Lia Addadi

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 ± 2 nNμm-2. The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.

Design strategies in mineralized biological materials

Organisms have been producing mineralized skeletons for the past n550 nmillion years. They have evolved many different strategies for nimproving nthese materials at almost all hierarchical levels from nAngstroms to nmillimetres. Key components of biological materials are the nmacromolecules, which are intimately involved in controlling nnucleation, ngrowth, shaping and adapting mechanical properties of the mineral nphase to nfunction. One interesting tendency that we have noted is that norganisms nhave developed several strategies to produce materials that have nmore nisotropic properties. Much can still be learned from studying the nprinciples of structure–function relations of biological nmaterials. nSome of this information may also provide new ideas for improved ndesign of nsynthetic materials.

Amorphous calcium carbonate transforms into calcite during sea urchin larval spicule growth

Sea urchin larvae form an endoskeleton composed of a pair of spicules. For more than a century it has been stated that each spicule comprises a single crystal of the CaCO3 mineral, calcite. We show that an additional mineral phase, amorphous calcium carbonate, is present in the sea urchin larval spicule, and that this inherently unstable mineral transforms into calcite with time. This observation significantly changes our concepts of mineral formation in this well–studied organism.

Hierarchical assembly of cell-matrix adhesion complexes

The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion-mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 microm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability.

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.

A pavement of pearl

How do pearls grow? Both pearls and mother of pearl are made of nacre, one of several substances that molluscs build their shells from. It consists of layers of aragonite crystal, sandwiched between fibrous organic layers that provide it with elasticity. But the whole organic matrix is constructed first, so why doesnt it get in the way of crystal growth? The answer is simple -- a scattering of small pores, which both allow material to be supplied for crystal growth and help align the crystals.

Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida.

Almost all mineralized tissues contain proteins that are unusually acidic. As they are also often intimately associated with the mineral phase, they are thought to fulfill important functions in controlling mineral formation. Relatively little is known about these important proteins, because their acidic nature causes technical difficulties during purification and characterization procedures. Much effort has been made to overcome these problems, particularly in the study of mollusk‐shell formation. To date about 16 proteins from mollusk‐shell organic matrices have been sequenced, but only two are unusually rich in aspartic and glutamic acids. Here we screened a cDNA library made from the mRNA of the shell‐forming cells of a bivalve, Atrina rigida, using probes for short Asp‐containing repeat sequences, and identified ten different proteins. Using more specific probes designed from one subgroup of conserved sequences, we obtained the full sequences of a family of seven aspartic acid‐rich proteins, which we named “Asprich”; a subfamily of the unusually acidic shell‐matrix proteins. Polyclonal antibodies raised against a synthetic peptide of the conserved acidic1 domain of these proteins reacted specifically with the matrix components of the calcitic prismatic layer, but not with those of the aragonitic nacreous layer. Thus the Asprich proteins are constituents of the prismatic layer shell matrix. We can identify different domains within these sequences, including a signal peptide characteristic of proteins destined for extracellular secretion, a conserved domain rich in aspartic acid that contains a sequence very similar to the calcium‐binding domain of Calsequestrin, and another domain rich in aspartic acid, that varies between the seven sequences. We also identified a domain with DEAD repeats that may have Mg‐binding capabilities. Although we do not know, as yet, the function of these proteins, their generally conserved sequences do indicate that they might well fulfill basic functions in shell formation.

Initial Stages of Cell-Matrix Adhesion Can Be Mediated and Modulated by Cell-Surface Hyaluronan

A conceptual temporal and spatial gap exists between the first encounter of a cell with an adhesive substrate and the advanced stages of focal adhesion formation. Although ample information is available on focal adhesions structure and function, the mechanism of the first interaction events and the nature of the molecules mediating them are largely unknown. In this paper we identify cell-surface-associated hyaluronan as a mediator and modulator of the first steps of adhesion of A6 and other cells to conventional tissue culture substrates as well as to the surfaces of calcium-(R,R)-tartrate tetrahydrate crystals. Treatment of A6 cells with hyaluronidase suppresses their rapid interactions with these adhesive substrates, and incubation of either the hyaluronidase-treated cells or the substrate with hyaluronan restores cell adhesion. In contrast, excess hyaluronan on both the cells and the substrate strongly inhibits adhesion. We thus propose that cell-surface-associated hyaluronan can mediate and modulate cell-matrix adhesion at the very first encounter with the substrate. It may promote it through the establishment of exquisitely stereospecific chemical interactions or inhibit it by virtue of steric exclusion and/or electrostatic repulsion.

Structural Differences Between Biogenic Amorphous Calcium Carbonate Phases Using X-ray Absorption Spectroscopy**

We compare the organization of the first coordination shells around the calcium ion in biogenic ACC phases from three different sources. The results show that although the three biogenic samples have the same chemical composition, which is referred to collectively under the name “amorphous calcium carbonate”, they are structurally different from one another. These differences may be attributed to the diverse modes of formation of such biogenic materials and may account for their known variations in stability.

Biologically Formed Amorphous Calcium Carbonate

Many organisms from a wide variety of taxa produce amorphous calcium carbonate (ACC), despite the fact that it is inherently unstable and relatively soluble in its pure state. These properties also make it difficult to detect and characterize ACC. Raman spectroscopy is a particularly useful method for investigating ACC because the sample can be examined wet, and extended X-ray absorption fine structure (EXAFS) analysis can provide detailed information on the short-range order. Other methods for characterizing ACC include infrared spectroscopy, thermogravimetric analysis and differential thermal analysis (TGA and DTA), transmission electron microscopy (TEM), and electron and X-ray diffraction. Because of the difficulties involved, we suspect that ACC is far more widely distributed than is presently known, and a comparison of EXAFS spectra shows that different biogenic ACC phases have different short-range order structures. We also suspect that ACC fulfils many different functions, including as a transient precursor phase during the formation of crystalline calcium carbonate.