Actinin-4 is preferentially involved in circular ruffling and macropinocytosis in mouse macrophages: analysis by fluorescence ratio imaging

We have applied fluorescence ratio imaging to the analysis of an actin-binding protein concentration relative to F-actin in macrophages, in order to explore the role of a novel (alpha)-actinin isoform, actinin-4, relative to that of the classical isoform, actinin-1. Conventional immunofluorescence images showed that both isoforms were enriched in F-actin-rich regions such as cell surface ruffles. However, ratio images further demonstrated that actinin-4 concentrations relative to F-actin were higher in peripheral inward curved ruffles and dorsal circular ruffles, presumed precursor forms of macropinosomes, than in straight linear ruffles, while actinin-1 concentrations were uniform among the different types of ruffles. Macropinosome pulse-labeling and chase experiments indicated that actinin-4 was also closely associated with newly formed macropinosomes and gradually dissociated with their maturation. Consistent with ratio imaging data, macrophages scrape-loaded with anti-actinin-4 showed a more reduced rate of macropinocytosis than those loaded with anti-actinin-1. Altogether, these results indicate that actinin-4 and actinin-1 contribute differently to F-actin dynamics, that actinin-4 is more preferentially involved in early stages of macropinocytosis than actinin-1. A similar redistribution of actinin-4 was also observed during phagocytosis, suggesting that actinin-4 may play the same role in the two mechanistically analogous types of endocytosis, i.e. macropinocytosis and phagocytosis.