Molecular machinery and signal transduction of phagocytosis and macropinocytosis

巨噬细胞的巨噬作用和吞噬作用是细胞表面的运动，它们分别摄取细胞外液和细胞外颗粒，在生物防御、免疫功能中起着重要作用。这些细胞表面运动是由f -肌动蛋白聚合、分解和重排介导的，并受到许多肌动蛋白相关蛋白和复杂的信号转导系统的巧妙控制。本研究调查了各种肌动蛋白结合蛋白和肌球蛋白亚类，旨在阐明控制吞噬和巨噬过程的机械分子机制和信号转导。本研究通过对α-肌动蛋白的比值成像，发现α-肌动蛋白的一种新型异构体actiin -4比其他异构体更优先参与大肌肽体的形成。研究还表明，肌动蛋白-4在巨噬小体的形成过程中发挥了捆绑作用，因为在细胞内引入针对肌动蛋白-4的特异性抗体可显著抑制巨噬细胞增多症。此外，我们利用MLCK抑制剂ML-7选择性抑制肌动蛋白，揭示了肌动蛋白II在吞噬和巨噬细胞增多症中的独特作用。在巨嗜红细胞增多过程中，ML-7抑制了包括圆形褶皱形成在内的总褶皱运动。在吞噬作用中，ML-7不抑制假足延伸形成吞噬杯。而吞噬杯挤压收缩明显受到抑制。利用磷酸化myosin II的特异性抗体，免疫细胞化学证实磷酸化myosin定位于褶边和吞噬杯上，当ML-7抑制MLCK时，磷酸化myosin显著减少。这表明肌球蛋白II是巨噬作用中圆形褶皱形成和吞噬作用中吞噬杯挤压所必需的。

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-4Furthermore, 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.