Contact

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Dr. Frank Schnorrer
Group Leader
Phone:+49 89 8578 - 2434

MPI of Biochemistry, Am Klopferspitz 18, 82152 Martinsried

www.biochem.mpg.de/schnorrer

Muscle Dynamics

Research Group "Muscle Dynamics"

The Biomechanics of Muscle Morphogenesis

Muscles are the major force producing organs of our body. They enable us to climb mountains, run marathons or swim across the Channel. The contractile units of all muscles are called sarcomeres. These are arrayed into linear chains known as myofibrils that span across each muscle fiber. The coordinated movement of myosin motors (thick filaments) along actin tracks (thin filaments) shortens each sarcomere and thus produces muscle force.

 

Muscle morphogenesis is a multi-step process. Myoblasts proliferate, migrate and fuse to myotubes. Myotube tips search for tendon cells, to which they establish stable force resistant attachments. Finally, myofibrils and sarcomeres assemble within each myofiber. The biomechanics of each myofiber is tuned to the functional needs of each particular muscle type in the body.

 

<p>Flight muscle morphogenesis and sarcomere scheme.</p> Zoom Image

Flight muscle morphogenesis and sarcomere scheme.

 

We combine the advantages of the Drosophila genetic tool box with high resolution in vivo imaging to functionally dissect the biomechanics of muscle morphogenesis. Questions we are particularly fascinated by include:

 

  • How do muscles establish force resistant attachments to tendons?
  • How are contractile myofibrils and sarcomeres built?
  • How is functional muscle diversity achieved?


Drosophila adult muscle development

Flight muscles (green) are attaching to tendons (orange), tension is built up and tendons form long extensions.

We use the Drosophila adult muscles, in particular the flight muscles to image muscle-tendon attachment and myofibrillogenesis in intact developing animals. We found that myotubes first attach to tendon cells at both myotube tips and only after being stably attached, myofibrillogenesis is triggered throughout the entire muscle. Using in vivo laser cutting experiments we discovered that mechanical tension is generated after muscles have attached to tendons. Interestingly, this tension build-up is required for ordered myofibrillogenesis. We hypothesized that tension is used as a molecular compass to direct myofibril assembly, assuring that each myofibril spans across the entire developing muscle fiber. This guarantees effective muscle contractions in the future. We are testing this hypothesis by applying molecular force sensors, which enable us to quantify tension across individual molecules within developing myofibrils.

 

<p>Flight muscles (green) stably attached to tendons (red). Myofibrils start to assemble.</p> Zoom Image

Flight muscles (green) stably attached to tendons (red). Myofibrils start to assemble.

 

To walk or to fly

<p>Wild type fibrillar flight muscles but not tubular leg muscles express a particular Titin-related isoform (green), which is lost upon knock-down of <em>spalt</em> or <em>arrest</em>.</p> Zoom Image

Wild type fibrillar flight muscles but not tubular leg muscles express a particular Titin-related isoform (green), which is lost upon knock-down of spalt or arrest.

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Flight muscles harbour a specialised ‘fibrillar’ contractile apparatus to power fast wing oscillations at 200 Hz. This requires a very stiff muscle fiber type, related to vertebrate heart muscle. In contrast, slowly moving leg muscles display a tubular muscle architecture, closely related to striated vertebrate body muscles. We identified the conserved transcription factor Spalt as myofibril selector gene that instructs fibrillar muscle morphogenesis by inducing expression and alternative splicing of key sarcomeric components. We found that downstream of Spalt the muscle specific splicing program is regulated by the RNA binding protein Arrest (Bruno). Thus as in vertebrate muscle, the biomechanics of a fiber-type specific contractile apparatus is regulated by transcription and alternative splicing of specific sarcomeric components. Currently, we are investigating the mechanism of fiber-type specifc alternative splicing as well as the individual impact of the regulated components on myofibril assembly and muscle biomechanics.

 

 CRISPRing the fly

<p>Endogenous Spalt protein tagged with GFP (green) by CRISPR-mediated HDR followed by RMCE is functional and localises to flight muscle nuclei. Trachea in white, myofibrils in red.</p> Zoom Image

Endogenous Spalt protein tagged with GFP (green) by CRISPR-mediated HDR followed by RMCE is functional and localises to flight muscle nuclei. Trachea in white, myofibrils in red.

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We have developed an efficient two-step genome engineering protocol to manipulate any Drosophila gene of interest at its endogenous locus. In step 1, we apply CRISPR/Cas9-mediated homology directed repair to replace a target region with a selectable marker (red fluorescent eyes), which is flanked by 2 attP sites. In step 2, we replace the inserted marker with any DNA of choice using ΦC31-mediated cassette exchange (RMCE). This enables flexible and efficient engineering of the locus, for example to generate a tagged allele, or to insert point mutations of choice.

 

The Drosophila TransgeneOme

<p>LamininA-GFP expression in the adult thorax. Trachea and motor neurons are particularly well visible.</p> Zoom Image

LamininA-GFP expression in the adult thorax. Trachea and motor neurons are particularly well visible.

 

In collaboration with the Tomancak, Sarov and VijayRaghavan labs we generated a genome-wide resource for the analysis of protein localisation in Drosophila. We tagged 10,000 proteins by inserting GFP into large genomic FlyFos clones. For about 900 clones we generated transgenic flies, which can be used to assess in vivo dynamics and subcellular localisation of the tagged proteins. For many of the tagged proteins functional antibodies or live imaging tools had not been available before. All transgenic lines are available from VDRC and thus can be used by the fly community. A preprint of the manuscript can be found here:

http://biorxiv.org/content/early/2015/10/04/028308

 
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