Potter D A, Tirnauer J S, Janssen R, Croall D E, Hughes C N, Fiacco K A, Mier J W, Maki M, Herman I M
Division of Hematology and Oncology, Tupper Research Institute, Department of Medicine, New England Medical Center, Boston, Massachusetts, USA.
J Cell Biol. 1998 May 4;141(3):647-62. doi: 10.1083/jcb.141.3.647.
Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.
先前的研究表明，钙依赖性蛋白酶（钙蛋白酶）在伤口愈合过程中参与肌动蛋白细胞骨架的重塑，并且在细胞迁移过程中具有活性。为了直接测试钙蛋白酶在细胞铺展中所起的作用，分离出了几种源自NIH-3T3的克隆细胞系，这些细胞系过表达钙蛋白酶的生物学抑制剂——钙蛋白酶抑制蛋白。相对于对照，这些细胞稳定地过表达钙蛋白酶抑制蛋白2至8倍，并且在形态、铺展、细胞骨架结构和生化特性方面与亲代细胞系和对照细胞系均有所不同。突变细胞的形态学特征包括无法伸出片状伪足，以及丝状伪足、延伸和回缩异常。野生型细胞在接种后30分钟内伸出薄片，而所有过表达钙蛋白酶抑制蛋白的细胞系均无法铺展并组装富含肌动蛋白的突起。经原位或体外测量，基因改变以过表达钙蛋白酶抑制蛋白的细胞显示出钙蛋白酶活性降低。由于钙蛋白酶受到抑制，ERM蛋白埃兹蛋白显著增加，而根蛋白或膜突蛋白则没有。为了证实钙蛋白酶活性的抑制与铺展缺陷有关，还分析了钙蛋白酶的药理学抑制剂。细胞渗透性抑制剂钙肽素和MDL 28,170可立即抑制铺展。丝状伪足形成和片状延伸等密切相关过程的失败表明，钙蛋白酶与肌动蛋白重塑和细胞铺展密切相关。
