Activated cofilin colocalises with Arp2/3 complex in apoptotic blebs during programmed cell death

Etoposide inhibits topoisomerase II and induces apoptosis in human epidermoid cancer cells (A431) and normal rat fibroblasts (NRK) as verified by apoptotic morphology and chromatin degradation. Here we examine changes in the localisation of actin, cofilin and the Arp2/3 complex during the apoptotic process in response to etoposide. Twenty-four hours after etoposide addition, a large number of cells of both lines exhibited nuclear and cytoplasmic fragmentation with the formation of numerous blebs typical of apoptosis. Etoposide exposure induces dissolution of stress fibres and an increase in actin and cofilin in membrane patches and apoptotic blebs. The actin is more peripherally located than the cofilin, similar to that reported for lamellipodia of highly motile keratocytes. By contrast, in control cells, cofilin is evenly distributed throughout the cytoplasm, though often enriched around the nucleus. The active form is inferred to be more peripherally localised and to be present in apoptotic blebs, since an antibody specific for phosphorylated cofilin did not stain the cell periphery nor apoptotic blebs. Although immunoblots of 2D gels demonstrate that the ratio of de-phosphorylated to phosphorylated cofilin does not change after etoposide treatment, this does not mean that there are no changes in the turnover of the active and inactive forms. Transfection of both cell lines with EGFP-containing constructs of wild-type cofilin and mutants resembling its activated (S3A) and inactivated (S3D) forms shows that the active form has a more peripheral localisation and is also present in the membrane blebs with a strong colocalisation with actin. We further show that Arp 2/3 also localises in apoptotic blebs and discuss the role of these proteins in apoptosis by analogy with actin-based protrusive motility in lamellipodia.