Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphthoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.