The role of integrin-linked kinase in the molecular architecture of focal adhesions.

Integrin-mediated focal adhesions (FAs) are large, multi-protein complexes that link the actin cytoskeleton to the extracellular matrix and take part in adhesion-mediated signaling. These adhesions are highly complex and diverse at the molecular level; thus, assigning particular structural or signaling functions to specific components is highly challenging. Here, we combined functional, structural and biophysical approaches to assess the role of a major FA component, namely, integrin-linked kinase (ILK), in adhesion formation. We show here that ILK plays a key role in the formation of focal complexes, early forms of integrin adhesions, and confirm its involvement in the assembly of fibronectin-bound fibrillar adhesions. Examination of ILK-null fibroblasts by cryo-electron tomography pointed to major structural changes in their FAs, manifested as disarray of the associated actin filaments and an increase in the packing density of FA-related particles. Interestingly, adhesion of the mutant cells to the substrate required a higher ligand density than in control cells. These data indicate that ILK has a key role in integrin adhesion assembly and sub-structure, and in the regulation of the FA-associated cytoskeleton.

Visualizing cellular processes at the molecular level by cryo-electron tomography.

The cellular landscape rapidly changes throughout the biological processes that transpire within a cell. For example, the cytoskeleton is remodeled within fractions of a second. Therefore, reliable structural analysis of the cell requires approaches that allow for instantaneous arrest of functional states of a given process while offering the best possible preservation of the delicate cellular structure. Electron tomography of vitrified but otherwise unaltered cells (cryo-ET) has proven to be the method of choice for three-dimensional (3D) reconstruction of cellular architecture at a resolution of 4-6 nm. Through the use of cryo-ET, the 3D organization of macromolecular complexes and organelles can be studied in their native environment in the cell. In this Commentary, we focus on the application of cryo-ET to study eukaryotic cells - in particular, the cytoskeletal-driven processes that are involved in cell movements, filopodia protrusion and viral entry. Finally, we demonstrate the potential of cryo-ET to determine structures of macromolecular complexes in situ, such as the nuclear pore complex.

Differential effect of actomyosin relaxation on the dynamic properties of focal adhesion proteins.

Treatment of cultured cells with inhibitors of actomyosin contractility induces rapid deterioration of stress fibers, and disassembly of the associated focal adhesions (FAs). In this study, we show that treatment with the Rho kinase inhibitor Y-27632, which blocks actomyosin contractility, induces disarray in the FA-associated actin bundles, followed by the differential dissociation of eight FA components from the adhesion sites. Live-cell microscopy indicated that the drug triggers rapid dissociation of VASP and zyxin from FAs (τ values of 7-8 min), followed by talin, paxillin and ILK (τ ~16 min), and then by FAK, vinculin and kindlin-2 (τ = 25-28 min). Examination of the molecular kinetics of the various FA constituents, using Fluorescence Recovery After Photobleaching (FRAP), in the absence of or following short-term treatment with the drug, revealed major changes in the kon and koff values of the different proteins tested, which are in close agreement with their differential dissociation rates from the adhesion sites. These findings indicate that mechanical, actomyosin-generated forces differentially regulate the molecular kinetics of individual FA-associated molecules, and thereby modulate FA composition and stability.

Dissecting the molecular architecture of integrin adhesion sites by cryo-electron tomography.

Focal adhesions are integrin-based multiprotein complexes, several micrometres in diameter, that mechanically link the extracellular matrix with the termini of actin bundles. The molecular diversity of focal adhesions and their role in cell migration and matrix sensing has been extensively studied, but their ultrastructural architecture is still unknown. We present the first three-dimensional structural reconstruction of focal adhesions using cryo-electron tomography. Our analyses reveal that the membrane-cytoskeleton interaction at focal adhesions is mediated through particles located at the cell membrane and attached to actin fibres. The particles have diameters of 25 +/- 5 nm, and an average interspacing of approximately 45 nm. Treatment with the Rho-kinase inhibitor Y-27632 induces a rapid decrease in particle diameter, suggesting that they are highly mechanosensitive. Our findings clarify the internal architecture of focal adhesions at molecular resolution, and provide insights into their scaffolding and mechanosensory functions.

Synthesis, two-dimensional assembly, and surface pressure-induced coalescence of ultranarrow PbS nanowires.

Ultranarrow (1.8 nm) PbS nanowires are synthesized in a single step, under benchtop conditions at relatively low temperature (90 degrees C). The nanowires exhibit a nearly perfect crystal lattice, high width uniformity, and tight side-by-side registry. Two-dimensional (2D) assembly over large areas (>15 microm2) is achieved using the Langmuir Blodgett method. The wire width can be readily controlled in the range 1.8-10 nm by a surface pressure-induced coalescence reaction, as monitored by transmission electron microscopy and Raman spectroscopy. The fluorescence of the 2D assembly shows strong polarization dependence along the long axis of the wires, making the system potentially suitable for orientation-sensitive devices.

Switchable assembly of ultra narrow CdS nanowires and nanorods.

We report on a simple route for the production of uniform and ultra narrow wurtzite CdS nanowires and nanorods. The nanorods are medium friendly (can exist in organic and aqueous phase) thus making them flexibly suitable for various applications. The centimeter range switchable ordering of the nanowires/rods into 3D microstrings by application of low magnitude DC electric field simply via two graphite electrodes is demonstrated. More sophisticated electrodes can be used for the same system to achieve more complex and fine patterns that can find potential use in nanoelectronics. The aligned microstrings (also wires/rods) show strong polarization dependence along their long axes. The polarized emission with respect to the unique c-axis makes the system suitable for orientation sensitive devices.