SILAC-based proteomic quantification of chemoattractant induced cytoskeleton dynamics on a second to minute timescale

Cytoskeletal dynamics during cell behaviours ranging from endocytosis and exocytosis to cell division and movement is controlled by a complex network of signalling pathways, the full details of which are as yet unresolved. Here we show that SILAC-based proteomic methods can be used to characterise the rapid chemoattractant induced dynamic changes in the actin-myosin cytoskeleton and regulatory elements on a proteome wide scale with a second to minute timescale resolution. This approach provides novel insights in the ensemble kinetics of key cytoskeletal constituents and association of known and novel identified binding proteins. We validate the proteomic data by detailed microscopy based analysis of in vivo translocation dynamics for key signalling factors. This rapid large-scale proteomic approach may be applied to other situations where highly dynamic changes in complex cellular compartments are expected to play a key role.

从胞吞作用、胞吐作用到细胞分裂与细胞运动等多种细胞行为过程中，细胞骨架动力学变化受复杂的信号通路网络调控，但其完整细节迄今尚未完全阐明。本研究证实，基于稳定同位素氨基酸细胞培养标记（Stable Isotope Labeling by Amino acids in Cell culture, SILAC）的蛋白质组学方法，可在全蛋白质组范围内以秒至分钟级时间分辨率，表征趋化剂诱导下肌动蛋白-肌球蛋白细胞骨架及其调控元件的快速动态变化。该方法可为关键细胞骨架组分的整体动力学特征，以及已知与新鉴定结合蛋白的相互作用提供全新见解。本研究通过对关键信号因子的体内易位动态开展详细的显微镜成像分析，验证了蛋白质组学数据的可靠性。这种快速、大规模的蛋白质组学方法，可推广应用于其他复杂细胞区室存在高度动态变化且该变化有望发挥关键作用的研究场景。