A "cell-free" system to study regulation of focal adhesions and of the connected actin cytoskeleton.

Cattelino, Anna (2000). A "cell-free" system to study regulation of focal adhesions and of the connected actin cytoskeleton. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000ff7e

Abstract

The exact composition of focal adhesions and the role of single components in their assembly and regulation during dynamic adhesive processes are poorly understood. I have characterised and used a "cell-free" system consisting of ventral plasma membranes (VPMs) prepared from adherent fibroblasts, and found that this subcellular fraction preserves intact focal adhesion components and the connected actin cytoskeleton. Moreover, VPMs show an accumulation of several tyrosine phosphorylated proteins, including tyrosine phosphorylated focal adhesion kinase and paxillin. In particular, a pool of higly phosphorylated paxillin is found in focal adhesions, suggesting an important role for the phosphorylated polypeptide in the mechanism of integrin-mediated adhesion.
With the aim of obtaining new important tools for my studies, I have produced and characterised monoclonal antibodies (mAbs) raised against VPM preparations.
The use of VPM preparations as a "cell-free" system has shown that changes in [Ca²⁺] can affect integrin behaviour within VPMs. I observed a correlation between integrin localisation and the functional state of the receptors, which can be reversibly modulated either by changes in free [Ca²⁺], or by function modulating anti-integrin pi mAbs. [Ca²⁺]-induced integrin redistribution is dependent on the presence of the pi cytoplasmic domain, whereas it is independent from the presence of filamentous actin (F- actin) and focal adhesion components in this experimental system, thus implicating the uncoupling of events relevants for focal adhesion assembly under "cell-free" conditions. Moreover, reconstitution experiments show that a-actinin colocalises and redistributes with pi receptors on VPMs depleted of actin, implicating binding of a-actinin to the receptors. Finally, I found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerisation is inhibited. These data attest the value of the system for further analysis of the molecular mechanisms regulating integrin function and focal adhesions.