The study of calmodulin (CaM) functions in living cells has been tackled up to date using cell-permeant CaM inhibitors or interference-RNA methods. CaM inhibitors may lack specificity and the siRNA interference approach is challenging, as all three CaM genes expressing an identical protein in mammals have to be blocked. Therefore, we recently introduced a novel genetic system using CRISPR/Cas9-mediated gene deletion and conditional CaM expression to study the function of CaM in HeLa cells. Here, we describe the effect of CaM downregulation on the basal and epidermal growth factor (EGF)-dependent 2D- and 3D-migration in HeLa cells. CaM downregulation inhibited cell migration on a 2D-surface in the absence but not in the presence of EGF. In contrast, CaM downregulation led to inhibition of 3D-migration across a porous membrane both in the absence and presence of EGF. CaM downregulation decreased the expression of Rac1, Cdc42 and RhoA, all known to play crucial roles in cell migration. These results show that EGF-dependent 2D- and 3D-migration utilize distinct CaM-regulated systems and identify several essential migratory proteins directly or indirectly regulated by CaM.

迄今为止，已经使用细胞渗透性CaM抑制剂或干扰RNA方法解决了对钙调蛋白（CaM）在活细胞中的功能的研究。CaM抑制剂可能缺乏特异性，而siRNA干扰方法具有挑战性，因为必须阻断哺乳动物中表达相同蛋白质的所有三个CaM基因。因此，我们最近介绍了一种使用CRISPR / Cas9介导的基因缺失和条件性CaM表达的新型遗传系统，以研究CaM在HeLa细胞中的功能。在这里，我们描述了CaM下调对HeLa细胞中基础和表皮生长因子（EGF）依赖性2D和3D迁移的影响。在不存在但不存在EGF的情况下，CaM下调抑制了2D表面上的细胞迁移。相反，在不存在和存在EGF的情况下，CaM下调都会抑制3D迁移穿过多孔膜。CaM的下调降低了Rac1，Cdc42和RhoA的表达，这些都已知在细胞迁移中起关键作用。这些结果表明，依赖EGF的2D和3D迁移利用了不同的CaM调控系统，并鉴定了由CaM直接或间接调控的几种必需的迁移蛋白。