Vinculin controls focal adhesion formation by direct interactions with talin and actin

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs

The Architecture of the Adhesive Apparatus of Cultured Osteoclasts: From Podosome Formation to Sealing Zone Assembly

BACKGROUND: Osteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood. PRINCIPAL FINDINGS: Combining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers. SIGNIFICANCE: The sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions

Conformational states during vinculin unlocking differentially regulate focal adhesion properties

Abstract Focal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the detailed molecular mechanisms underlying FA mechanosensitivity are still unclear. Besides talin, another key candidate for regulating FA-associated mechanosensing, is vinculin, a prominent FA component, which possesses either closed (“auto-inhibited”) or open (“active”) conformation. A direct experimental demonstration, however, of the conformational transition between the two states is still absent. In this study, we combined multiple structural and biological approaches to probe the transition from the auto-inhibited to the active conformation, and determine its effects on FA structure and dynamics. We further show that the transition from a closed to an open conformation requires two sequential steps that can differentially regulate FA growth and stability

Sealing zone.

<p>(A, B) Frames from a movie taken by time-lapse microscopy from live GFP-actin osteoclasts plated on bone. (A) Four frames taken at 10 minutes intervals showing dynamic reorganization of actin belts. (B)One minute temporal ratio figure. Blue pixels represent new structures and red pixels faded structures. Note the high rate of dynamic reorganization. (C–E)Osteoclast ventral membrane on bone. (C) SEM overview of the ventral membrane of a cell plated on bone. The central area (arrow head) presumably corresponds to the ruffled border. The sealing zone (double arrow) is thicker than on glass. (D, E) Higher magnification views (from yellow box in (E)) of the podosomes forming the sealing zone. (F) Osteoclasts were plated on bone slices and removed three days later, leaving numerous resorption pits on the surface.</p

Structural relations between podosome ring and core domains.

<p>(A) Osteoclast ventral membranes were labeled for paxillin and actin, and simultaneously prepared for HR-SEM. On the left is the actin labeling, followed by paxillin labeling and by their merged picture. On the right is the corresponding SEM micrograph. (B) Higher magnification view of the area in the yellow rectangle in (A). (C) Higher magnification view of the area in the yellow rectangle in (B). (D) Merged actin/paxillin labeling from the same area as in (C). (E) Merged image between (C) and (D). The correlation between SEM and immunofluorescence shows paxillin association with podosome radial actin fibers, reaching up to but not co-localizing with the central bundle. (F, G, H) Osteoclast ventral membranes were labeled for paxillin with 15nm colloidal gold particles and visualized by back scattering signal (F) or secondary electron detector (G, H). (F) and (G) show the same area. The colloidal gold particles in (G, H) are marked with brownish dots, showing paxillin in association with actin fibers in close proximity with the ventral membrane.</p