Publications by authors named "Alexei Grichine"

53 Publications

Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying signaling pathway usage.

J Cell Sci 2021 Jan 25;134(2). Epub 2021 Jan 25.

Institute for Advanced Biosciences, Centre de Recherche Université Grenoble Alpes, Inserm U 1209, CNRS UMR 5309, 38706 La Tronche, France

Upon activation by different transmembrane receptors, the same signaling protein can induce distinct cellular responses. A way to decipher the mechanisms of such pleiotropic signaling activity is to directly manipulate the decision-making activity that supports the selection between distinct cellular responses. We developed an optogenetic probe (optoSRC) to control SRC signaling, an example of a pleiotropic signaling node, and we demonstrated its ability to generate different acto-adhesive structures (lamellipodia or invadosomes) upon distinct spatio-temporal control of SRC kinase activity. The occurrence of each acto-adhesive structure was simply dictated by the dynamics of optoSRC nanoclusters in adhesive sites, which were dependent on the SH3 and Unique domains of the protein. The different decision-making events regulated by optoSRC dynamics induced distinct downstream signaling pathways, which we characterized using time-resolved proteomic and network analyses. Collectively, by manipulating the molecular mobility of SRC kinase activity, these experiments reveal the pleiotropy-encoding mechanism of SRC signaling.
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http://dx.doi.org/10.1242/jcs.254599DOI Listing
January 2021

Pyclen-Based Ln(III) Complexes as Highly Luminescent Bioprobes for and One- and Two-Photon Bioimaging Applications.

J Am Chem Soc 2020 06 21;142(22):10184-10197. Epub 2020 May 21.

Univ Brest, UMR CNRS 6521 CEMCA, 6 Avenue Victor le Gorgeu, 29200 Brest, France.

In addition to the already described ligand , two pyclen-based lanthanide chelators, and , bearing two specific picolinate two-photon antennas (tailor-made for each targeted metal) and one acetate arm arranged in a dissymmetrical manner, have been synthesized, to form a complete family of lanthanide luminescent bioprobes: [Eu], [Sm], [Yb], [Tb], and [Dy]. Additionally, the symmetrically arranged regioisomer was also synthesized as well as its [Eu] complex to highlight the astonishing positive impact of the dissymmetrical -distribution of the functional chelating arms. The investigation clearly shows the high performance of each bioprobe, which, depending on the complexed lanthanide, could be used in various applications. Each presents high brightness, quantum yields, and lifetimes. Staining of the complexes into living human breast cancer cells was observed. In addition, two-photon microscopy was performed for the first time on a living zebrafish model with [Eu]. No apparent toxicity was detected on the growth of the zebrafish, and images of high quality were obtained.
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http://dx.doi.org/10.1021/jacs.0c03496DOI Listing
June 2020

Cationic Biphotonic Lanthanide Luminescent Bioprobes Based on Functionalized Cross-Bridged Cyclam Macrocycles.

Chemphyschem 2020 05 7;21(10):1036-1043. Epub 2020 Apr 7.

Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, F-69342, Lyon, France.

Cationic lanthanide complexes are generally able to spontaneously internalize into living cells. Following our previous works based on a diMe-cyclen framework, a second generation of cationic water-soluble lanthanide complexes based on a constrained cross-bridged cyclam macrocycle functionalized with donor-π-conjugated picolinate antennas was prepared with europium(III) and ytterbium(III). Their spectroscopic properties were thoroughly investigated in various solvents and rationalized with the help of DFT calculations. A significant improvement was observed in the case of the Eu complex, while the Yb analogue conserved photophysical properties in aqueous solvent. Two-photon (2P) microscopy imaging experiments on living T24 human cancer cells confirmed the spontaneous internalization of the probes and images with good signal-to-noise ratio were obtained in the classic NIR-to-visible configuration with the Eu luminescent bioprobe and in the NIR-to-NIR with the Yb one.
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http://dx.doi.org/10.1002/cphc.202000085DOI Listing
May 2020

Live imaging of single platelets at work.

Platelets 2020 Jul 27;31(5):551-558. Epub 2019 Dec 27.

Institute for Advanced Biosciences, University Grenoble Alpes , Grenoble, France.

Although live imaging of dynamic processes in platelets is a challenging task, several important observations have been published during the last 20 years. We will discuss the amazing insights that have been achieved, the difficulties that can be encountered as well as some questions still open and the future technical perspectives.
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http://dx.doi.org/10.1080/09537104.2019.1708886DOI Listing
July 2020

A "Multi-Heavy-Atom" Approach toward Biphotonic Photosensitizers with Improved Singlet-Oxygen Generation Properties.

Chemistry 2019 Jul 30;25(38):9026-9034. Epub 2019 May 30.

Laboratoire de Chimie de l'ENS de Lyon, Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, 69342, Lyon, France.

Two trispicolinate 1,4,7-triazacyclonane (TACN)-based ligands bearing three picolinate biphotonic antennae were synthetized and their Yb and Gd complexes isolated. One series differs from the other by the absence (L )/presence (L ) of bromine atoms on the antenna backbone, offering respectively improved optical and singlet-oxygen generation properties. Photophysical properties of the ligands, complexes and micellar Pluronic suspensions were investigated. Complexes exhibit high two-photon absorption cross-section combined either with NIR emission (Yb) or excellent O generation (Gd). The very large intersystem crossing efficiency induced by the combination of bromine atom and heavy rare-earth element was corroborated with theoretical calculations. The O generation properties of L Gd micellar suspension under two-photon activation leads to tumour cell death, suggesting the potential of such structures for theranostic applications.
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http://dx.doi.org/10.1002/chem.201901047DOI Listing
July 2019

Polyanionic Polydentate Europium Complexes as Ultrabright One- or Two-photon Bioprobes.

Chemphyschem 2018 Sep 10. Epub 2018 Sep 10.

Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Laboratoire de Chimie, F69342, Lyon, France.

A family of europium (III) complexes based on a polydentate ligand functionalized by charge-transfer antennae presents remarkable one- and two-photon photophysical proper-ties in water or buffer. A detailed analysis of their emission properties suggests that the wrapping of the ligand around the central rare-earth ion results in an overall Cs symmetry in agreement with the theoretical simulation and that about 65-70 % of the emission intensity is concentrated in the hypersensitive D → F transition at 615 nm. Their brightness is excellent, in the range of the best lanthanide bioprobes making them very attractive for bio-imaging experiments.
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http://dx.doi.org/10.1002/cphc.201800557DOI Listing
September 2018

Combining a pyclen framework with conjugated antenna for the design of europium and samarium luminescent bioprobes.

Chem Commun (Camb) 2018 Jun;54(48):6173-6176

Université de Brest, UMR-CNRS 6521, UFR des Sciences et Techniques 6 avenue Victor le Gorgeu, C.S. 93837, 29238, Brest, Cedex 3, France.

The first pyclen based ligand bearing two picolinate intra-ligand charge transfer transition antennae and one acetate arm organized in a dissymmetric manner was synthesized for Eu(iii) and Sm(iii) complexation. The europium complex presents an excellent brightness and biphotonic imaging of T24-cells has been performed using Eu(iii) and the less common Sm(iii) bioprobes.
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http://dx.doi.org/10.1039/c8cc02035cDOI Listing
June 2018

In Vitro Dermal Safety Assessment of Silver Nanowires after Acute Exposure: Tissue vs. Cell Models.

Nanomaterials (Basel) 2018 Apr 11;8(4). Epub 2018 Apr 11.

Univ. Grenoble Alpes, Univ. Savoie Mont Blanc, CNRS, IRD, IFSTTAR, ISTerre, 38000 Grenoble, France.

Silver nanowires (AgNW) are attractive materials that are anticipated to be incorporated into numerous consumer products such as textiles, touchscreen display, and medical devices that could be in direct contact with skin. There are very few studies on the cellular toxicity of AgNW and no studies that have specifically evaluated the potential toxicity from dermal exposure. To address this question, we investigated the dermal toxicity after acute exposure of polymer-coated AgNW with two sizes using two models, human primary keratinocytes and human reconstructed epidermis. In keratinocytes, AgNW are rapidly and massively internalized inside cells leading to dose-dependent cytotoxicity that was not due to Ag⁺ release. Analysing our data with different dose metrics, we propose that the number of NW is the most appropriate dose-metric for studies of AgNW toxicity. In reconstructed epidermis, the results of a standard skin irritation assay classified AgNW as non-irritant to skin and we found no evidence of penetration into the deeper layer of the epidermis. The findings show that healthy and intact epidermis provides an effective barrier for AgNW, although the study does not address potential transport through follicles or injured skin. The combined cell and tissue model approach used here is likely to provide an important methodology for assessing the risks for skin exposure to AgNW from consumer products.
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http://dx.doi.org/10.3390/nano8040232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923562PMC
April 2018

Twisted Charge-Transfer Antennae for Ultra-Bright Terbium(III) and Dysprosium(III) Bioprobes.

Chemistry 2018 Mar 5;24(14):3408-3412. Epub 2018 Feb 5.

Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Laboratoire de Chimie, 69342, Lyon, France.

The design of original twisted charge transfer antennae in which a non-planar geometry is enforced thanks to one or two bulky ortho-Me substituents allows us to prepare the corresponding ultra-bright Tb and Dy bioprobes. The brightness of the Tb derivative compares well with that of the benchmark Tb-Lumi4 complex. The first bio-imaging experiments with a Dy luminescent bioprobe are also reported.
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http://dx.doi.org/10.1002/chem.201705933DOI Listing
March 2018

Terbium(III) Luminescent Complexes as Millisecond-Scale Viscosity Probes for Lifetime Imaging.

J Am Chem Soc 2017 06 31;139(23):7693-7696. Epub 2017 May 31.

Laboratoire de Chimie, École Normale Supérieure de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1 , 46 allée d'Italie, 69364 Lyon Cedex 07, France.

Fluorescent probes that are able to directly measure viscosity are attractive candidates for the study of intracellular environments. We report a new class of luminescent rotors, based on the sensitized emission of a terbium(III) complex. A 4-fold increase in both quantum yield and luminescence lifetime was observed in viscous media for the studied complexes, with a lifetime ranging from 0.23 to 0.89 ms over a broad range of viscosities (0.6-1200 cP). The presented approach, relying on the millisecond-scale luminescence lifetime of the lanthanide ions, was applied to fixed T24 cancer cells using temporal sampling lifetime imaging microscopy.
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http://dx.doi.org/10.1021/jacs.7b02951DOI Listing
June 2017

Near infrared two photon imaging using a bright cationic Yb(iii) bioprobe spontaneously internalized into live cells.

Chem Commun (Camb) 2017 May;53(44):6005-6008

Univ. Lyon, Ecole Normale Supérieure de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Laboratoire de Chimie, 46 allée d'Italie, 69364 Lyon, France.

An Yb(iii) complex based on a dimethyl cyclen macrocyclic ligand functionalized by charge transfer antennae was prepared. This cationic [YbL3] complex is stable and soluble in water and presents interesting photophysical nonlinear properties. It is spontaneously internalized and accumulates in live cells. High quality images have been obtained both in a classical NIR-to-vis configuration and in the more challenging NIR-to-NIR one.
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http://dx.doi.org/10.1039/c7cc02835kDOI Listing
May 2017

Keto-polymethines: a versatile class of dyes with outstanding spectroscopic properties for and two-photon microscopy imaging.

Chem Sci 2017 Jan 3;8(1):381-394. Epub 2016 Aug 3.

ENS Lyon , Université de Lyon 1 , CNRS Laboratoire de chimie de l'ENS Lyon , UMR 5182 CNRS, 46 allée d'Italie , 69364 Lyon , France . Email: ; Email:

The synthesis of keto-heptamethine derivatives has been expanded to various new symmetrical and asymmetrical structures, including an unprecedented di-anionic keto-polymethine. The spectroscopic behavior of these new dyes has been systematically and thoroughly investigated, revealing that the formation of hydrogen bond interactions with protic solvents is responsible for a dramatic enhancement of the fluorescence quantum yield in the far-red spectral region. The existence of these strong hydrogen-bond interactions was further confirmed by molecular dynamics simulations. These bis-dipolar polymethines exhibit large two-photon absorption (TPA) cross-sections ( in GM) in the near-infrared, making them ideal candidates for NIR-to-NIR two-photon microscopy imaging applications. We demonstrate that the molecular engineering of the hydrophilic/hydrophobic balance enables targeting of different cellular components, such as cytoplasm or cell membranes. Addition of appropriate substituents provides the molecule with high-water-solubility, affording efficient two-photon probes for angiography.
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http://dx.doi.org/10.1039/c6sc02488bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365052PMC
January 2017

Microvasculature on a chip: study of the Endothelial Surface Layer and the flow structure of Red Blood Cells.

Sci Rep 2017 03 24;7:45036. Epub 2017 Mar 24.

Univ. Grenoble Alpes, LIPHY, F-38000 Grenoble, France.

Microvasculatures-on-a-chip, i.e. in vitro models that mimic important features of microvessel networks, have gained increasing interest in recent years. Such devices have allowed investigating pathophysiological situations involving abnormal biophysical interactions between blood cells and vessel walls. Still, a central question remains regarding the presence, in such biomimetic systems, of the endothelial glycocalyx. The latter is a glycosaminoglycans-rich surface layer exposed to blood flow, which plays a crucial role in regulating the interactions between circulating cells and the endothelium. Here, we use confocal microscopy to characterize the layer expressed by endothelial cells cultured in microfluidic channels. We show that, under our culture conditions, endothelial cells form a confluent layer on all the walls of the circuit and display a glycocalyx that fully lines the lumen of the microchannels. Moreover, the thickness of this surface layer is found to be on the order of 600 nm, which compares well with measurements performed ex or in vivo on microcapillaries. Furthermore, we investigate how the presence of endothelial cells in the microchannels affects their hydrodynamic resistance and the near-wall motion of red blood cells. Our study thus provides an important insight into the physiological relevance of in vitro microvasculatures.
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http://dx.doi.org/10.1038/srep45036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364477PMC
March 2017

Genetically Encoded Fluorescent Biosensors to Explore AMPK Signaling and Energy Metabolism.

Exp Suppl 2016;107:491-523

Laboratory of Fundamental and Applied Bioenergetics (LBFA) and SFR Environmental and Systems Biology (BEeSy), University Grenoble Alpes, Grenoble, France.

Maintenance of energy homeostasis is a basic requirement for cell survival. Different mechanisms have evolved to cope with spatial and temporal mismatch between energy-providing and -consuming processes. Among these, signaling by AMP-activated protein kinase (AMPK) is one of the key players, regulated by and itself regulating cellular adenylate levels. Further understanding its complex cellular function requires deeper insight into its activation patterns in space and time at a single cell level. This may become possible with an increasing number of genetically encoded fluorescent biosensors, mostly based on fluorescence resonance energy transfer, which have been engineered to monitor metabolic parameters and kinase activities. Here, we review basic principles of biosensor design and function and the advantages and limitations of their use and provide an overview on existing FRET biosensors to monitor AMPK activation, ATP concentration, and ATP/ADP ratios, together with other key metabolites and parameters of energy metabolism.
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http://dx.doi.org/10.1007/978-3-319-43589-3_20DOI Listing
June 2017

Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

FASEB J 2017 02 31;31(2):663-673. Epub 2016 Oct 31.

Groupe de Recherche et D'etude du Processus Inflammatoire (GREPI), EA 7408, Université Grenoble Alpes, Saint-Martin-d'Hères, France;

The phagocyte NADPH oxidase 2 (Nox2) is an enzymatic complex that is involved in innate immunity, notably via its capacity to produce toxic reactive oxygen species. Recently, a proteomic analysis of the constitutively active Nox2 complex, isolated from neutrophil fractions, highlighted the presence of 6-phosphofructo-2-kinase (PFK-2). The purpose of this work was to study the relationship between PFK-2 and NADPH oxidase in neutrophils. Data have underlined a specific association of the active phosphorylated form of PFK-2 with Nox2 complex in stimulated neutrophils. In its active form, PFK-2 catalyzes the production of fructose-2,6-bisphosphate, which is the main allosteric activator of phosphofructo-1-kinase, the limiting enzyme in glycolysis. Pharmacologic inhibition of PFK-2 phosphorylation and cell depletion in PFK-2 by a small interfering RNA strategy led to a decrease in the glycolysis rate and a reduction in NADPH oxidase activity in stimulated cells. Surprisingly, alteration of Nox2 activity impacted the glycolysis rate, which indicated that Nox2 in neutrophils was not only required for reactive oxygen species production but was also involved in supporting the energetic metabolism increase that was induced by inflammatory conditions. PFK-2 seems to be a strategic element that links NADPH oxidase activation and glycolysis modulation, and, as such, is proposed as a potential therapeutic target in inflammatory diseases.-Baillet, A., Hograindleur, M.-A., El Benna, J., Grichine, A., Berthier, S., Morel, F., Paclet, M.-H. Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.
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http://dx.doi.org/10.1096/fj.201600720RDOI Listing
February 2017

Cationic Two-Photon Lanthanide Bioprobes Able to Accumulate in Live Cells.

Inorg Chem 2016 Jul 1;55(14):7020-5. Epub 2016 Jul 1.

Université de Brest, UMR-CNRS 6521, UFR des Sciences et Techniques 6 avenue Victor le Gorgeu, C.S. 93837, 29238, Brest Cedex 3, France.

An original cationic water-soluble cyclen-based Eu(III) complex [EuL(1)](+) featuring a chromophore-functionalized antenna to increase the two-photon (2P) absorption properties was synthesized. The photophysical properties were thoroughly studied in various solvents and rationalized with the help of theoretical calculations. The complex exhibits an optimized 2P absorption cross section. Finally, 2P microscopy imaging experiments on living T24 human cancer cells highlighted the spontaneous internalization and the biological stability of this 2P bioprobe in vitro. Macrocyclic-based antennas open new perspectives for future optimization of the photophysical properties and allows envisaging the design of Eu, Tb, Yb, and Sm bioprobes. This result also opens the way for the design of functional two-photon Ln complexes able to monitor intracellular physicochemical parameters.
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http://dx.doi.org/10.1021/acs.inorgchem.6b00891DOI Listing
July 2016

The impact of cardiac ischemia/reperfusion on the mitochondria-cytoskeleton interactions.

Biochim Biophys Acta 2016 06 11;1862(6):1159-71. Epub 2016 Mar 11.

University Grenoble Alpes, Laboratory of Fundamental and Applied Bioenergetics, INSERM U1055, Grenoble, France; Hospital of the University Grenoble Alpes, Department Thorax (EFCR), France. Electronic address:

Cardiac ischemia-reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation (OxPhos) and compartmentalized intracellular energy transfer via the phosphocreatine/creatine kinase (CK) network. The restriction of ATP/ADP diffusion at the level of the mitochondrial outer membrane (MOM) is an essential element of compartmentalized energy transfer. In adult cardiomyocytes, the MOM permeability to ADP is regulated by the interaction of voltage-dependent anion channel with cytoskeletal proteins, particularly with β tubulin II. The IR-injury alters the expression and the intracellular arrangement of cytoskeletal proteins. The objective of the present study was to investigate the impact of IR on the intracellular arrangement of β tubulin II and its effect on the regulation of mitochondrial respiration. Perfused rat hearts were subjected to total ischemia (for 20min (I20) and 45min (I45)) or to ischemia followed by 30min of reperfusion (I20R and I45R groups). High resolution respirometry and fluorescent confocal microscopy were used to study respiration, β tubulin II and mitochondrial arrangements in cardiac fibers. The results of these experiments evidence a heterogeneous response of mitochondria to IR-induced damage. Moreover, the intracellular rearrangement of β tubulin II, which in the control group colocalized with mitochondria, was associated with increased apparent affinity of OxPhos for ADP, decreased regulation of respiration by creatine without altering mitochondrial CK activity and the ratio between octameric to dimeric isoenzymes. The results of this study allow us to highlight changes of mitochondrial interactions with cytoskeleton as one of the possible mechanisms underlying cardiac IR injury.
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http://dx.doi.org/10.1016/j.bbadis.2016.03.009DOI Listing
June 2016

Improving Surgical Resection of Metastatic Liver Tumors With Near-Infrared Optical-Guided Fluorescence Imaging.

Surg Innov 2016 Aug 24;23(4):354-9. Epub 2015 Nov 24.

INSERM-UJF U823, Institut Albert Bonniot, Grenoble, France.

Objective The aim of this study was to investigate the feasibility and future clinical applications of near-infrared (NIR) fluorescence imaging to guide liver resection surgery for metastatic cancer to improve resection margins. Summary Background Data A subset of patients with metastatic hepatic tumors can be cured by surgery. The degree of long-term and disease-free survival is related to the quality of surgery, with the best resection defined as "R0" (complete removal of all tumor cells, as evidenced by microscopic examination of the margins). Although intraoperative ultrasonography can evaluate the surgical margins, surgeons need a new tool to perfect the surgical outcome. Methods A preliminary study was performed on 3 patients. We used NIR imaging postoperatively "ex vivo" on the resected liver tissue. The liver tumors were preoperatively labelled by intravenously injecting the patient with indocyanine green (ICG), a NIR fluorescent agent (24 hours before surgery, 0.25 mg/kg). Fluorescent images were obtained using a miniaturized fluorescence imaging system (FluoStic, Fluoptics, Grenoble, France). Results After liver resection, the surgical specimens from each patient were sliced into 10-mm sections in the operating room and analyzed with the FluoStic. All metastatic tumors presented rim-type fluorescence. Two specimens had incomplete rim fluorescence. The pathologist confirmed the presence of R1 margins (microscopic residual resection), even though the ultrasonographic analysis indicated that the result was R0. Conclusions Surgical liver resection guided by NIR fluorescence can help detect potentially uncertain anatomical areas that may be missed by preoperative imaging and by ultrasonography during surgery. These preliminary results will need to be confirmed in a larger prospective patient series.
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http://dx.doi.org/10.1177/1553350615618287DOI Listing
August 2016

Unexpected Efficiency of a Luminescent Samarium(III) Complex for Combined Visible and Near-Infrared Biphotonic Microscopy.

Chemistry 2015 Dec 22;21(49):17757-61. Epub 2015 Oct 22.

University of Lyon 1, ENS Lyon, CNRS UMR 5182, 46 allée d'Italie, 69364 Lyon (France).

An original samarium(III) complex based on a triazacyclononane platform functionalized with a charge-transfer antenna chromophore exhibited optimized brightness and was successfully used as an emissive species for two-photon microscopy experiments in both the visible and near-infrared spectral ranges.
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http://dx.doi.org/10.1002/chem.201503711DOI Listing
December 2015

Two-photon multiplexing bio-imaging using a combination of Eu- and Tb-bioprobes.

Dalton Trans 2015 Mar;44(11):4918-24

Laboratoire de Chimie UMR UMR 5182 CNRS-University Lyon- ENS Lyon, 46 allée d'Italie, 69364 Lyon, France.

During the course of our research studies aiming to combine the unique photophysical properties of lanthanide complexes and the intrinsic advantage of a nonlinear two-photon excitation, we described the proof-of-concept of a biphotonic multiplexing bio-imaging experiment. To that end, a new Tb-luminescent bioprobe [TbL(2)] was designed using the TACN-trispicolinate platform. The biphotonic imaging of T24 fixed cells stained either with [TbL(2)] or with the already described [EuL(1)] complexes is reported. Finally the biphotonic multiplexing experiment was performed using a combination of both probes using the spectral mode of the two-photon microscope.
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http://dx.doi.org/10.1039/c4dt03115fDOI Listing
March 2015

Relative contribution of c1q and apoptotic cell-surface calreticulin to macrophage phagocytosis.

J Innate Immun 2014 15;6(4):426-34. Epub 2014 Feb 15.

Institut de Biologie Structurale (IBS), Immune Response to Pathogens and Altered Self (IRPAS) Group, Université Grenoble Alpes, Grenoble, France.

C1q has been shown to recognize apoptotic cells, to enhance their uptake and to modulate cytokine release by phagocytes and thus promote immune tolerance. Surface-exposed calreticulin (CRT), known as a C1q receptor, is also considered to be an early eat-me signal that enhances the phagocytosis of apoptotic cells and is capable of eliciting an immunogenic response. However, the molecular mechanisms that trigger these functions are not clear. We hypothesized that CRT and C1q might act together in these processes. We first showed, by means of fluorescence resonance energy transfer (FRET), that CRT interacts with the C1q globular region at the surface of early apoptotic cells. Next, we pointed out that knockdown of CRT on early apoptotic HeLa cells impairs the enhancement effect of C1q on their uptake by THP-1 monocyte-derived macrophages. Furthermore, a deficiency of CRT induces contrasting effects on cytokine release by THP-1 macrophages, increasing interleukin (IL)-6 and monocyte chemotactic protein 1/CCL2 and decreasing IL-8. Remarkably, these effects were greatly reduced when apoptotic cells were opsonized by C1q, which counterbalanced the effect of the CRT deficiency. These results demonstrate that CRT-C1q interaction is involved in the C1q bridging function and they highlight the particular ability of C1q to control the phagocyte inflammatory status, i.e. by integrating the molecular changes that could occur at the surface of dying cells.
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http://dx.doi.org/10.1159/000358834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6741593PMC
May 2015

Motor-driven marginal band coiling promotes cell shape change during platelet activation.

J Cell Biol 2014 Jan 13;204(2):177-85. Epub 2014 Jan 13.

Institut Albert Bonniot, Institut National de la Santé et de la Recherche Médicale U823; and 2 Techniques de l'Ingénierie Médicale et de la Complexité-Informatique, Mathématiques et Applications, Grenoble, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5525; Université Joseph Fourier-Grenoble 1, F-38041 Grenoble, France.

Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes.
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http://dx.doi.org/10.1083/jcb.201306085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3897189PMC
January 2014

Essential function of dynamin in the invasive properties and actin architecture of v-Src induced podosomes/invadosomes.

PLoS One 2013 9;8(12):e77956. Epub 2013 Dec 9.

Department of Medicine, Harvard Medical School, Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts, United States of America ; Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts, United States of America.

The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077956PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3857171PMC
September 2014

Role of mitochondria-cytoskeleton interactions in respiration regulation and mitochondrial organization in striated muscles.

Biochim Biophys Acta 2014 Feb 2;1837(2):232-45. Epub 2013 Nov 2.

INSERM U1055, Laboratory of Fundamental and Applied Bioenergetics, Joseph Fourier University, Grenoble, France; Department of Rehabilitation and Physiology, University Hospital Grenoble, France. Electronic address:

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein tubulin βII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase-phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, βII tubulin. We found that in oxidative soleus skeletal muscle the high apparent Km for ADP is associated with low MOM permeability and high expression of non-polymerized βII tubulin. Very low expression of non-polymerized form of βII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent Km for ADP).
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http://dx.doi.org/10.1016/j.bbabio.2013.10.011DOI Listing
February 2014

Correction of cell-induced optical aberrations in a fluorescence fluctuation microscope.

Opt Lett 2013 Jul;38(14):2401-3

University of Grenoble 1/CNRS, LIPhy UMR 5588, Grenoble, France.

We describe the effect of optical aberrations on fluorescence fluctuations microscopy (FFM), when focusing through a single living cell. FFM measurements are performed in an aqueous fluorescent solution and prove to be a highly sensitive tool to assess the optical aberrations introduced by the cell. We demonstrate an adaptive optics (AO) system to remove the aberration-related bias in the FFM measurements. Our data show that AO is not only useful when imaging deep in tissues but also when performing FFM measurements through a single cellular layer. This work paves the way for the application of FFM to complex three-dimensional multicellular samples.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967118PMC
http://dx.doi.org/10.1364/OL.38.002401DOI Listing
July 2013

Matters of the heart in bioenergetics: mitochondrial fusion into continuous reticulum is not needed for maximal respiratory activity.

J Bioenerg Biomembr 2013 Aug;45(4):319-31

Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia.

Mitochondria are dynamic structures for which fusion and fission are well characterized for rapidly dividing cells in culture. Based on these data, it has recently been proposed that high respiratory activity is the result of fusion and formation of mitochondrial reticulum, while fission results in fragmented mitochondria with low respiratory activity. In this work we test the validity of this new hypothesis by analyzing our own experimental data obtained in studies of isolated heart mitochondria, permeabilized cells of cardiac phenotype with different mitochondrial arrangement and dynamics. Additionally, we reviewed published data including electron tomographic investigation of mitochondrial membrane-associated structures in heart cells. Oxygraphic studies show that maximal ADP-dependent respiration rates are equally high both in isolated heart mitochondria and in permeabilized cardiomyocytes. On the contrary, these rates are three times lower in NB HL-1 cells with fused mitochondrial reticulum. Confocal and electron tomographic studies show that there is no mitochondrial reticulum in cardiac cells, known to contain 5,000-10,000 individual, single mitochondria, which are regularly arranged at the level of sarcomeres and are at Z-lines separated from each other by membrane structures, including the T-tubular system in close connection to the sarcoplasmic reticulum. The new structural data in the literature show a principal role for the elaborated T-tubular system in organization of cell metabolism by supplying calcium, oxygen and substrates from the extracellular medium into local domains of the cardiac cells for calcium cycling within Calcium Release Units, associated with respiration and its regulation in Intracellular Energetic Units.
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http://dx.doi.org/10.1007/s10863-012-9494-4DOI Listing
August 2013

Ytterbium-based bioprobes for near-infrared two-photon scanning laser microscopy imaging.

Angew Chem Int Ed Engl 2012 Jul 23;51(27):6622-5. Epub 2012 May 23.

University Lyon 1, ENS Lyon, CNRS UMR 5182, 46 allée d'Italie, 69364 Lyon, France.

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http://dx.doi.org/10.1002/anie.201202212DOI Listing
July 2012

Regulation of respiration in muscle cells in vivo by VDAC through interaction with the cytoskeleton and MtCK within Mitochondrial Interactosome.

Biochim Biophys Acta 2012 Jun 4;1818(6):1545-54. Epub 2012 Jan 4.

INSERM U1055, Laboratory of Fundamental and Applied Bioenergetics, Joseph Fourier University, Grenoble, France.

This review describes the recent experimental data on the importance of the VDAC-cytoskeleton interactions in determining the mechanisms of energy and metabolite transfer between mitochondria and cytoplasm in cardiac cells. In the intermembrane space mitochondrial creatine kinase connects VDAC with adenine nucleotide translocase and ATP synthase complex, on the cytoplasmic side VDAC is linked to cytoskeletal proteins. Applying immunofluorescent imaging and Western blot analysis we have shown that β2-tubulin coexpressed with mitochondria is highly important for cardiac muscle cells mitochondrial metabolism. Since it has been shown by Rostovtseva et al. that αβ-heterodimer of tubulin binds to VDAC and decreases its permeability, we suppose that the β-tubulin subunit is bound on the cytoplasmic side and α-tubulin C-terminal tail is inserted into VDAC. Other cytoskeletal proteins, such as plectin and desmin may be involved in this process. The result of VDAC-cytoskeletal interactions is selective restriction of the channel permeability for adenine nucleotides but not for creatine or phosphocreatine that favors energy transfer via the phosphocreatine pathway. In some types of cancer cells these interactions are altered favoring the hexokinase binding and thus explaining the Warburg effect of increased glycolytic lactate production in these cells. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
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http://dx.doi.org/10.1016/j.bbamem.2011.12.034DOI Listing
June 2012

Epithelial protein lost in neoplasm (EPLIN) interacts with α-catenin and actin filaments in endothelial cells and stabilizes vascular capillary network in vitro.

J Biol Chem 2012 Mar 22;287(10):7556-72. Epub 2011 Dec 22.

INSERM U882, F-38054 Grenoble, France.

Adherens junctions are required for vascular endothelium integrity. These structures are formed by the clustering of the homophilic adhesive protein VE-cadherin, which recruits intracellular partners, such as β- and α-catenins, vinculin, and actin filaments. The dogma according to which α-catenin bridges cadherin·β-catenin complexes to the actin cytoskeleton has been challenged during the past few years, and the link between the VE-cadherin·catenin complex and the actin cytoskeleton remains unclear. Recently, epithelial protein lost in neoplasm (EPLIN) has been proposed as a possible bond between the E-cadherin·catenin complex and actin in epithelial cells. Herein, we show that EPLIN is expressed at similar levels in endothelial and epithelial cells and is located at interendothelial junctions in confluent cells. Co-immunoprecipitation and GST pulldown experiments provided evidence that EPLIN interacts directly with α-catenin and tethers the VE-cadherin·catenin complex to the actin cytoskeleton. In the absence of EPLIN, vinculin was delocalized from the junctions. Furthermore, suppression of actomyosin tension using blebbistatin triggered a similar vinculin delocalization from the junctions. In a Matrigel assay, EPLIN-depleted endothelial cells exhibited a reduced capacity to form pseudocapillary networks because of numerous breakage events. In conclusion, we propose a model in which EPLIN establishes a link between the cadherin·catenin complex and actin that is independent of actomyosin tension. This link acts as a mechanotransmitter, allowing vinculin binding to α-catenin and formation of a secondary molecular bond between the adherens complex and the cytoskeleton through vinculin. In addition, we provide evidence that the EPLIN clutch is necessary for stabilization of capillary structures in an angiogenesis model.
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http://dx.doi.org/10.1074/jbc.M111.328682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293581PMC
March 2012