Shedding light on myogenesis: using optogenetics to investigate myoblast differentiation and muscle regeneration
1) Barbara Di Ventura (PI; University of Heidelberg), coworker:
2) Olivier Kassel (PI; Karlsruhe Institute of Technology, KIT), coworker
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.
Logic of interactions within the joint project
The Kassel lab investigates the temporal control of myogenic differentiation. The challenge is to specifically manipulate signalling components with a high temporal and spatial precision. The Di Ventura lab develops innovative optogenetic tools particularly suited to address this challenge. Both labs work together to adapt and further develop these tools for the particular demands of the biological system, with the global aim to dissect the temporal control of myogenesis and of muscle regeneration in vivo.
Publications Kassel/DiVentura relevant for the proposal
Wehler P, Di Ventura B (2019) Engineering optogenetic control of endogenous p53 protein levels. Appl Sci 9(10): 2095
Di Ventura B, Mootz HD (2018) Switchable inteins for conditional protein splicing. Biol chem 7:10624
Wehler P, Niopek D, Eils R, Di Ventura B (2016) Optogenetic control of nuclear protein import in living cells using light-inducible nuclear localization signals (LINuS). Curr Protoc Chem Biol 8:131-145
Niopek D, Wehler P, Roensch J, Eils R, Di Ventura B (2016) Optogenetic control of nuclear protein export. Nat Comm 7:10624
Waldhauer MC, Schmitz SN, Ahlmann-Eltze C, Gleixner JG, Schmelas CC, Huhn AG, Bunne C, Büscher M, Horn M, Klughammer N, Kreft J, Schäfer E, Bayer PA, Krämer SG, Neugebauer J, Wehler P, Mayer MP, Eils R, Di Ventura B (2015) Backbone circularization of Bacillus subtilis family 11 xylanase increases its thermostability and its resistance against aggregation. Mol BioSyst, doi: 10.1039/C5MB00341E
Niopek D, Benzinger D, Roensch J, Draebing T, Wehler P, Eils R, Di Ventura B (2014) Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat Comm 5:4404
Kemler D, Dahley O, Roßwag S, Litfin M, Kassel O (2016) The LIM domain protein nTRIP6 acts as a co-repressor for the transcription factor MEF2C in myoblasts. Sci Rep 6:7746.
Diefenbacher ME, Reich D, Dahley O, Kemler D, Litfin M, Herrlich P, Kassel O (2014) The LIM Domain Protein nTRIP6 Recruits the Mediator Complex to AP-1-Regulated Promoters. PLoS ONE 9: e97549
Röder IV, Strack S, Reischl M, Dahley O, Khan MM, Kassel O, Zaccolo M, Rudolf R (2012) Participation of Myosin va and pka type I in the regeneration of neuromuscular junctions. PLoS ONE 7: e40860
Diefenbacher ME, Litfin M, Herrlich P, Kassel O (2010) The nuclear isoform of the LIM domain protein Trip6 integrates activating and repressing signals at the promoter-bound glucocorticoid receptor. Mol Cell Endocrinol 320: 58-66
Kassel O, Schneider S, Heilbock C, Litfin M, Göttlicher M, Herrlich P (2004) A nuclear isoform of the focal adhesion LIM-domain protein Trip6 integrates activating and repressing signals at AP- 1- and NF-kappaB-regulated promoters. Genes Dev 18: 2518-2528