INDIRECT ASSOCIATION OF EZRIN WITH F-ACTIN - ISOFORM SPECIFICITY AND CALCIUM SENSITIVITY

Whereas it has been demonstrated that muscle and nonmuscle isoactins a re segregated into distinct cytoplasmic domains, the mechanism regulat ing subcellular sorting is unknown (Herman, 1993a). To reveal whether isoform-specific actin-binding proteins function to coordinate these e vents, cell extracts derived from motile (E(m)) versus stationary (E(s )) cytoplasm were selectively and sequentially fractionated over filam entous isoactin affinity columns prior to elution with a KCl step grad ient. A polypeptide of interest, which binds specifically to beta-acti n filament columns, but not to muscle actin columns has been conclusiv ely identified as the ERM family member, ezrin. We studied ezrin-beta interactions in vitro by passing extracts (E(m)) over isoactin affinit y matrices in the presence of Ca2+-containing versus Ca2+-free buffers , with or without cytochalasin D. Ezrin binds and can be released from beta-actin Sepharose-4B in the presence of Mg2+/EGTA and 100 mM NaCl (at 4 degrees C and room temperature), but not when affinity fractiona tion of E(m) is carried out in the presence of 0.2 mM CaCl2 or 2 mu M cytochalasin D. N-acetyl-(leucyl)(2)-norleucinal and E64, two specific inhibitors of the calcium-activated protease, calpain I, protect ezri n binding to beta actin in the presence of calcium. Moreover, biochemi cal analysis of endothelial lysates reveals that a calpain I cleavage product of ezrin emerges when cell locomotion is stimulated in respons e to monolayer injury. Immunofluorescence analysis of leading lamellae reveals that anti-ezrin and anti-beta-actin IgGs can be simultaneousl y co-localized, extending the results of isoactin affinity fractionati on of E(m)-derived extracts and suggesting that ezrin and beta-actin i nteract in vivo. To test the hypothesis that ezrin binds directly to b eta-actin, we performed three sets of studies under a wide range of ph ysiological conditions (pH 7.0-8.5) using purified pericyte ezrin and either alpha- or beta-actin. These included co-sedimentation, isoactin affinity fractionation, and co-immunoprecipitation. Results of these experiments reveal that purified ezrin does not directly bind to beta- actin filaments, either in solution or while isoactins are covalently cross-linked to Sepharose-4B. This is in contrast to our finding that ezrin and beta-actin could be co-immunoprecipitated or co-sedimented f rom E(m)-derived cell lysates. To explore whether calcium transients o ccur in cellular domains enriched in ezrin and beta-actin, we mapped c ellular free calcium in endothelial monolayers crawling in response to injury. Confocal imaging of fluo-3 fluorescence followed by simultane ous double antibody staining reveals a transient rise of free calcium within ezrin-beta-actin-enriched domains in the majority of motile cel ls bordering the wound edge. These results support the notion that cal cium and calpain I modulate ezrin and beta-actin interactions during f orward protrusion formation.