Gough A H, Taylor D L
Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
J Cell Biol. 1993 Jun;121(5):1095-107. doi: 10.1083/jcb.121.5.1095.
Calmodulin is a calcium transducer that activates key regulatory and structural proteins through calcium-induced binding to the target proteins. A fluorescent analog of calmodulin in conjunction with ratio imaging, relative to a volume indicator, has demonstrated that calmodulin is uniformly distributed in serum-deprived fibroblasts and there is no immediate change in the distribution upon stimulation with complete serum. The same fluorescent analog of calmodulin together with steady state fluorescence anisotropy imaging microscopy has been used to define the temporal and spatial changes in calmodulin binding to cellular targets during stimulation of serum-deprived fibroblasts and in polarized fibroblasts during wound healing. In serum-deprived fibroblasts, which exhibit a low free calcium ion concentration, a majority of the fluorescent analog of calmodulin remained unbound (fraction bound, fB < 10%). However, upon stimulation of the serum-deprived cells with complete serum, calmodulin binding (maximum fB approximately 95%) was directly correlated with the time course of the elevation and decline of the free calcium ion concentration, while the contraction of stress fibers continued for an hour or more. Calmodulin binding was also elevated in the leading lamellae of fibroblasts (maximum FB approximately 50%) during the lamellar contraction phase of wound healing and was spatially correlated with the contraction of transverse fibers containing myosin II. Highly polarized and motile fibroblasts exhibited the highest anisotropy (calmodulin binding) in the retracting tails and in association with contracting transverse fibers in the cortex of the cell. These results suggest that local activation of myosin II-based contractions involves the local binding of calmodulin to target proteins. The results also demonstrate a powerful yet simple mode of light microscopy that will be valuable for mapping molecular binding of suitably labeled macromolecules in living cells.
钙调蛋白是一种钙传感器，它通过钙诱导与靶蛋白结合来激活关键的调节蛋白和结构蛋白。相对于体积指示剂，钙调蛋白的荧光类似物与比率成像相结合，已证明钙调蛋白在血清饥饿的成纤维细胞中均匀分布，在用完全血清刺激后其分布没有立即变化。钙调蛋白的同一荧光类似物与稳态荧光各向异性成像显微镜一起，已被用于确定血清饥饿的成纤维细胞受到刺激时以及伤口愈合过程中极化的成纤维细胞中钙调蛋白与细胞靶标的结合在时间和空间上的变化。在血清饥饿的成纤维细胞中，其游离钙离子浓度较低，大部分钙调蛋白荧光类似物仍未结合（结合分数，fB < 10%）。然而，在用完全血清刺激血清饥饿细胞后，钙调蛋白结合（最大fB约为95%）与游离钙离子浓度升高和下降的时间进程直接相关，而应力纤维的收缩持续了一个小时或更长时间。在伤口愈合的片状收缩阶段，成纤维细胞的前缘片状结构中钙调蛋白结合也升高（最大FB约为50%），并且在空间上与含有肌球蛋白II的横向纤维的收缩相关。高度极化且活跃的成纤维细胞在收缩尾部以及与细胞皮质中收缩的横向纤维相关联处表现出最高的各向异性（钙调蛋白结合）。这些结果表明，基于肌球蛋白II的收缩的局部激活涉及钙调蛋白与靶蛋白的局部结合。这些结果还展示了一种强大而简单的光学显微镜模式，这对于绘制活细胞中适当标记的大分子的分子结合情况将是有价值的。
