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VIII - Kassel/Di Ventura

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

Participants:

1) Barbara Di Ventura (PI; University of Heidelberg), coworker:

2) Olivier Kassel (PI; Karlsruhe Institute of Technology, KIT), coworker

Abstract:

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

Skeletal muscle regeneration relies on the process of myogenesis. Upon muscle injury, resident adult muscle stem cells are activated and proliferate to give rise to a population of progenitor cells, the myoblasts. Myoblasts then further differentiate into myocytes, fuse to form myotubes which finally mature into muscle fibres. Myogenesis is regulated by various signalling pathways that must be activated or inhibited in a highly temporally controlled manner. Interestingly, the activity of the cytosolic protein kinase PKR is dynamically regulated and appears to play a crucial role in myogenesis. However, the precise function of PKR at specific stages of differentiation is unclear. Our preliminary work has shown that PKR regulates the expression of the transcriptional co-regulator nTRIP6, which plays a key role in the temporal control of differentiation. Given the complex temporal regulation of myogenesis, the use of conventional tools to study the precise contribution of PKR to nTRIP6 regulation and to stage-specific differentiation poses a major challenge. The high temporal and spatial precision of optogenetics provides an ideal solution, as it would allow the manipulation of PKR in the cytosol of selected cells, at specific time points during differentiation. We have developed an innovative tool, called LEXY, for controlling by light the export of proteins from the nucleus into the cytosol. Furthermore, we have successfully used the split intein system to reassemble two protein fragments into a functional protein. The aim of this project is to develop a new optogenetic tool combining LEXY and split inteins to study the function of PKR in myogenesis. LIPS (Light-Induced Protein trans-Splicing) will allow a tight control of the function of endogenous PKR by blue light. To this aim, we will engineer an inhibitor and an activator of PKR and precisely control their activity by LIPS, in selected cells, at specific stages of myoblast differentiation. We will investigate PKR function in myogenesis in vitro and in vivo in a zebrafish model of muscle regeneration. More generally, our innovative tool will allow the precise dissection of the temporal and spatial control of myogenesis, a system not amenable so far to optogenetics.

 

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

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: 27746.

Niopek D, Wehler P, Roensch J, Eils R, Di Ventura B (2016) Optogenetic control of nuclear protein export. Nat Commun 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 Commun 5: 4404

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

Diefenbacher M, Sekula S, Heilbock C, Maier JV, Litfin M, van Dam H, Castellazzi M, Herrlich P & Kassel O (2008) Restriction to Fos family members of Trip6-dependent coactivation and glucocorticoid receptor-dependent trans-repression of activator protein-1. Mol Endocrinol 22: 1767–1780

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