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

Abstract

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 Ca 2ϩ concentrations induce quasi-reversible diffusion of ␤1 integrins out of focal adhesions, whereas low Ca 2ϩ 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 Ca 2ϩ -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.

Liprin-α1 promotes cell spreading on the extracellular matrix by affecting the distribution of activated integrins

“…1A). In ventral plasma membranes (VPMs) prepared from ECMattached transfected cells (Cattelino et al, 1997;Cattelino et al, 1999), liprin-α1 remained associated with the substrate-bound plasma membrane (Fig. 1B).…”

“…VPMs were prepared from chicken embryo fibroblasts (CEFs) or COS7 cells on 10 μg ml -1 FN, as described (Cattelino et al, 1999). Briefly, VPMs were prepared with a jet of ice-cold buffer with anti-proteases and anti-phosphatases (20 mM HEPESNaOH pH 7.6, 0.3 mM PMSF, 10 mM NaF, 1 mM NaV), and used for immunofluorescence.…”

Smooth Muscle Actin Determines Mechanical Force-induced p38 Activation

“…These adhesion complexes are evidently more resistant to latrunculin and are not subject to the depolymerization of filaments induced by swinholide. Indeed, low-dose latrunculin B treatment may selectively preserve more stable actin filaments that insert into cell attachments on the cell surface (43,44).…”

Disruption of Focal Adhesions by Integrin Cytoplasmic Domain-associated Protein-1α

“…Purification of Ventral Plasma Membranes-The purification of HeLa, GD25-␤ 1 , or NIH3T3 ventral plasma membranes was performed as previously described by Cattelino et al (34). The cells were grown overnight on fibronectin-coated coverslips in complete medium.…”