PDP2018-01

Muscle building: bridging molecular order to macroscopic morphogenesis

Abstract

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.

Keywords

Muscle; sarcomere; Drosophila; mechanical tension; polarization resolved microscopy; stem cells

Objectives

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.

Interdisciplinarity

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.

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