作　　者： ; ; ;

机构地区： 重庆大学生物医学工程联合学院

出　　处： 《生物物理学报》 2010年第2期125-137,共13页

摘　　要： 轻链钙调蛋白结合蛋白(light-chain Caldesmon,l-CaD)是一种重要的肌动蛋白结合蛋白,普遍存在于众多非肌肉细胞中。体外研究证明,l-CaD能通过与肌动蛋白的结合起到促进原肌动蛋白(G-actin)聚合、稳定肌动蛋白纤维(F-actin)结构的作用。在磷酸化作用下,l-CaD能从肌动蛋白纤维上脱离并促进肌动蛋白纤维的解聚。该研究拟考察l-CaD在细胞内对细胞肌动蛋白骨架的调节作用,阐明l-CaD对细胞运动能力的影响,作者将天然低表达l-CaD的人源性乳腺癌细胞MCF-7作为细胞模型,在MCF-7胞内以基因转染的方式高表达外源野生型l-CaD及其磷酸化突变株A1234-CaD(不可磷酸化CaD)、D1234-CaD(完全磷酸化CaD)。首先,通过激光共聚焦扫描,探讨了l-CaD对细胞骨架重排的调节;其次,通过细胞迁移transwell阵列,检测了l-CaD对细胞迁移能力的影响;最后,在单细胞层次上测定了细胞基底牵张力、胰酶刺激下的细胞基底脱附能力,并进一步检测了l-CaD对细胞迁移子过程中细胞伸张、收缩的影响。研究结果显示,l-CaD在胞内对细胞骨架的形成有显著的调控作用。非磷酸化l-CaD主要富集在细胞骨架上,增强了细胞骨架的强度,导致细胞基底牵张力以及对胰酶的耐受性增强,但对细胞的迁移能力有显著的抑制作用;磷酸化l-CaD跟细胞骨架结合能力很弱,对细胞的运动能力没有显著影响。通过磷酸化,l-CaD起到了一个"蛋白开关"的作用,通过控制细胞骨架的解聚、重排来调节细胞的运动能力。 Light chain（l-CaD） is an important actin binding protein, presents almost all types of vertebrate cells.In vitro studies showed that l-CaD can bind to G-actin to accelerate the polymerization of the G-actin into F-actin and stabilize the formed F-actin.Upon phosphorylation, the l-CaD will disassociate from the F-acin to facilitate its disassembly into G-actin.In order to explore whether the l-CaD mediate the cell mobility by its effect on cytoskeleton, Human breast cancer cell line MCF-7 was chosen as experimental model in which the expression level of l-CaD is very low.By transfection, wild type l-CaD and its phosphorylation mutant（A1234-CaD unphosphorylatable CaD;D1234-CaD phosphorylated CaD） were force-expressed in MCF-7 cells.First, the effect of the l-CaD on cytoskeleton structure was tested by confocal images;and then the transwell assay was performed to address the influence of l-CaD on cell migration activities;Furthermore, the cell contractility and cell detachment which are sub-steps of cell migration were addressed by Fourier-Transform traction microscopy on single cell level and trypsin stimulation respectively.The results showed that l-CaD had significant effect on cytoskeleton structure of MCF-7 cells.The unphosphorylated l-CaD concentrated on cytoskeleton, increased its intensity which generated more cell contractility and inhibited the response of the cells to the trypsin stimulation.Because of the stabilization effect on cytoskeleton by unphosphorylated l-CaD, the rearrangement of the cytoskeleton was inhibited which resulted in the decrease of the cell migration activities;The phosphorylated l-CaD has very low affinity to the cytoskeleton, so its effect on cell mobility was not significant compared to the controlled cells.Taken together, upon phosphorylation, the l-CaD acted like a ＂protein switch＂, it will mediate the cell mobility by its effect on cytoskeleton disassembly and rearrangement.

关 键 词： 钙调蛋白结合蛋白 细胞骨架 细胞迁移 细胞基底牵张力

领　　域： [生物学] [生物学]