Analyzing focal adhesion structure by atomic force microscopy

Abstract

Atomic force microscopy (AFM) can produce high-resolution topographic images of biological samples in physiologically relevant environments and is therefore well suited for the imaging of cellular surfaces. In this work we have investigated focal adhesion complexes by combined fluorescence microscopy and AFM. To generate high-resolution AFM topographs of focal adhesions, REF52 (rat embryo fibroblast) cells expressing YFP-paxillin as a marker for focal adhesions were de-roofed and paxillin-positive focal adhesions subsequently imaged by AFM. The improved resolution of the AFM topographs complemented the optical images and offered ultrastructural insight into the architecture of focal adhesions. Focal adhesions had a corrugated dorsal surface formed by microfilament bundles spaced 127±50 nm (mean±s.d.) apart and protruding 118±26 nm over the substratum. Within focal adhesions microfilaments were sometimes branched and arranged in horizontal layers separated by 10 to 20 nm. From the AFM topographs focal adhesion volumes could be estimated and were found to range from 0.05 to 0.50 μm3. Furthermore, the AFM topographs show that focal adhesion height increases towards the stress-fiber-associated end at an angle of about 3°. Finally, by correlating AFM height information with fluorescence intensities of YFP-paxillin and F-actin staining, we show that the localization of paxillin is restricted to the ventral half of focal adhesions, whereas F-actin-containing microfilaments reside predominantly in the membrane-distal half.

Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes

“…4 A and B) suggest this latter possibility. Interestingly, recent atomic force microscopy images of ''de-roofed'' fibroblasts cells also reveal adhesions consisting of many fibrillar-like structures decorated with numerous globular aggregates, all of dimensions (20-80 nm) similar to those seen here (34). However, a more disquieting option is that aggregation occurs because of the PA-FP tags, and that untagged proteins would not exhibit this behavior.…”

Extracellular matrix-induced transforming growth factor-β receptor signaling dynamics

“…Integrin b1 and TbR1 receptors can coexist and their recruitment can be further induced by collagen binding to the same membrane domains, as determined using immunofluorescence and co-IP. The colocalization of some of these complexes with Vinculin indicates selective signaling recruitment within the plasma membrane at sites of nascent ligand receptor interactions with the ECM (Evans and Matsudaira, 2006;Franz and Muller, 2005;Gonzales et al, 2001;Parsons et al, 1994;Petit and Thiery, 2000;Ziegler et al, 2006). The novel observation is that TGF-b receptors can also be incorporated into these domains.…”

Toward correlating structure and mechanics of platelets

“…37 To understand the interplay of mechanical forces and adhesion in platelets, the mechanical measurements need to be quantitatively correlated with the structural data. The two techniques can be used in tandem-AFM for nano-mechanics and cryo-ET to look at exactly those regions for structural insight.…”