In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis.
In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis.
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通过改进的FRAP分析，斑马鱼胚胎中骨骼肌肌营养不良蛋白的体内动力学。
DOI：
10.7554/elife.06541
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发表时间：
2015-10-13
期刊：
影响因子：
7.7
通讯作者：
Hughes SM
中科院分区：
文献类型：
--
作者：
Bajanca F;Gonzalez-Perez V;Gillespie SJ;Beley C;Garcia L;Theveneau E;Sear RP;Hughes SM
肌营养不良蛋白形成肌膜和细胞骨架之间的重要联系，其扰动导致肌营养不良症。我们首次分析了Dystrophin在体内的结合动力学。在宿主斑马鱼胚胎的成熟纤维中，我们的分析揭示了一个可扩散的Dystrophin和复合物结合在纤维膜上的池。结合建模、改进的FRAP方法和漂白的直接半定量分析，表明存在两种具有广泛不同结合寿命的膜结合肌营养不良蛋白群体：稳定、紧密结合的库和具有高周转率的动态结合库，与细胞质交换。池。这三个群体在人类和斑马鱼中一致地发现了在野生型或dmdta 222 a/ta 222 a斑马鱼胚胎中过表达的肌营养不良蛋白，其缺乏肌营养不良蛋白，以及在表达内源性驱动的标记的斑马鱼肌营养不良蛋白的Gt（dmd-Citrine）ct 90 a中。这些结果导致一个新的模型肌营养不良蛋白膜协会在发展中的肌肉，并强调我们的方法作为一个有价值的战略，在体内分析复杂的蛋白质动力学。DOI：http://dx.doi.org/10.7554/eLife.06541.001一种名为Dystrophin的蛋白质在维持肌肉细胞收缩和放松时的结构完整性方面起着关键作用。编码Dystrophin的基因突变可导致几种不同类型的肌营养不良症，这是一组肌肉逐渐减弱的疾病。肌营养不良蛋白的一些突变可导致轻度症状，可能影响生活质量，但不会危及生命。然而，在更严重的情况下，患者在儿童时期失去行走能力，预期寿命缩短。目前还没有治愈这些疾病的方法，而且我们对肌营养不良蛋白如何起作用的理解仍然存在很大的差距，这使得开发有效的治疗方法变得更加困难。斑马鱼经常被用作研究肌肉萎缩症的模型。在这项研究中，Bajanca等人将人类Dystrophin引入斑马鱼肌肉细胞，并使用数学建模和称为“光漂白后荧光恢复”的方法相结合来分析其行为。在这些实验中，人类肌营养不良蛋白被附着在一个在显微镜下发绿色荧光的标签上，这使得它很容易被看到，并在活体动物的细胞内进行真实的跟踪。Bajanca等人观察到，肌营养不良蛋白可以在很长一段时间内与包围细胞的膜保持牢固的结合，或者与膜短暂地相互作用。Bajanca等人对斑马鱼中天然存在的Dystrophin蛋白进行了进一步的实验，并观察到它的行为与人类蛋白相似，这表明这种行为可能对蛋白质的工作能力很重要。Bajanca等人的发现揭示了肌营养不良蛋白在活的肌肉细胞中表现出复杂的行为。一些抗肌萎缩蛋白分子牢固地附着在膜上的事实支持了以前的发现，即这种蛋白质为细胞提供了机械稳定性。然而，在肌肉细胞内存在一组更移动的肌营养不良蛋白分子的发现表明，这种蛋白质可能还起着其他作用。因此，这些发现为研究开辟了新的途径，可能有助于未来新治疗方法的发展。DOI：http://dx.doi.org/10.7554/eLife.06541.002网站
Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics. DOI: http://dx.doi.org/10.7554/eLife.06541.001 A protein called Dystrophin plays a key role in maintaining the structural integrity of muscle cells as they contract and relax. Mutations in the gene that encodes Dystrophin can cause several different types of muscular dystrophy, a group of diseases in which muscle progressively weakens. Some mutations in Dystrophin can lead to mild symptoms that may affect the quality of life but are not life threatening. However, in more serious cases, patients lose the ability to walk in childhood and have shortened life expectancy. There is no cure for these diseases, and there are still big gaps in our understanding of how Dystrophin works, which makes it more difficult to develop efficient therapies. The zebrafish is often used as a model to study muscular dystrophies. In this study, Bajanca et al. introduced human Dystrophin into zebrafish muscle cells and analysed its behaviour using a combination of mathematical modelling and a method known as ‘fluorescence recovery after photobleaching’. In these experiments, the human Dystrophin was attached to a tag that fluoresces green under a microscope, which allowed it to be easily seen and be followed in real time inside the cells of live animals. Bajanca et al. observed that Dystrophin could either remain firmly associated with the membrane that surrounds the cell over long periods of time or interact briefly with the membrane. Bajanca et al. carried out further experiments with the Dystrophin protein naturally found in zebrafish and observed that it behaved in a similar manner to the human protein, suggesting this behaviour is likely to be important for the ability of the protein to work. Bajanca et al.'s findings reveal that Dystrophin displays complex behaviour in living muscle cells. The fact that some Dystrophin molecules are firmly attached to the membrane support previous findings that this protein provides mechanical stability to the cells. However, the discovery that there is a group of more mobile Dystrophin molecules within muscle cells suggests that this protein may also play other roles. Therefore, these findings open a new avenue for research that may contribute to the development of new therapy approaches in future. DOI: http://dx.doi.org/10.7554/eLife.06541.002