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

揭示肌生成：利用光遗传学研究成肌细胞分化和肌肉再生

• 批准号: 315384510
• 负责人: Dr. Olivier Kassel
• 金额: --
• 依托单位: Zentrum für Biologische Signalstudien (BIOOS)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/315384510?language=en
• 关键词: Shedding; light; myogenesis; using; optogenetics

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.

骨骼肌再生依赖于肌肉发生的过程。肌肉损伤后，成体肌肉干细胞被激活，并产生一组增殖的前体细胞，即成肌细胞。然后，这些细胞进一步分化为固定的前体细胞，即肌细胞，肌细胞融合形成肌管，肌管最终成熟为肌肉纤维。肌肉发生受到各种信号通路的调控，这些信号通路以高度时间控制的方式被激活或抑制。此外，这些途径中的一些在肌肉发生的不同阶段发挥着不同的功能。例如，雷帕霉素-复合体1(MTORC1)途径的机制靶点是动态调节的，似乎在整个肌肉发生过程中发挥着关键作用。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，如光遗传学所提供的。在SPP的第一阶段，我们已经表征了肌发生过程中mTORC1活动的动态。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的结构性活性和显性负突变体的形式来操纵内源性mTORC1活性。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通过尚未确定的复合体的核功能来调节的。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用于光诱导蛋白质反式剪接方面取得了实质性进展，我们称之为LIPs。Lips可以通过蓝光控制内含素介导的分裂，将感兴趣蛋白(POI)的两个非活性片段重组为功能蛋白。考虑到我们的第一个设计遭受了实质性的泄漏，即重建已经在黑暗中进行，我们已经实施了其他几层控制。这些新设计的原理验证实验表明了系统的健壮性。在第二阶段，我们将把LIPs应用于Rheb突变体，以研究mTORC1在体外肌肉发生和斑马鱼胚胎肌肉再生中的阶段特异性作用。此外，我们将进一步发展嘴唇，以增加其紧密性，并允许局部光诱导的POI重建以亚细胞的空间精度。最后，使用最合适的嘴唇设计，我们将剖析mTORC1在斑马鱼胚胎肌肉生长调节中的胞浆和核功能。我们还将研究光介导的更多POI的重建，并展示LIP的普遍适用性。因此，嘴唇将是光遗传工具箱中的一个重要补充。