用于细胞核钙成像的新型钙指示剂的构建与鉴定(英文)

Engineering of GCaMP6-based Calcium Indicators for Nuclear Calcium Imaging

细胞核钙离子是基因转录等细胞核反应过程重要的调控因子.然而,细胞核内钙离子信号的调控机制尚不清楚.缺乏稳定的、敏感的细胞核钙指示剂,是导致其调控机制难以研究的重要原因之一.针对这一问题,设计了能够在细胞核内特异性表达的、具有核定位功能的钙指示剂.以基因编码钙指示剂(GECIs)家族成员GCa MP6为模板,首先融合了对钙离子不敏感的红色荧光蛋白td Tomato来对局部的钙信号进行量化,其次融合了核定位信号(NLS),使GCa MP6能够特异定位于细胞核中.结果表明,NLS-GCa MP6-td Tomato能够在细胞核中有效发挥作用,并且在钙敏感性与动力学上,也与GCa MP6相当.这一新型细胞核钙指示剂将为研究细胞核钙离子的功能及其调控机制提供新的方法与途径.

Nuclear calcium（ Ca2 ＋） is an important regulator of gene expression and many other nuclear processes. However,the mechanisms regulating nuclear Ca2 ＋signaling remain controversial,due to the lack of specific and sensitive Ca2 ＋indicators for nuclear Ca2 ＋imaging. To generate nuclear-targeted calcium indicators,we fused nuclear localization signal（ NLS） sequences to GCa MP6, the newly developed and highly sensitive genetically-encoded calcium indicators. We also fused a Ca2 ＋-insensitive fluorescent protein td Tomato to GCa MP6 for identification of nuclei and in situ calibration of local Ca2 ＋signal. The engineered GCa MP6 variants showed robust nuclear targeting and minimum cytosolic localization. Moreover,their sensitivity and kinetics are comparable to those of the parental GCa MP6 variants. These Ca2 ＋indicators will permit unambiguous nuclear Ca2 ＋imaging in studying diverse cellular processes in vitro and in vivo.