Map of -actinin velocities in an MEF cell.

The linkage between the substratum and the actin cytoskeleton underlies a cell's ability to assemble and disassemble adhesions and thus, is crucial for regulating the formation of protrusions and cell migration. Paul Wiseman and colleagues now describe, in the Journal of Cell Science, the application of a novel image analysis tool to examine the movement of adhesion-related proteins and actin in migrating cells. Detailed protein velocity maps reveal differences in the efficiency of the linkage between integrin and actin among different cell types and indicate that the efficiency of linkage increases as actin and adhesions become more organized.

Integrins are key mediators of the linkage between the extracellular matrix (ECM) and actin. Previous studies indicate that the bond between the ECM and integrins is stronger than the link between integrins and the cytoskeleton as integrin-containing 'footprints' are often observed on the substratum after the rear region of a migrating cell retracts. Many other adhesion-related proteins such as talin, vinculin, focal adhesion kinase (FAK), Src and paxillin are also likely to be important regulators of the ECM-actin linkage, but the complexity of their interactions in both space and time has made it particularly difficult to asses their role in this linkage, until now.

By using spatio-temporal image correlation spectroscopy (STICS), the authors generated maps of molecular transport and quantified coupling between adhesion components and actin. STICS works by calculating the spatial-temporal correlation function from a series of images taken over time and directly reflects the movement of fluorescently-labelled macromolecules in live cells. An immobile filtering algorithm was developed to remove the contributions of large static structures, such as adhesions or actin filaments, and facilitate the visualization of proteins moving within or along these structures.

The authors first confirmed the validity of the technique by measuring the rates of mRFP-actin and -actinin-EGFP flow in mouse embryonic fibroblasts (MEFs). The values obtained, both in the absence and presence of retrograde flow inhibitors, were comparable to those measured by fluorescence speckle microscopy.

In CHO cells, under migration-promoting conditions, the movements of a-actinin and actin were highly coupled, but EGFP-fused paxillin, FAK, talin and vinculin had a much lower directional correlation with mRFP-actin. 5-integrin moved slowly and showed little directional correlation with the actin flow, supporting the notion of a strong interaction between 5-integrin and the ECM. In 3T3 cells and MEFs, which have highly organized actin filaments and larger adhesions, the movements of paxilin, talin and FAK were more tightly coupled to actin. The influence of the organization of actin filaments and adhesions on the linkage to the ECM was further corroborated when CHO cells were plated on high concentrations of fibronectin. Not only did the cells form more organized adhesions and actin stress fibres, there was also marked increase in the directional correlation for both talin and paxillin movement relative to actin movement.

Taken together, these results indicate that the efficiency of linkage between the ECM and actin is regulated by adhesion strength. The authors propose that ECM-actin linkage is regulated at two levels: first through interactions between integrins and adhesion-related proteins, and second between adhesion-related proteins and the actin cytoskeleton. The general applicability of STICS holds promise as a powerful new tool for dissecting protein movements in other contexts such as during membrane trafficking or protein transport in polarized cell systems.

Author: Monica Hoyos-Flight