaging. In addition to generating super-resolved images, uPAINT can provide dynamical information on a single live cell with large statistics revealing localization-specific diffusion properties of membrane biomolecules. Interestingly, any membrane biomolecule that can be labeled with a fluorescent ligand can be studied, making uPAINT an extremely versatile method.
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http://dx.doi.org/10.1007/978-1-62703-137-0_7DOI Listing
March 2013

Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions.

Nat Cell Biol 2012 Oct 30;14(10):1057-67. Epub 2012 Sep 30.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, UMR 5297, F-33000 Bordeaux, France.

Integrins in focal adhesions (FAs) mediate adhesion and force transmission to extracellular matrices essential for cell motility, proliferation and differentiation. Different fibronectin-binding integrins, simultaneously present in FAs, perform distinct functions. Yet, how integrin dynamics control biochemical and biomechanical processes in FAs is still elusive. Using single-protein tracking and super-resolution imaging we revealed the dynamic nano-organizations of integrins and talin inside FAs. Integrins reside in FAs through free-diffusion and immobilization cycles. Integrin activation promotes immobilization, stabilized in FAs by simultaneous connection to fibronectin and actin-binding proteins. Talin is recruited in FAs directly from the cytosol without membrane free-diffusion, restricting integrin immobilization to FAs. Immobilized β3-integrins are enriched and stationary within FAs, whereas immobilized β1-integrins are less enriched and exhibit rearward movements. Talin is enriched and mainly stationary, but also exhibited rearward movements in FAs, consistent with stable connections with both β-integrins. Thus, differential transmission of actin motion to fibronectin occurs through specific integrins within FAs.
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http://dx.doi.org/10.1038/ncb2588DOI Listing
October 2012

Quantum-yield-optimized fluorophores for site-specific labeling and super-resolution imaging.

J Am Chem Soc 2011 Jun 11;133(21):8090-3. Epub 2011 May 11.

Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt/M., Germany.

Single-molecule applications, saturated pattern excitation microscopy, and stimulated emission depletion (STED) microscopy demand bright as well as highly stable fluorescent dyes. Here we describe the synthesis of quantum-yield-optimized fluorophores for reversible, site-specific labeling of proteins or macromolecular complexes. We used polyproline-II (PPII) helices as sufficiently rigid spacers with various lengths to improve the fluorescence signals of a set of different trisNTA-fluorophores. The improved quantum yields were demonstrated by steady-state and fluorescence lifetime analyses. As a proof of principle, we characterized the trisNTA-PPII-fluorophores with respect to in vivo protein labeling and super-resolution imaging at synapses of living neurons. The distribution of His-tagged AMPA receptors (GluA1) in spatially restricted synaptic clefts was imaged by confocal and STED microscopy. The comparison of fluorescence intensity profiles revealed the superior resolution of STED microscopy. These results highlight the advantages of biocompatible and, in particular, small and photostable trisNTA-PPII-fluorophores in super-resolution microscopy.
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http://dx.doi.org/10.1021/ja200967zDOI Listing
June 2011

Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density.

Biophys J 2010 Aug;99(4):1303-10

Centre National de la Recherche Scientifique UMR 5091, Cellular Physiology of the Synapse, Bordeaux, France.

Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.
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http://dx.doi.org/10.1016/j.bpj.2010.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920718PMC
August 2010

Multi-level molecular clutches in motile cell processes.

Trends Cell Biol 2009 Sep 26;19(9):475-86. Epub 2009 Aug 26.

CNRS UMR 5091, Institut Magendie, Université Bordeaux 2, 33077 Bordeaux, France.

To trigger cell motility, forces generated by the cytoskeleton must be transmitted physically to the external environment through transmembrane adhesion molecules. One model put forward twenty years ago to describe this process is the molecular clutch by which a modular interface of adaptor proteins mediates a dynamic mechanical connection between the actin flow and cell adhesion complexes. Recent optical imaging experiments have identified key clutch molecules linked to specific chemical and mechanical signal transduction pathways, particularly regarding integrins in migrating cells, IgCAMs in neuronal growth cones, and cadherins at intercellular junctions. We propose here the concept of a multi-level clutch as a useful analogy to grasp the complexity of the dynamic molecular interactions involved in a panel of motile behaviors and shapes.
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http://dx.doi.org/10.1016/j.tcb.2009.07.001DOI Listing
September 2009

Neurexin/neuroligin interaction kinetics characterized by counting single cell-surface attached quantum dots.

Biophys J 2009 Jul;97(2):480-9

Physiologie Cellulaire de la synapse, Centre National de la Recherche Scientifique and University of Bordeaux, Bordeaux, France.

We report what to our knowledge is a new method to characterize kinetic rates between cell-surface-attached adhesion molecules. Cells expressing specific membrane receptors are surface-labeled with quantum dots coated with their respective ligands. The progressive diminution in the total number of surface-diffusing quantum dots tracked over time collectively reflects intrinsic ligand/receptor interaction kinetics. The probability of quantum dot detachment is modeled using a stochastic analysis of bond formation and dissociation, with a small number of ligand/receptor pairs, resulting in a set of coupled differential equations that are solved numerically. Comparison with the experimental data provides an estimation of the kinetic rates, together with the mean number of ligands per quantum dot, as three adjustable parameters. We validate this approach by studying the calcium-dependent neurexin/neuroligin interaction, which plays an important role in synapse formation. Using primary neurons expressing neuroligin-1 and quantum dots coated with purified neurexin-1beta, we determine the kinetic rates between these two binding partners and compare them with data obtained using other techniques. Using specific molecular constructs, we also provide interesting information about the effects of neurexin and neuroligin dimerization on the kinetic rates. As it stands, this simple technique should be applicable to many types of biological ligand/receptor pairs.
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http://dx.doi.org/10.1016/j.bpj.2009.04.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711312PMC
July 2009

Activity-independent and subunit-specific recruitment of functional AMPA receptors at neurexin/neuroligin contacts.

Proc Natl Acad Sci U S A 2008 Dec 19;105(52):20947-52. Epub 2008 Dec 19.

Centre National de la Recherche Scientifique UMR 5091, Physiologie Cellulaire de la Synapse, Institut François Magendie, Université Bordeaux 2, 33077 Bordeaux, France.

A combination of cell culture and animal studies has recently shown that adhesion between neurexins and neuroligins played important roles in synapse initiation, maturation, and function. Binding of neurexin-1beta to neuroligin-1 triggers the postsynaptic clustering of the scaffold postsynaptic density protein 95, but the composition and timing of accumulation of glutamate receptors at those nascent contacts remain unclear. Using glutamate iontophoresis and patch-clamp recordings, we identified functional AMPA receptors (AMPARs) and NMDA receptors at postsynaptic density protein 95 clusters induced by neurexin-1beta coated microspheres on primary hippocampal neurons. The recruitment of AMPARs occurred as early as 2 h after initial contact, and was not blocked by TTX/2-amino-5-phosphovaleric acid (APV) treatment. The differential recruitment of recombinant subunits GluR1 and GluR2, as well as the absence of rectification in voltage/current curves, further indicate that neurexin/neuroligin contacts primarily recruit GluR2-containing AMPARs. Finally, by using glutamate un-caging and calcium imaging, we show that AMPARs participate in calcium entry at neurexin-1beta induced post-synapses, most likely through the activation of voltage-gated calcium channels. Such rapid and activity-independent accumulation of functional AMPARs at neurexin-1beta-induced postsynapses points to a new role of AMPARs in synaptogenesis.
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http://dx.doi.org/10.1073/pnas.0804007106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634880PMC
December 2008