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

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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.