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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 caldesmon (l-CaD) is an important actin-binding protein that is ubiquitous in many non-muscle cells. In vitro studies have shown that l-CaD can promote the actin (G-actin) polymerization, stabilizing actin fiber structure (F-actin) through the combination with actin. Under phosphorylation, l-CaD can detach from actin fibers and promote depolymerization of actin fibers. The aim of this study is to investigate the regulatory effect of l-CaD on cellular actin cytoskeleton and to elucidate the effect of l-CaD on cell motility. The authors used MCF-7, a naturally-derived human breast cancer cell line with low expression of l-CaD Cell model, exogenous wild-type l-CaD and its phosphorylation mutant A1234-CaD (not phosphorylated CaD), D1234-CaD (fully phosphorylated CaD) were highly expressed in the intracellular of MCF-7 by gene transfection. Firstly, we investigated the regulation of cytoskeletal rearrangements by l-CaD by laser scanning confocal microscope. Secondly, the effect of l-CaD on cell migration was examined by transwell array. Finally, Cell basal tension, basal cell detachment ability under pancreatic stimulation, and further examined the effect of l-CaD on cell expansion and contraction during cell migration. The results show that l-CaD in the intracellular cytoskeleton formation has a significant regulatory role. Non-phosphorylated l-CaD is mainly enriched in the cytoskeleton and enhances the strength of the cytoskeleton, resulting in basal cell stretch and pancreatic enzyme resistance enhancement, but significantly inhibited cell migration; phosphorylation L-CaD binds to cytoskeleton weakly and has no significant effect on cell motility. Through phosphorylation, l-CaD plays a “protein switch ” role, by controlling the cytoskeletal depolymerization, rearrangement to regulate cell motility.