Department of Medical Protein Research, VIB, 9000 Ghent, Belgium.
Cell Mol Life Sci. 2010 May;67(9):1519-35. doi: 10.1007/s00018-010-0266-1. Epub 2010 Feb 7.
RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca(2+)-activated gelsolin (K (d) approximately 10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca(2+) (K (d) approximately 5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.
RNA 干扰极大地促进了我们对基因功能的理解，但最近的报告揭示了其不良的副作用。重组骆驼单域抗体（VHH）为研究蛋白质功能提供了一种有吸引力的手段，而不会影响基因表达。我们制备了针对凝溶胶（gelsolin）的 VHH（GsnVHH），凝溶胶是一种多功能的肌动蛋白结合蛋白，可控制细胞肌动蛋白组织和迁移。在哺乳动物细胞中，GsnVHH 诱导凝溶胶向线粒体或核的定位，揭示了包括游离凝溶胶和肌动蛋白结合的凝溶胶复合物在内的不同亚群。GsnVHH 13 特异性识别 Ca(2+) 激活的凝溶胶（K (d) 约为 10 nM），而 GsnVHH 11 则无论 Ca(2+) 如何结合凝溶胶（K (d) 约为 5 nM），但完全阻止其与 G-actin 的相互作用。两种 GsnVHH 都能追踪 EGF 刺激的 MCF-7 细胞中膜皱襞中的凝溶胶，并延迟细胞迁移，而不影响凝溶胶对 F-actin 的切断/加帽或成核活性。我们得出结论，VHH 代表了一种阻断结构蛋白的有效方法，而凝溶胶的肌动蛋白成核比之前预期的更为复杂。
