Model of a focal adhesion's ultrastructure, showing three x–y views at increasing distance from the extracellular matrix (a–c). (a) Above the matrix-attached plasma membrane are the focal-adhesion-associated particles. (b) Inside the focal adhesion, particles are shown interacting with short actin filaments. (c) The well-structured, bundled actin network is involved in applying force to the adhesion site. Nevertheless, the unbundled actin network is seen in the outermost layer of the focal adhesion.From Nature Cell Biology 12, 909-915 (2010). doi:10.1038/ncb2095

Identifying the components of focal adhesions — regions that link the actin cytoskeleton to the extracellular matrix — has been relatively straightforward, but determining the three-dimensional (3D) organization of these molecularly crowded structures has posed much more of a challenge. Now, a group of researchers, led by Ohad Medalia, has succeeded in gaining insights into the molecular architecture of focal adhesions, discovering the presence of particles that mediate the membrane–cytoskeleton interactions at these regions.

The discovery of these particles, reported in Nature Cell Biology, was made using a combination of fluorescence microscopy and cryo-electron tomography. The architecture of the actin–membrane interface was visualized by generating, from tomographic slices, a 3D surface-rendering of the site of adhesion of cells expressing fluorescently tagged paxillin cultured on an electron-microscope grid coated with fibronectin. The small, doughnut-shaped structures were typically — but not exclusively — concentrated within focal adhesions. Despite being oriented uniformly (parallel to the substrate and 30–70 nm above it), the particles were clustered, rather than showing a uniform distribution along the adhesion site; at the periphery of focal adhesions, inter-particle spacing was 20–40 nm, as opposed to 40–70 nm in the middle. The overall morphology of the particles was similar, although variations in diameter (20–30 nm), position and number of interacting actin fibres were seen. However, the interaction between these heterogeneous particles and aligned actin bundles appeared to be indirect, through short filaments. Indeed, further analysis revealed that the particles interacted directly both with the cytoplasmic face of the plasma membrane and the ends of short actin filaments, implying a potential function for these focal-adhesion-associated particles in mediating membrane–cytoskeleton interactions and responding to force.

Next, they investigated the nature of these particles in more detail: they 'unroofed' the cells by shearing off the dorsal membrane, rapidly fixed the exposed focal adhesions, immunogold-labelled for vinculin and then carried out cryo-electron tomography. High-magnification images showed the gold labels to be very near the particles, indicating a tight association of vinculin with these structures, although the location of the gold labelling bore no relation to the position of the attached fibres.

The positioning of the particles at key locations between the plasma membrane and the contractile machinery led Medalia's group to test the potential effect of altered local mechanical force. They inhibited cellular contractility using, among other inhibitors, the Rho kinase inhibitor Y-27632, and showed that, although the appearance, number and interspacing of the particles was largely unaffected by the treatment, their diameter rapidly decreased.

Altogether, this study provides a much needed insight into the architecture of focal adhesions, leading to the following model of their structure: an outer domain of the plasma membrane, containing integrins; particles connected to the cytoplasmic domain of the plasma membrane; short, tangential filaments that interact with the particles; and bundles of actin filaments associated with the shorter filaments. Improved knowledge of the structure of focal adhesions will undoubtedly shed further light on their regulation and function.

Author: Katrin Legg - Copyright © 2010 Nature Publishing Group, a division of MacMillan Publishers Limited; used with permission