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Vol. 10, Issue 2, 373-391, February 1999

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Anna Cattelino,^* Chiara Albertinazzi,^* Mario Bossi,^* David R. Critchley, and Ivan de Curtis^*

 ^*Cell Adhesion Unit, Department for Biological and Technological Research, San Raffaele Scientific Institute, 20132 Milan, Italy; and  Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca²⁺ concentrations induce quasi-reversible diffusion of 1 integrins out of focal adhesions, whereas low Ca²⁺ concentrations induce irreversible recruitment of 1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated 1 receptors show that the cytoplasmic portion of 1 is required for low Ca²⁺-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified -actinin colocalizes and redistributes with 1 receptors on ventral plasma membranes depleted of actin, implicating binding of -actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

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   Corresponding author.

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Molecular Biology of the Cell
Vol. 10, 373-391, February 1999
Copyright © 1999 by The American Society for Cell Biology

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