Muscle building: bridging molecular order to macroscopic morphogenesis
Sarcomeres are force‐generating and load-bearing devices of muscle. Each sarcomere contains a pseudo-crystalline order of bipolar actin, myosin and titin filaments. During development sarcomeres assemble into long periodic chains called myofibrils that span the entire muscle. How sarcomeric components organize into molecularly ordered structures (molecular order) while assembling long myofibrils (macroscopic order) is an important unsolved question. Here, we will use genetic, mechanical and advanced imaging approaches to tackle this question. The recruited postdoc will implement a live imaging approach based on polarization resolved microscopy to probe the molecular order of sarcomeric components in developing Drosophila muscles in vivo and human iPS-derived muscles in vitro, and test the role of tension for order generation. The originality of the project relies on the integration of quantitative approaches to bridge from the molecular to the macroscopic scale to explain how large muscle fibers build their contractile machines.
Muscle; sarcomere; Drosophila; mechanical tension; polarization resolved microscopy; stem cells
Aim 1: The PostDoc will develop and apply live probes for measuring molecular order of actin, myosin and titin and map the ordering process locally and across myofibrils in Drosophila and human muscles.
Aim 2: The PostDoc will manipulate tension optically (laser severing) or genetically (titin mutations) and quantify the consequences for order generation. This will be essential to bridge from protein order and mechanical tension to macroscopic morphogenesis of myofibrils.
Proposed approach (experimental / theoretical / computational)
Biology: We quantified build-up of actin order in Drosophila muscles using fixed probes (Loison et al., PLoS Biology 2018). Here, we will design new live probes to quantify order dynamics of muscle myosin and titin, and we will apply live actin probes established in the Brasselet lab (Mavrakis et al., in prep). Our strategy will truncate linkers or introduce rigid linkers to preserve molecular order or the protein. In particular, inserting a fluorophore into titin, which is under strong mechanical tension, will test the link between order and tension. CRISPR-based tools to insert probes are well established the Schnorrer lab (Zhang et al., G3 2014).
Physics: We will use polarization resolved microscopy to probe molecular order, using a fast confocal imaging system combined with a polarization-resolved system, developed by the Brasselet lab (Kress et al., Biophysical J 2013). We will optically manipulate tension using laser microlesions. To our knowledge, this study will be the first attempt to probe and manipulate the dynamics of molecular order in living tissues.
The success of this project depends on a tight collaboration between the group of Frank Schnorrer, a biologist at the IBDM at the Luminy campus in Marseille, and the group of Sophie Brasselet, a physicist at the Institute Fresnel in Marseille. This interdisciplinary consortium provides complementary expertise in genetics, molecular biology, mechanics and advanced quantitative imaging. The two group leaders have a long experience in interdisciplinary research and already collaborated successfully (Loison et al., PLoS Biology 2018). The proposed project is only feasible by continuous interactions between both collaborating groups in order to link the biology and genetics of myofibrillogenesis with a molecular understanding of force-‐driven self-‐organization of supramolecular complexes across different dimensions -‐ micrometer sized sarcomeres and centimeter large muscle fibers. Establishing this link in Drosophila muscles in vivo and human iPS-‐derived muscles in vitro should enable future modeling approaches of myofibrillogenesis.