Biomedical subjects

Mireille Lambert

Publications and source records attributed to Mireille Lambert.

12 recordsLinked to original sources

PubMed2006

Regulation of cell-cell junctions by the cytoskeleton.

A major form of animal cell-cell adhesion results from the dynamic association of cadherin molecules, cytosolic catenins and actin microfilaments. Cadherins dynamically regulate the cytoskeleton. In turn, the actin cytoskeleton contributes to cadherin molecule oligomerization at cell contacts and to cell reshaping in response to environmental changes. Over the past two years, this evolutionarily conserved adhesion system has been intensively revisited in both its structural and functional aspects; this is illustrated by the remarkable progress in the determination of physical parameters of cadherin bonds (including force measurement) and the new insights into the role of alpha-catenin and the regulation of actin dynamics at cadherin contacts. Other recent studies uncover the important contribution of acto-myosin, microtubules and cell tension to adherens junction formation, cell differentiation and tissue reshaping/remodeling. An open challenge is now to integrate these new data with the diversity of cadherin adhesive complexes.

Actins↗

PubMed2006

Traction forces exerted through N-cadherin contacts.

BACKGROUND INFORMATION: Mechanical forces play an important role in the organization, growth and function of living tissues. The ability of cells to transduce mechanical signals is governed by two types of microscale structures: focal adhesions, which link cells to the extracellular matrix, and adherens junctions, which link adjacent cells through cadherins. Although many studies have examined forces induced by focal adhesions, there is little known about the role of adherens junctions in force-regulation processes. The present study focuses on the determination of force transduction through cadherins at a single cell level. RESULTS: We characterized for the first time the distribution of forces developed by the cell through cadherin contacts. A N-cadherin (neural cadherin)-Fc chimaera, which mimicks the cell adhesion molecule N-cadherin, was immobilized on a muFSA (micro-force sensor array), comprising a dense array of vertical elastomer pillars, which were used both as a cell culture support for N-cadherin-expressing C2 myogenic cells and as detectors for force mapping. We coated the top of the pillars on which cells adhere and recruit adhesion complexes and F-actin. Individual pillar bending allowed the measurement of forces that mainly developed at the cell edge and directed toward their centre. Similar force distribution and amplitude were detected with an unrelated cell line of neuronal origin. Further comparison with forces applied by cells on pillars coated with fibronectin indicates that mechanical stresses transduced through both types of adhesions were comparable in distribution, orientation and amplitude. CONCLUSIONS: These results present a versatile method to measure and map forces exerted by cell-cell adhesion complexes. They show that cells transduce mechanical stress through cadherin contacts which are of the same order as magnitude of those previously characterized for focal adhesions. Altogether, they emphasize the mechanotransduction role of cytoskeleton-linked adhesion receptors of the cadherin family in tissue cohesion and reshaping.

Actins↗

PubMed2005

Regulation of N-cadherin dynamics at neuronal contacts by ligand binding and cytoskeletal coupling.

N-cadherin plays a key role in axonal outgrowth and synaptogenesis, but how neurons initiate and remodel N-cadherin-based adhesions remains unclear. We addressed this issue with a semiartificial system consisting of N-cadherin coated microspheres adhering to cultured neurons transfected for N-cadherin-GFP. Using optical tweezers, we show that growth cones are particularly reactive to N-cadherin coated microspheres, which they capture in a few seconds and drag rearward. Such strong coupling requires an intact connection between N-cadherin receptors and catenins. As they move to the basis of growth cones, microspheres slow down while gradually accumulating N-cadherin-GFP, demonstrating a clear delay between bead coupling to the actin flow and receptor recruitment. Using FRAP and photoactivation, N-cadherin receptors at bead-to-cell contacts were found to continuously recycle, consistently with a model of ligand-receptor reaction not limited by membrane diffusion. The use of N-cadherin-GFP receptors truncated or mutated in specific cytoplasmic regions show that N-cadherin turnover is exquisitely regulated by catenin partners. Turnover rates are considerably lower than those obtained previously in single molecule studies, demonstrating an active regulation of cadherin bond kinetics in intact cells. Finally, spontaneous neuronal contacts enriched in N-cadherin exhibited similar turnover rates, suggesting that such dynamics of N-cadherin may represent an intrinsic mechanism underlying the plasticity of neuronal adhesions.

Actin Cytoskeleton↗

PubMed2005

A novel function for cadherin-11 in the regulation of motor axon elongation and fasciculation.

We previously observed that cadherin-11, a type II cadherin, is expressed in growing motor and sensory axons in the mouse embryo. Here, we assessed its functional involvement in the regulation of axon elongation and fasciculation by evaluating the activity of a specific cadherin-11 homophilic ligand, cad11-Fc (cadherin-11 extracellular region fused to Fc fragment of IgG), on the length and organization of motor axons outgrowing from embryonic ventral spinal cord explants. Cad11-Fc substrate enhanced axon growth and prevented interactions occurring between growing axons, providing evidences for a role of cadherin-11 in the control of growth cone progression. Comparison of cadherin-11 with N-cadherin, a type I cadherin concomitantly expressed by motor axons, revealed similarities in their functional properties, including the ability to reorganize the actin cytoskeleton through interactions with catenins, but differences in their axon growth-promoting activity, arguing for subtle differences in their contributions to peripheral nerve elongation.

Animals↗

PubMed2004

N-cadherin activation substitutes for the cell contact control in cell cycle arrest and myogenic differentiation: involvement of p120 and beta-catenin.

N-cadherin is expressed throughout skeletal myogenesis and has been proposed to be involved in the differentiation program of myogenic precursors. Here, we further characterize the N-cadherin involvement and its mechanism of action at the onset of differentiation, through controlled N-cadherin activation by plating isolated C2 myoblasts on surfaces coated with a chimeric Ncad-Fc homophilic ligand (N-cadherin ectodomain fused to the immunoglobulin G Fc fragment). We show that N-cadherin activation substitutes for the cell density in myogenic differentiation by promoting myogenin and troponin T expression. In addition, N-cadherin adhesion participates to the associated cell cycle arrest through the nuclear accumulation of cyclin-dependent kinase inhibitors p21 and p27. Mouse primary myoblast cultures exhibited similar responses to N-cadherin as C2 cells. RNA interference knockdowns of the N-cadherin-associated cytoplasmic proteins p120 and beta-catenin produced opposite effects on the differentiation pathway. p120 silencing resulted in a decreased myogenic differentiation, associated with a reduction in cadherin-catenin content, which may explain its action on myogenic differentiation. beta-Catenin silencing led to a stimulatory effect on myogenin expression, without any effect on cell cycle. Our results demonstrate that N-cadherin adhesion may account for cell-cell contact-dependent cell cycle arrest and differentiation of myogenic cells, involving regulation through p120 and beta-catenins.

Animals↗

PubMed2004

[Regulation of cell adhesion by the cadherin cytoplasmic domain].

Juxtacrine cell interactions associated to cadherin-mediated cell-cell adhesion play a major role in the organization and homeostasis of tissues. Here, we review the intracellular molecules and regulations controlling the formation of cell-cell contacts initiated by homophilic interactions of cadherin ectodomain. These regulations involve proteins associated to cadherin cytoplasmic tail, named catenins, their association to the actin cytoskeleton and the stability of these complexes at the cell membrane. The underlying molecular mechanisms, which participate in the formation of dynamic cell-cell contacts, are intensively investigated.

Actins↗

PubMed2003

Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways.

Cell adhesion molecules of the cadherin family contribute to the regulation of cell shape and fate by mediating strong intercellular adhesion through Ca2+-dependent interaction of their ectodomain and association of their cytoplasmic tail to actin. However, the mechanisms co-ordinating cadherinmediated adhesion with the reorganization of the actin cytoskeleton remain elusive. Here, the formation of de novo contacts was dissected by spreading cells on a highly active N-cadherin homophilic ligand. Cells responded to N-cadherin activation by extending lamellipodium and organizing cadherin-catenin complexes and actin filaments in cadherin adhesions. Lamellipodium protrusion, associated with actin polymerization at the leading edge sustained the extension of cadherin contacts through a phosphoinositide 3-kinase (PI 3-kinase)-Rac1 pathway. Cadherin adhesions were formed by PI 3-kinase-independent, Rac1-dependent co-recruitment of adhesion complexes and actin filaments. The expression and localization of p120 at the plasma membrane, associated with an increase in membrane-associated Rac1 was required for both cell responses, consistent with a major role of p120 in signalling pathways initiated by cadherin activation and contributing to Rac1-dependent contact extension and maturation. These results provide additional information on the mechanisms by which cadherin coordinates adhesion with dynamic changes in the cytoskeleton to control cell shape and intercellular junction organization.

Animals↗

PubMed2003

Homoassociation of VE-cadherin follows a mechanism common to "classical" cadherins.

Vascular endothelial cadherin (VE-cadherin/cadherin5) is specifically expressed in adherens junctions of endothelial cells and exerts important functions in cell-cell adhesion as well as signal transduction. To analyze the mechanism of VE-cadherin homoassociation, the ectodomains CAD1-5 were connected by linker sequences to the N terminus of the coiled-coil domain of cartilage matrix protein (CMP). The chimera VECADCMP were expressed in mammalian cells. The trimeric coiled-coil domain leads to high intrinsic domain concentrations and multivalency promoting self-association. Ca(2+)-dependent homophilic association of VECADCMP was detected in solid phase assays and cross-linking experiments. A striking analogy to homoassociation of type I ("classical") cadherins like E, N or P-cadherin was observed when interactions in VECADCMP and between these trimeric proteins were analyzed by electron microscopy. Ca(2+)-dependent ring-like and double ring-like arrangements suggest interactions between domains 1 and 2 of the ectodomains, which may be correlated with lateral and adhesive contacts in the adhesion process. Association to complexes composed of two VECADCMP molecules was also demonstrated by chemical cross-linking. No indication for an antiparallel association of VECAD ectodomains to hexameric complexes as proposed by Legrand et al. was found. Instead the data suggest that homoassociation of VE-cadherin follows the conserved mechanism of type I cadherins.

Animals↗

PubMed2002

Recruitment of the kainate receptor subunit glutamate receptor 6 by cadherin/catenin complexes.

Kainate receptors modulate synaptic transmission by acting either at presynaptic or at postsynaptic sites. The precise localization of kainate receptors as well as the mechanisms of targeting and stabilization of these receptors in neurons are largely unknown. We have generated transgenic mice expressing the kainate receptor subunit glutamate receptor 6 (GluR6) bearing an extracellular myc epitope (myc-GluR6), in forebrain neurons, in which it assembles with endogenous kainate receptor subunits. In transgenic mice crossed with GluR6-deficient mice, myc-GluR6 efficiently rescues the missing subunit. Immunoprecipitation of transgenic brain extracts with anti-myc antibodies demonstrates an interaction with cadherins, beta-catenin, and p120 catenin, as well as with the associated proteins calcium calmodulin-dependent serine kinase and Velis, but not with alpha-catenin. In glutathione S-transferase-pulldown experiments, beta-catenin interacts, although indirectly, with the last 14 aa of GluR6. Transfected myc-GluR6 colocalizes with beta-catenin at cell-cell junctions in non-neuronal cells. Finally, activation of N-cadherins by ligand-covered latex beads recruits GluR6 to cadherin/catenin complexes. These results suggest an important role for cadherin/catenin complexes in the stabilization of kainate receptors at the synaptic membrane during synapse formation and remodeling.

Animals↗

PubMed2002

Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton.

Cadherin receptors are key morphoregulatory molecules during development. To dissect their mode of action, we developed an approach based on the use of myogenic C2 cells and beads coated with an Ncad-Fc ligand, allowing us to mimic cadherin-mediated adhesion. We used optical tweezers and video microscopy to investigate the dynamics of N-cadherin anchoring within the very first seconds of bead-cell contact. The analysis of the bead movement by single-particle tracking indicated that N-cadherin molecules were freely diffusive in the first few seconds after bead binding. The beads rapidly became diffusion-restricted and underwent an oriented rearward movement as a result of N-cadherin anchoring to the actin cytoskeleton. The kinetics of anchoring were dependent on ligand density, suggesting that it was an inducible process triggered by active cadherin recruitment. This anchoring was inhibited by the dominant negative form of Rac1, but not that of Cdc42. The Rac1 mutant had no effect on cell contact formation or cadherin-catenin complex recruitment, but did inhibit actin recruitment. Our results suggest that cadherin anchoring to the actin cytoskeleton is an adhesion-triggered, Rac1-regulated process enabling the transduction of mechanical forces across the cell membrane; they uncover novel aspects of the action of cadherins in cell sorting, cell migration, and growth cone navigation.

Actins↗

PubMed2002

Complementary expression and regulation of cadherins 6 and 11 during specific steps of motoneuron differentiation.

Cadherins constitute one of the major classes of adhesion receptors participating in the architectural organization of embryonic tissues. We previously identified type II cadherins 6 and 11, whose mRNA expression was tightly regulated during mouse neuromuscular development. Here we determine the regulation of expression and the localization of the corresponding proteins in relation to spinal neuron differentiation and peripheral nerve outgrowth. Cadherin-11 expression initially appeared in motor columns before extending to the whole spinal cord, dorsal root ganglia, and sensory-motor peripheral nerves. Then, its expression decreased in nervous tissues and became predominant in mesenchymes. Cadherin-6 was exclusively expressed in floor and roof plates, motor columns, and motor peripheral nerves, including motor axons and Schwann cell precursors. Compared to cadherin-11, its expression in motor columns was delayed and restricted to certain subpopulations of motoneurons. These results strongly implicate cadherins 6 and 11 in the control of spinal motoneuron differentiation and segregation and in axoaxonal, axoglial, and glio-glial interactions during sensory-motor nerve progression.

Animals↗

PubMed2002

Cadherin-based cell adhesion in neuromuscular development.

The organisation and differentiation of striated skeletal muscles and their innervation is a particularly complex process implicating cells of mesodermic (myoblasts and fibroblasts) and neuroectoderrmic origin (neurons and glial cells). Myogenic and motor neuron precursors, the two major cell types participating in the formation of the neuromuscular axis, migrate, segregate, reassociate and differentiate in a coordinated fashion. The subsequent organisation of muscle cells and the establishment of muscle innervation rely on a complex tissular and cellular architectural organisation, which cannot be understood without taking into account juxtacrine cell interactions, and especially cell adhesion. Cell adhesion receptors of the cadherin family are widely expressed and dynamically regulated in space and time throughout neuromuscular development. A single cell expresses in general more than one cadherin at its surface and it is the combination of these molecules and their level of expression that determine their action within a given cell population. We focused in this review on the expression and roles of classical cadherins in relation to muscle cell and motoneuron differentiation. We also review the latest results on the mode of action of cadherins allowing to propose cellular and molecular cues on the mechanisms by which these cell adhesion receptors control muscle and neuronal cell shape, migration and differentiation.

Animals↗