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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]

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

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]

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]

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]

Various genes related to early events in human gustation have recently been discovered, yet a thorough understanding of taste transduction is hampered by gaps in our knowledge of the signaling chain. As a first step toward gaining additional insight, the expression specificity of genes in human taste tissue needs to be determined. To this end, a fungiform papillae cDNA library has been generated and analyzed. For validation of the library, taste-related gene probes were used to detect known molecules. Subsequently, DNA sequence analysis was performed to identify further candidates. Of 987 clones sequenced, clustering results in 288 contigs. Comparison of these contigs with genomic databases reveals that 207 contigs (71.9%) match known genes, 16 (5.6%) match hypothetical genes, eight (2.8%) match repetitive sequences and 57 (19.8%) have no or low similarity to annotated genes. The results indicate that despite a high level of redundancy, this human fungiform cDNA library contains specific taste markers and is valuable for investigation of both known and novel taste-related genes. [ABSTRACT FROM AUTHOR]

.We report in this paper two simple and effective methods to decorate glass surfaces that enable protein micropatterning and subsequent spatially controlled adhesion of cells. The first method combines simultaneously the potentialities of two existing techniques, namely microcontact printing (μCP) and microfluidic networks (μFN) to achieve dual protein patterning in a single step. The second method is mainly based on the well-known property of poly(ethylene glycol) (PEG) to resist against protein adsorption. Both approaches were used to produce heterogeneous surfaces on which micron-size or submicronic streptavidin-coated lines alternate with cell-repellent areas. We first describe the implementation of the two methods and discuss the main pitfalls to avoid. Then, using these templates, we have monitored the kinetics of attachment of individual biotinylated (i.e. "attractant" towards streptavidin) red blood cells by directly measuring the propagation velocity of the adhesion front. Depending on the surface density of biotin, we found two distinct regimes, in agreement with existing theoretical models. [ABSTRACT FROM AUTHOR]

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]

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]

A correction to the article "Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions" that was published in the September 30, 2012 issue is presented.

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]

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]

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. [ABSTRACT FROM AUTHOR]

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]

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]

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]