Morphological analysis of spherical extrusion induced in okadaic acid-treated cells by PEG-cholesterol

PEG-胆固醇诱导大田酸处理细胞球形挤压的形态学分析

• 批准号: 13670007
• 负责人: BABA Takeshi
• 金额: $ 2.62万
• 依托单位: University of Yamanashi, Faculty of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670007/
• 关键词: PEG-cholesterol; okadaic acid; actin microfilament; lipid raft; K562 cell; endocytosis; PEG-コレストロール; ダイナミン

Interconnection between surface microdomains and the actin cytoskeleton is vital to various cellular activities. We studied the responses of okadaic acid (OKA)-treated K562 leukemia cells to crosslinking of membrane microdomains. Although OKA alone induced clustering of surface-bound F-actin, addition of a biotinylated poly(ethylene glycol) derivative of cholesterol (bPEG-Chol) and subsequent binding of streptavidin (SA) further induced accumulation of the clusters, resulting in the formation of a spherical cell extrusion. This extrusion was also induced by direct crosslinking of a raft marker, CD59, and ganglioside GM1. In addition, we found that knockout of the gene encoding Fyn kinase inhibited formation of the spherical extrusion in murine T-cells. In bPEG-Chol/SA-treated cells, CD59, ganglioside GM1, and clathrin/AP-2 were all accumulated on the surface of the actin-rich extrusion, whereas dynamin and transferrin receptors were unaffected. Intermediate filaments, mitochondria, and other vesicles also accumulated. These results suggest that crosslinking of membrane domains exaggerates the linkage between actin and a defined set of membrane proteins in OKA-treated cells.

表面微结构域和肌动蛋白细胞骨架之间的互连对于各种细胞活动至关重要。我们研究了冈田酸 (OKA) 处理的 K562 白血病细胞对膜微区交联的反应。虽然单独的 OKA 诱导表面结合的 F-肌动蛋白聚集，但添加生物素化的胆固醇聚乙二醇衍生物 (bPEG-Chol) 以及随后的链霉亲和素 (SA) 结合进一步诱导聚集，导致形成球形细胞挤出。这种挤压也是由筏标记 CD59 和神经节苷脂 GM1 的直接交联引起的。此外，我们发现敲除 Fyn 激酶编码基因可抑制小鼠 T 细胞球形挤压的形成。在 bPEG-Chol/SA 处理的细胞中，CD59、神经节苷脂 GM1 和网格蛋白/AP-2 均积聚在富含肌动蛋白的挤出物表面，而动力蛋白和转铁蛋白受体不受影响。中间丝、线粒体和其他囊泡也积累起来。这些结果表明，在 OKA 处理的细胞中，膜结构域的交联夸大了肌动蛋白和一组确定的膜蛋白之间的联系。