Shedding light on myogenesis: using optogenetics to investigate myoblast differentiation and muscle regeneration

骨骼肌再生依赖于肌生成过程。在肌肉损伤后，常驻的成体肌肉干细胞被激活，并产生增殖祖细胞群，即成肌细胞。然后这些细胞进一步分化成固定的前体细胞，肌细胞，融合形成肌管，最终成熟为肌纤维。肌生成受多种信号通路的调控，这些信号通路的激活或抑制受到高度的时间控制。此外，其中一些通路在肌肉形成的不同阶段发挥不同的功能。例如，雷帕霉素复合物1 （mTORC1）通路的机制靶点是动态调节的，似乎在整个肌生成过程中起着至关重要的作用。由于其复杂的时间调控，研究mTORC1在特定阶段的精确功能需要很高的时间和空间精度，例如光遗传学。在SPP的第一阶段，我们描述了肌生成过程中mTORC1活性的动态。此外，我们已经在体外和斑马鱼胚胎肌肉工具中验证了以mTORC1激活剂RHEB的组成活性和显性负突变的形式操纵内源性mTORC1活性的方法。令人惊讶的是，我们的结果表明mTORC1对肌肉生长的影响可能是由复合体的一个尚未表征的核功能介导的。与此同时，我们在开发一种创新的基于AsLOV2结构域的光遗传工具和光诱导蛋白反式剪接方面取得了实质性进展，我们称之为LIPS。LIPS允许通过蓝光控制分裂间质介导的重组，将感兴趣蛋白（POI）的两个无活性片段重组为功能蛋白。考虑到我们的第一个设计存在大量的漏洞，即重构已经在黑暗中发生，我们已经实现了其他几个控制层。这些新设计的原理验证实验表明了系统的鲁棒性。在第二阶段，我们将把LIPS应用于RHEB突变体，以研究mTORC1在体外肌肉形成和斑马鱼胚胎肌肉再生中的阶段性作用。此外，我们将进一步开发LIPS以增加其密封性，并允许局部光诱导POI重构具有亚细胞空间精度。最后，使用最合适的LIPS设计，我们将解剖mTORC1在斑马鱼胚胎肌肉生长调节中的胞质和细胞核功能。我们还将研究进一步poi的光介导重构，并展示LIPS的一般适用性。因此，LIPS将是光遗传学工具箱的重要补充。

Skeletal muscle regeneration relies on the process of myogenesis. Upon muscle injury, resident adult muscle stem cells are activated and give rise to a population of proliferating progenitor cells, the myoblasts. These then further differentiate into committed precursor cells, the myocytes, that fuse to form myotubes, which finally mature into muscle fibres. Myogenesis is regulated by various signalling pathways that are activated or inhibited in a highly temporally controlled manner. Furthermore, some of these pathways exert different functions in different stages of myogenesis. For example the mechanistic target of rapamycin-complex 1 (mTORC1) pathway is dynamically regulated and appears to play a crucial role in the overall process of myogenesis. Given its complex temporal regulation, studying the precise function of mTORC1 at specific stages requires high temporal and spatial precision, such as that provided by optogenetics. During the first period of the SPP, we have characterised the dynamics of mTORC1 activity during myogenesis. Furthermore, we have validated in vitro and in the zebrafish embryo muscle tools to manipulate endogenous mTORC1 activity, in the form of a constitutively active and a dominant negative mutant of the mTORC1 activator RHEB. Surprisingly, our results suggest that the effect of mTORC1 on muscle growth might be mediated by a yet uncharacterized nuclear function of the complex. In parallel, we have made substantial progress towards the development of an innovative AsLOV2 domain-based optogenetic tool for Light-Induced Protein trans-Splicing which we called LIPS. LIPS allows the control by blue light of the split intein-mediated reconstitution of two inactive fragments of a protein of interest (POI) into a functional protein. Given that our first designs suffered from substantial leakiness, i.e. reconstitution occurring already in the dark, we have implemented several other layers of control. Proof-of-principle experiments with these new designs showed the robustness of the system. In the second period, we will apply LIPS to the RHEB mutants in order to investigate the stage-specific role of mTORC1 in myogenesis in vitro and in muscle regeneration in the zebrafish embryo. Moreover, we will further develop LIPS to increase its tightness and to allow localized light-induced POI reconstitution with sub-cellular spatial precision. Finally, using the most appropriate LIPS design, we will dissect the cytosolic and nuclear functions of mTORC1 in the regulation of muscle growth in the zebrafish embryo. We will also investigate the light-mediated reconstitution of further POIs and show the general applicability of LIPS. Therefore, LIPS will represent an important addition to the optogenetic toolbox.