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Molecular machinery and signal transduction of phagocytosis and macropinocytosis

吞噬作用和巨胞饮作用的分子机制和信号转导

基本信息

批准号：
12670017
负责人：
NOBUKAZU Araki
金额：
$ 1.73万
依托单位：
Kagawa Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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.

巨噬细胞的巨吞作用和吞噬作用是细胞表面的运动，分别用于摄取细胞外液和颗粒，在生物防御、免疫功能中起重要作用。这些细胞表面的运动是由肌动蛋白的聚合、解聚和重排介导的，而这些运动是由许多肌动蛋白相关蛋白和复杂的信号转导系统巧妙地控制的。本研究对肌动蛋白结合蛋白和肌球蛋白亚类进行了研究，通过肌动蛋白/肌动蛋白比值成像发现肌动蛋白-4（actinin-4）是α-actinin的一种新亚型，它优先参与巨胞饮体的形成。另外，由于肌动蛋白4的特异性抗体的引入显著地抑制了巨胞饮作用，因此肌动蛋白4在巨胞饮体形成中起捆绑作用。此外，我们通过使用选择性抑制肌球蛋白的MLCK抑制剂ML-7，揭示了肌球蛋白II在吞噬作用和巨胞饮作用中的不同作用。在巨胞饮的过程中，总皱褶运动，包括圆形皱褶的形成被ML-7抑制。在吞噬作用中，ML-7不抑制伪足的延伸形成吞噬杯。而吞噬杯的挤压收缩则受到明显抑制。使用磷酸化肌球蛋白II的特异性抗体，免疫细胞化学证实，磷酸化肌球蛋白定位于皱褶和吞噬杯，并且当MLCK被ML-7抑制时，磷酸化肌球蛋白显著减少。这些表明，肌球蛋白II是所需的圆形皱褶形成巨胞饮，吞噬杯挤压吞噬作用。