|Budget Amount *help
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2002: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Macropinocytosis and phagocytosis observed in macrophages are cell surface movements for respectively taking in extracellular fluid and particles, and play the important role in biological defense, immune function. These cell surface movements are mediated by F-actin polymerization, disassembly, and rearrangement, which are ingeniously controlled by many actin-related proteins and complicated signal transduction system. This study surveyed various actin-binding proteins and myosin subclasses for the purpose of the elucidation of mechanical molecular mechanism and signal transduction, which control the processes of phagocytosis and macropinocytosis.
In this study, we found that actinin-4, a novel isoform of α-actinin, was preferentially involved in the macropinosome formation than other isoforms by ratio imaging of actinin/F-actin. It was also indicated that actinin-4 functioned in bundling in macropinosome formation, because macropinocytosis was significantly suppressed by the intracellular introduction of the specific antibody for actinin-4
Furthermore, we revealed the distinct role of myosin II in phagocytosis and macropinocytosis, by using ML-7, a MLCK inhibitor which selectively inhibits myosin. In the process of macropinocytosis, total ruffle motion including circular ruffle formation was suppressed by ML-7. In phagocytosis, the extension of pseudopodia to form phagocytic cup was not inhibited by ML-7. However, the squeezing constriction of phagocytic cup was markedly inhibited. Using specific antibody for phosphorylated myosin II, it was immunocytochemically confirmed that phosphorylated myosin localized on ruffles and phagocytic cups, and it remarkably decreased, when MLCK was inhibited by ML-7. These suggested that myosin II is required for circular ruffle formation in macropinocytosis, and for phagocytic cup squeezing in phagocytosis.