36 results on '"Rossier, Olivier"'
Search Results
2. Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions
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Orré, Thomas, Joly, Adrien, Karatas, Zeynep, Kastberger, Birgit, Cabriel, Clément, Böttcher, Ralph T., Lévêque-Fort, Sandrine, Sibarita, Jean-Baptiste, Fässler, Reinhard, Wehrle-Haller, Bernhard, Rossier, Olivier, and Giannone, Grégory
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- 2021
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3. Cell stretching is amplified by active actin remodelling to deform and recruit proteins in mechanosensitive structures
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Massou, Sophie, Nunes Vicente, Filipe, Wetzel, Franziska, Mehidi, Amine, Strehle, Dan, Leduc, Cecile, Voituriez, Raphaël, Rossier, Olivier, Nassoy, Pierre, and Giannone, Grégory
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- 2020
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4. A super-resolution platform for correlative live single-molecule imaging and STED microscopy
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Inavalli, V. V. G. Krishna, Lenz, Martin O., Butler, Corey, Angibaud, Julie, Compans, Benjamin, Levet, Florian, Tønnesen, Jan, Rossier, Olivier, Giannone, Gregory, Thoumine, Olivier, Hosy, Eric, Choquet, Daniel, Sibarita, Jean-Baptiste, and Nägerl, U. Valentin
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- 2019
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5. Localization-based super-resolution imaging meets high-content screening
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Beghin, Anne, Kechkar, Adel, Butler, Corey, Levet, Florian, Cabillic, Marine, Rossier, Olivier, Giannone, Gregory, Galland, Rémi, Choquet, Daniel, and Sibarita, Jean-Baptiste
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- 2017
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6. Talin and kindlin cooperate to control the density of integrin clusters.
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Pernier, Julien, Dos Santos, Marcelina Cardoso, Souissi, Mariem, Joly, Adrien, Narassimprakash, Hemalatha, Rossier, Olivier, Giannone, Grégory, Helfer, Emmanuèle, Sengupta, Kheya, and Le Clainche, Christophe
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INTEGRINS ,EXTRACELLULAR matrix ,CELL adhesion ,FOCAL adhesions ,ACTOMYOSIN ,DENSITY ,LIPIDS - Abstract
Focal adhesions are composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Adhesion depends on the activation of integrins. Talin and kindlin proteins are intracellular activators of integrins that bind to β-integrin cytoplasmic tails. Integrin activation and clustering through extracellular ligands guide the organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we used a biomimetic in vitro system, made of giant unilamellar vesicles, containing transmembrane integrins (herein aIIbβ3), with purified talin (talin-1), kindlin (kindlin-2, also known as FERMT2) and actomyosin. Here, we show that talin and kindlin individually have the ability to cluster integrins. Talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin increases integrin-talin-actomyosin coupling. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion. [ABSTRACT FROM AUTHOR]
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- 2023
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7. The cancer glycocalyx mechanically primes integrin-mediated growth and survival
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Paszek, Matthew J., DuFort, Christopher C., Rossier, Olivier, Bainer, Russell, Mouw, Janna K., Godula, Kamil, Hudak, Jason E., Lakins, Jonathon N., Wijekoon, Amanda C., Cassereau, Luke, Rubashkin, Matthew G., Magbanua, Mark J., Thorn, Kurt S., Davidson, Michael W., Rugo, Hope S., Park, John W., Hammer, Daniel A., Giannone, Gregory, Bertozzi, Carolyn R., and Weaver, Valerie M.
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Glycoproteins -- Properties ,Glycocalyx -- Properties -- Physiological aspects ,Integrins -- Growth -- Properties ,Cancer cells -- Physiological aspects ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
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., The composition of cell surface glycans and glycoproteins changes markedly and in tandem with cell fate transitions occurring in embryogenesis, tissue development, stem-cell differentiation and diseases such as cancer (1-3). [...]
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- 2014
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8. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking
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Rossier, Olivier and Giannone, Grégory
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- 2016
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9. Micropatterned "adherent/repellent" glass surfaces for studying the spreading kinetics of individual red blood cells onto protein-decorated substrates
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Cuvelier, Damien, Rossier, Olivier, Bassereau, Patricia, and Nassoy, Pierre
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- 2003
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10. Influence of submerged aquatic vegetation on size class distribution of perch (Perca fluviatilis) and roach (Rutilus rutilus) in the littoral zone of Lake Geneva (Switzerland)
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Rossier, Olivier, Castella, Emmanuel, and Lachavanne, Jean-Bernard
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- 1996
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11. Spatial and temporal separation of littoral zone fishes of Lake Geneva (Switzerland-France)
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Rossier, Olivier
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- 1995
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12. Analysis of a Human Fungiform Papillae cDNA Library and Identification of Taste-related Genes
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Rossier, Olivier, Cao, Jie, Huque, Taufiqul, Spielman, Andrew I., Feldman, Roy S., Medrano, Juan F., Brand, Joseph G., and le Coutre, Johannes
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- 2004
13. The α1a and α1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action
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Cotecchia, Susanna, Rossier, Olivier, Fanelli, Francesca, Leonardi, Amedeo, and De Benedetti, Pier G.
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- 2000
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14. The inner life of integrin adhesion sites: From single molecules to functional macromolecular complexes.
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Orré, Thomas, Rossier, Olivier, and Giannone, Grégory
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INTEGRINS , *EXTRACELLULAR matrix , *SINGLE molecules , *CELL proliferation , *PROTEIN engineering - Abstract
Abstract 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. [ABSTRACT FROM AUTHOR]
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- 2019
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15. 5th Indo-Pacific fish conference : abstracts
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Rossier, Olivier, Kulbicki, Michel, and Mou-Tham, Gérard
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POISSON MARIN ,VARIATION TEMPORELLE ,LAGON ,FACTEUR BIOTIQUE ,PLANTE AQUATIQUE ,ASSOCIATION D'ESPECES - Published
- 1997
16. Novel imaging methods and force probes for molecular mechanobiology of cytoskeleton and adhesion.
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Nunes Vicente, Filipe, Chen, Tianchi, Rossier, Olivier, and Giannone, Grégory
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MOLECULAR probes , *HIGH resolution imaging , *CYTOSKELETON , *CELL anatomy , *CELL imaging , *CELL motility - Abstract
Detection and conversion of mechanical forces into biochemical signals is known as mechanotransduction. From cells to tissues, mechanotransduction regulates migration, proliferation, and differentiation in processes such as immune responses, development, and cancer progression. Mechanosensitive structures such as integrin adhesions, the actin cortex, ion channels, caveolae, and the nucleus sense and transmit forces. In vitro approaches showed that mechanosensing is based on force-dependent protein deformations and reorganizations. However, the mechanisms in cells remained unclear since cell imaging techniques lacked molecular resolution. Thanks to recent developments in super-resolution microscopy (SRM) and molecular force sensors, it is possible to obtain molecular insight of mechanosensing in live cells. We discuss how understanding of molecular mechanotransduction was revolutionized by these innovative approaches, focusing on integrin adhesions, actin structures, and the plasma membrane. Mechanotransduction is involved in a variety of cell and tissue processes. For this, mechanosensitive structures, such as integrin adhesions, the cytoskeleton, or the plasma membrane, are key to sense, integrate, and transmit mechanical forces. Novel imaging methods and molecular sensors have unveiled how molecular mechanosensing occurs in these structures within the context of live cells. These include super-resolution imaging, single particle tracking, and molecular force sensors. Super-resolution microscopy and single protein tracking have revealed the 3D nanoscale organization and dynamics of integrin adhesion structures in various cell types, which are divided into functional nanolayers with specific paths for protein diffusion. Forces on actin regulators and F-actin trigger conformational changes controlling actin regulator function and binding, and therefore F-actin assembly and architecture. In migrating cells, mechanical plasticity emerges from global geometrical reorganization of actin networks under loads, or forces from elongating actin filaments controlling locally actin regulators dynamics and functions. Cell surface mechanics are emerging as a key parameter controlling cell motility, division, and differentiation. Variations in cortical tension and membrane-to-cortex attachment regulate the timing of differentiation while the nanoscale architecture of the actin cortex is linked to cortical tension. [ABSTRACT FROM AUTHOR]
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- 2023
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17. Integrins ?1 and ?3 exhibit distinct dynamic nanoscale organizations inside focal adhesions.
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Rossier, Olivier, Octeau, Vivien, Sibarita, Jean-Baptiste, Leduc, Cécile, Tessier, Béatrice, Nair, Deepak, Gatterdam, Volker, Destaing, Olivier, Albigès-Rizo, Corinne, Tampé, Robert, Cognet, Laurent, Choquet, Daniel, Lounis, Brahim, and Giannone, Grégory
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INTEGRINS , *FOCAL adhesions , *EXTRACELLULAR matrix , *CELL motility , *CELL proliferation , *CELL differentiation , *FIBRONECTIN-binding proteins , *HIGH resolution imaging - Abstract
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 ?3-integrins are enriched and stationary within FAs, whereas immobilized ?1-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 ?-integrins. Thus, differential transmission of actin motion to fibronectin occurs through specific integrins within FAs. [ABSTRACT FROM AUTHOR]
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- 2012
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18. Force generated by actomyosin contraction builds bridges between adhesive contacts.
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Rossier, Olivier M., Gauthier, Nils, Biais, Nicolas, Vonnegut, Wynn, Fardin, Marc-Antoine, Avigan, Philip, Heller, Evan R., Mathur, Anurag, Ghassemi, Saba, Koeckert, Michael S., Hone, James C., and Sheetz, Michael P.
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ACTOMYOSIN , *FIBRONECTINS , *MYOSIN , *POLYMERIZATION , *EXTRACELLULAR matrix , *ACTIN - Abstract
Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another. [ABSTRACT FROM AUTHOR]
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- 2010
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19. THE α1b-ADRENERGIC RECEPTOR SUBTYPE: MOLECULAR PROPERTIES AND PHYSIOLOGICAL IMPLICATIONS.
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Cotecchia, Susanna, Björklöf, Katja, Rossier, Olivier, Stanasila, Laura, Greasley, Peter, and Fanelli, Francesca
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ALPHA adrenoceptors ,ADRENERGIC receptors - Abstract
The aim of this review is to summarize some of the main findings from our laboratory as well as from others concerning the biochemical, molecular, and functional properties of the α1b-adrenergic receptor. Experimental and computational mutagenesis of the α1b-adrenergic receptor have been instrumental in elucidating some of the molecular mechanisms underlying receptor activation and receptor coupling to Gq. The knockout mouse model lacking the α1b-adrenergic receptor has highlighted the potential implication of this receptor subtype in variety of functions including the regulation of blood pressure, glucose homeostasis, and the rewarding response to drugs of abuse. [ABSTRACT FROM AUTHOR]
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- 2002
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20. Integrin-Functionalised Giant Unilamellar Vesicles via Gel-Assisted Formation: Good Practices and Pitfalls.
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Souissi, Mariem, Pernier, Julien, Rossier, Olivier, Giannone, Gregory, Le Clainche, Christophe, Helfer, Emmanuèle, and Sengupta, Kheya
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MEMBRANE proteins ,PROTEIN models ,BEST practices ,INTEGRINS ,PHYSICS ,BIOCHEMISTRY ,LIPOSOMES - Abstract
Giant unilamellar vesicles (GUV) are powerful tools to explore physics and biochemistry of the cell membrane in controlled conditions. For example, GUVs were extensively used to probe cell adhesion, but often using non-physiological linkers, due to the difficulty of incorporating transmembrane adhesion proteins into model membranes. Here we describe a new protocol for making GUVs incorporating the transmembrane protein integrin using gel-assisted swelling. We report an optimised protocol, enumerating the pitfalls encountered and precautions to be taken to maintain the robustness of the protocol. We characterise intermediate steps of small proteoliposome formation and the final formed GUVs. We show that the integrin molecules are successfully incorporated and are functional. [ABSTRACT FROM AUTHOR]
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- 2021
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21. Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion.
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Mehidi, Amine, Rossier, Olivier, Schaks, Matthias, Chazeau, Anaël, Binamé, Fabien, Remorino, Amanda, Coppey, Mathieu, Karatas, Zeynep, Sibarita, Jean-Baptiste, Rottner, Klemens, Moreau, Violaine, and Giannone, Grégory
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RHO GTPases , *HIGH resolution imaging , *CELL migration , *GUANOSINE triphosphatase , *DIFFUSION , *MICROFILAMENT proteins - Abstract
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. • Rac1 immobilization at the lamellipodium tip correlates with its activation • Rac1 immobilization depends on effector binding, including WRC • RhoA does not display selective immobilization at the lamellipodium tip • Local Rac1 activation at the lamellipodium tip triggers membrane protrusion Mehidi et al. use single protein tracking with loss- or gain-of-function mutants of Rho GTPases to reveal that transient Rac1 immobilization at the lamellipodium tip correlates with its activation and binding to effectors. Short-lived activations ensure a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion. [ABSTRACT FROM AUTHOR]
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- 2019
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22. Cytoskeletal coherence requires myosin-IIA contractility.
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Yunfei Cai, Rossier, Olivier, Gauthier, Nils C., Biais, Nicolas, Fardin, Marc-Antoine, Xian Zhang, Miller, Lawrence W., Ladoux, Benoit, Cornish, Virginia W., and Sheetz, Michael P.
- Subjects
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CYTOSKELETON , *MYOSIN , *RNA , *CYTOPLASM , *ACTIN , *POLYMERIZATION - Abstract
Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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23. Integrin-based adhesion compartmentalizes ALK3 of the BMPRII to control cell adhesion and migration.
- Author
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Guevara-Garcia, Amaris, Fourel, Laure, Bourrin-Reynard, Ingrid, Sales, Adria, Oddou, Christiane, Pezet, Mylène, Rossier, Olivier, Machillot, Paul, Chaar, Line, Bouin, Anne-Pascale, Giannone, Gregory, Destaing, Olivier, Picart, Catherine, and Albiges-Rizo, Corinne
- Subjects
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CELL adhesion , *FOCAL adhesions , *CELL receptors , *HIGH resolution imaging , *EXTRACELLULAR matrix , *CELL migration , *INTEGRINS - Abstract
The spatial organization of cell-surface receptors is fundamental for the coordination of biological responses to physical and biochemical cues of the extracellular matrix. How serine/threonine kinase receptors, ALK3-BMPRII, cooperate with integrins upon BMP2 to drive cell migration is unknown. Whether the dynamics between integrins and BMP receptors intertwine in space and time to guide adhesive processes is yet to be elucidated. We found that BMP2 stimulation controls the spatial organization of BMPRs by segregating ALK3 from BMPRII into β3 integrin-containing focal adhesions. The selective recruitment of ALK3 to focal adhesions requires β3 integrin engagement and ALK3 activation. BMP2 controls the partitioning of immobilized ALK3 within and outside focal adhesions according to single-protein tracking and super-resolution imaging. The spatial control of ALK3 in focal adhesions by optogenetics indicates that ALK3 acts as an adhesive receptor by eliciting cell spreading required for cell migration. ALK3 segregation from BMPRII in integrin-based adhesions is a key aspect of the spatio-temporal control of BMPR signaling. [ABSTRACT FROM AUTHOR]
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- 2022
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24. Lamellipodial Actin Mechanically Links Myosin Activity with Adhesion-Site Formation
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Giannone, Grégory, Dubin-Thaler, Benjamin J., Rossier, Olivier, Cai, Yunfei, Chaga, Oleg, Jiang, Guoying, Beaver, William, Döbereiner, Hans-Günther, Freund, Yoav, Borisy, Gary, and Sheetz, Michael P.
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CELL motility , *MYOSIN , *ELECTRON microscopy , *CELL physiology - Abstract
Summary: Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. [Copyright &y& Elsevier]
- Published
- 2007
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25. Structural determinants involved in the activation and regulation of G protein-coupled receptors: lessons from the alpha1-adrenegic receptor subtypes
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Cotecchia, Susanna, Stanasila, Laura, Diviani, Dario, Björklöf, Katja, Rossier, Olivier, and Fanelli, Francesca
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G proteins , *ADRENERGIC receptors , *DRUG receptors , *MEMBRANE proteins , *RHODOPSIN - Abstract
The aim of a large number of studies on G protein-coupled receptors was centered on understanding the structural basis of their main functional properties. Here, we will briefly review the results obtained on the α1-adrenergic receptor subtypes belonging to the rhodopsin-like family of receptors. These findings contribute, on the one hand, to further understand the molecular basis of adrenergic transmission and, on the other, to provide some generalities on the structure-functional relationship of G protein-coupled receptors. [Copyright &y& Elsevier]
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- 2004
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26. Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.
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Martins CS, Taveneau C, Castro-Linares G, Baibakov M, Buzhinsky N, Eroles M, Milanović V, Omi S, Pedelacq JD, Iv F, Bouillard L, Llewellyn A, Gomes M, Belhabib M, Kuzmić M, Verdier-Pinard P, Lee S, Badache A, Kumar S, Chandre C, Brasselet S, Rico F, Rossier O, Koenderink GH, Wenger J, Cabantous S, and Mavrakis M
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- Humans, Cell Membrane metabolism, Cytoskeleton metabolism, Microscopy, Actins metabolism, Septins analysis
- Abstract
Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane., (© 2022 Silva Martins et al.)
- Published
- 2023
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27. Single-Protein Tracking to Study Protein Interactions During Integrin-Based Migration.
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Radhakrishnan AV, Chen T, Nunes Vicente JF, Orré T, Mehidi A, Rossier O, and Giannone G
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- Actins genetics, Actins metabolism, Animals, Cell Adhesion, Cell Line, Transformed, Cryptochromes genetics, Cryptochromes metabolism, Cytoskeleton metabolism, Cytoskeleton ultrastructure, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Fibroblasts metabolism, Fibroblasts ultrastructure, Gene Expression, Integrins genetics, Mice, Microscopy instrumentation, Myosins genetics, Myosins metabolism, Neuropeptides genetics, Neuropeptides metabolism, Protein Binding, Pseudopodia metabolism, Pseudopodia ultrastructure, T-Lymphoma Invasion and Metastasis-inducing Protein 1 genetics, T-Lymphoma Invasion and Metastasis-inducing Protein 1 metabolism, Transcription Factors genetics, Transcription Factors metabolism, rac1 GTP-Binding Protein genetics, rac1 GTP-Binding Protein metabolism, Cell Movement, Integrins metabolism, Microscopy methods, Optogenetics methods, Protein Interaction Mapping methods, Single Molecule Imaging methods
- Abstract
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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- 2021
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28. Using Single-Protein Tracking to Study Cell Migration.
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Orré T, Mehidi A, Massou S, Rossier O, and Giannone G
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- Animals, Cell Line, Humans, Mice, Models, Biological, Cell Movement physiology, Protein Transport physiology
- Abstract
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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- 2018
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29. Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.
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Chamma I, Rossier O, Giannone G, Thoumine O, and Sainlos M
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- Amino Acid Sequence, Animals, COS Cells, Cell Membrane metabolism, Cricetinae, Mice, Models, Molecular, Protein Conformation, Fluorescent Dyes chemistry, Staining and Labeling methods, Streptavidin chemistry
- Abstract
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.
- Published
- 2017
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30. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions.
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Rossier O, Octeau V, Sibarita JB, Leduc C, Tessier B, Nair D, Gatterdam V, Destaing O, Albigès-Rizo C, Tampé R, Cognet L, Choquet D, Lounis B, and Giannone G
- Subjects
- Animals, Fibronectins metabolism, Mice, Microfilament Proteins metabolism, Protein Binding, Talin metabolism, Focal Adhesions metabolism, Integrin beta1 metabolism, Integrin beta3 metabolism
- Abstract
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.
- Published
- 2012
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31. Signaling network triggers and membrane physical properties control the actin cytoskeleton-driven isotropic phase of cell spreading.
- Author
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Rangamani P, Fardin MA, Xiong Y, Lipshtat A, Rossier O, Sheetz MP, and Iyengar R
- Subjects
- Animals, Computer Simulation, Gene Knockout Techniques, Mice, Models, Biological, Polymerization, Surface Properties, Wiskott-Aldrich Syndrome Protein metabolism, cdc42 GTP-Binding Protein metabolism, Actins metabolism, Cell Membrane metabolism, Cell Movement, Cytoskeleton metabolism, Signal Transduction
- Abstract
Cell spreading is regulated by signaling from the integrin receptors that activate intracellular signaling pathways to control actin filament regulatory proteins. We developed a hybrid model of whole-cell spreading in which we modeled the integrin signaling network as ordinary differential equations in multiple compartments, and cell spreading as a three-dimensional stochastic model. The computed activity of the signaling network, represented as time-dependent activity levels of the actin filament regulatory proteins, is used to drive the filament dynamics. We analyzed the hybrid model to understand the role of signaling during the isotropic phase of fibroblasts spreading on fibronectin-coated surfaces. Simulations showed that the isotropic phase of spreading depends on integrin signaling to initiate spreading but not to maintain the spreading dynamics. Simulations predicted that signal flow in the absence of Cdc42 or WASP would reduce the spreading rate but would not affect the shape evolution of the spreading cell. These predictions were verified experimentally. Computational analyses showed that the rate of spreading and the evolution of cell shape are largely controlled by the membrane surface load and membrane bending rigidity, and changing information flow through the integrin signaling network has little effect. Overall, the plasma membrane acts as a damper such that only ∼5% of the actin dynamics capability is needed for isotropic spreading. Thus, the biophysical properties of the plasma membrane can condense varying levels of signaling network activities into a single cohesive macroscopic cellular behavior., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
32. Mechanisms controlling cell size and shape during isotropic cell spreading.
- Author
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Xiong Y, Rangamani P, Fardin MA, Lipshtat A, Dubin-Thaler B, Rossier O, Sheetz MP, and Iyengar R
- Subjects
- Actin Cytoskeleton physiology, Actins, Adenosine Diphosphate pharmacology, Animals, Cell Adhesion drug effects, Cell Membrane physiology, Cell Polarity physiology, Cell Shape drug effects, Cell Size, Cells, Cultured, Cellular Structures drug effects, Cytoskeleton physiology, Epithelial Cells physiology, Membrane Fluidity physiology, Molecular Motor Proteins, Cell Movement physiology, Cell Shape physiology, Cytochalasin D pharmacology, Fibroblasts physiology, Movement physiology, Nucleic Acid Synthesis Inhibitors pharmacology
- Abstract
Cell motility is important for many developmental and physiological processes. Motility arises from interactions between physical forces at the cell surface membrane and the biochemical reactions that control the actin cytoskeleton. To computationally analyze how these factors interact, we built a three-dimensional stochastic model of the experimentally observed isotropic spreading phase of mammalian fibroblasts. The multiscale model is composed at the microscopic levels of three actin filament remodeling reactions that occur stochastically in space and time, and these reactions are regulated by the membrane forces due to membrane surface resistance (load) and bending energy. The macroscopic output of the model (isotropic spreading of the whole cell) occurs due to the movement of the leading edge, resulting solely from membrane force-constrained biochemical reactions. Numerical simulations indicate that our model qualitatively captures the experimentally observed isotropic cell-spreading behavior. The model predicts that increasing the capping protein concentration will lead to a proportional decrease in the spread radius of the cell. This prediction was experimentally confirmed with the use of Cytochalasin D, which caps growing actin filaments. Similarly, the predicted effect of actin monomer concentration was experimentally verified by using Latrunculin A. Parameter variation analyses indicate that membrane physical forces control cell shape during spreading, whereas the biochemical reactions underlying actin cytoskeleton dynamics control cell size (i.e., the rate of spreading). Thus, during cell spreading, a balance between the biochemical and biophysical properties determines the cell size and shape. These mechanistic insights can provide a format for understanding how force and chemical signals together modulate cellular regulatory networks to control cell motility., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
33. Cytoskeletal coherence requires myosin-IIA contractility.
- Author
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Cai Y, Rossier O, Gauthier NC, Biais N, Fardin MA, Zhang X, Miller LW, Ladoux B, Cornish VW, and Sheetz MP
- Subjects
- Animals, Blotting, Western, Cells, Cultured, Cytoskeleton drug effects, Fluorescent Antibody Technique, Heterocyclic Compounds, 4 or More Rings pharmacology, Mice, NIH 3T3 Cells, Nonmuscle Myosin Type IIA genetics, Nonmuscle Myosin Type IIA metabolism, rho-Associated Kinases antagonists & inhibitors, Actins metabolism, Cytoskeleton metabolism, Nonmuscle Myosin Type IIA physiology
- Abstract
Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.
- Published
- 2010
- Full Text
- View/download PDF
34. Plasma membrane area increases with spread area by exocytosis of a GPI-anchored protein compartment.
- Author
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Gauthier NC, Rossier OM, Mathur A, Hone JC, and Sheetz MP
- Subjects
- Animals, Endocytosis, Golgi Apparatus metabolism, HeLa Cells, Homeostasis, Humans, Lysosomes metabolism, Mice, Microtubules metabolism, NIH 3T3 Cells, Cell Compartmentation, Cell Membrane metabolism, Cell Movement, Exocytosis, Glycosylphosphatidylinositols metabolism
- Abstract
The role of plasma membrane (PM) area as a critical factor during cell motility is poorly understood, mainly due to an inability to precisely follow PM area dynamics. To address this fundamental question, we developed static and dynamic assays to follow exocytosis, endocytosis, and PM area changes during fibroblast spreading. Because the PM area cannot increase by stretch, spreading proceeds by the flattening of membrane folds and/or by the addition of new membrane. Using laser tweezers, we found that PM tension progressively decreases during spreading, suggesting the addition of new membrane. Next, we found that exocytosis increases the PM area by 40-60% during spreading. Reducing PM area reduced spread area, and, in a reciprocal manner, reducing spreadable area reduced PM area, indicating the interconnection between these two parameters. We observed that Golgi, lysosomes, and glycosylphosphatidylinositol-anchored protein vesicles are exocytosed during spreading, but endoplasmic reticulum and transferrin receptor-containing vesicles are not. Microtubule depolymerization blocks lysosome and Golgi exocytosis but not the exocytosis of glycosylphosphatidylinositol-anchored protein vesicles or PM area increase. Therefore, we suggest that fibroblasts are able to regulate about half of their original PM area by the addition of membrane via a glycosylphosphatidylinositol-anchored protein compartment.
- Published
- 2009
- Full Text
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35. Mutagenesis and modelling of the alpha(1b)-adrenergic receptor highlight the role of the helix 3/helix 6 interface in receptor activation.
- Author
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Greasley PJ, Fanelli F, Rossier O, Abuin L, and Cotecchia S
- Subjects
- Amino Acid Substitution, Animals, COS Cells, Chlorocebus aethiops, Cricetinae, Models, Molecular, Protein Structure, Secondary, Receptors, Adrenergic, alpha-1 chemistry, Receptors, Adrenergic, alpha-1 genetics, Transfection, Mutagenesis, Receptors, Adrenergic, alpha-1 metabolism
- Abstract
Computer simulations on a new model of the alpha1b-adrenergic receptor based on the crystal structure of rhodopsin have been combined with experimental mutagenesis to investigate the role of residues in the cytosolic half of helix 6 in receptor activation. Our results support the hypothesis that a salt bridge between the highly conserved arginine (R143(3.50)) of the E/DRY motif of helix 3 and a conserved glutamate (E289(6.30)) on helix 6 constrains the alpha1b-AR in the inactive state. In fact, mutations of E289(6.30) that weakened the R143(3.50)-E289(6.30) interaction constitutively activated the receptor. The functional effect of mutating other amino acids on helix 6 (F286(6.27), A292(6.33), L296(6.37), V299(6.40,) V300(6.41), and F303(6.44)) correlates with the extent of their interaction with helix 3 and in particular with R143(3.50) of the E/DRY sequence.
- Published
- 2002
- Full Text
- View/download PDF
36. The alpha1b-adrenergic receptor subtype: molecular properties and physiological implications.
- Author
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Cotecchia S, Björklöf K, Rossier O, Stanasila L, Greasley P, and Fanelli F
- Subjects
- Adrenergic alpha-Agonists pharmacology, Animals, Blood Pressure drug effects, Blood Pressure physiology, Glucose metabolism, Homeostasis, Humans, Mice, Mice, Knockout, Phosphorylation, Signal Transduction, Receptors, Adrenergic, alpha-1 physiology
- Abstract
The aim of this review is to summarize some of the main findings from our laboratory as well as from others concerning the biochemical, molecular, and functional properties of the alpha1b-adrenergic receptor. Experimental and computational mutagenesis of the alpha1b-adrenergic receptor have been instrumental in elucidating some of the molecular mechanisms underlying receptor activation and receptor coupling to Gq. The knockout mouse model lacking the alpha1b-adrenergic receptor has highlighted the potential implication of this receptor subtype in variety of functions including the regulation of blood pressure, glucose homeostasis, and the rewarding response to drugs of abuse.
- Published
- 2002
- Full Text
- View/download PDF
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