TEAM

Muscle Dynamics

Group leader : F. Schnorrer

We investigate the biomechanics of muscle morphogenesis, particularly focusing on how the contractile apparatus develops its quasi crystalline regularity across the muscle.

PUBLIC SUMMARY
Muscles are the major force producing organs in our body. They enable us to climb high peaks, ran marathons or swim across the Channel. All muscles share a similar contractile machine called a sarcomere. Large muscles contain thousands of these sarcomeres arrayed in series to form long chains called myofibrils. These linear myofibrils span across the entire muscle cell.

Drosophila flight muscles (blue) house many parallel myofibrils of many hundred sarcomeres.

Drosophila flight muscles (blue) house many parallel myofibrils of many hundred sarcomeres.

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 to convert myotubes to myofibers. The biomechanics of these myofibrils is tuned to the particular functional needs of each muscle type in the body.

Flight muscle morphogenesis and sarcomere scheme.

Flight muscle morphogenesis and sarcomere scheme.

We combine the advantages of the Drosophila genetic toolbox 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?

 

FOR SCIENTISTS
Drosophila adult muscle development

Movie showing the flight muscles (green), which 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 (Weitkunat et al. 2014).

Quantifying forces across molecules in vivo

(A-C) Functional principle of FRET-based tension sensors. We insert these sensors into proteins to determine the force across proteins by quantifying the life time of the fluorescence (FLIM). (D) A tissue under mechanical strain: flight muscles (green) pull on stably attached to tendons (red). Regular myofibrils (green) start to assemble.

(A-C) Functional principle of FRET-based tension sensors. We insert these sensors into proteins to determine the force across proteins by quantifying the life time of the fluorescence (FLIM). (D) A tissue under mechanical strain: flight muscles (green) pull on stably attached to tendons (red). Regular myofibrils (green) start to assemble.

During myofibril and sarcomere assembly a quasi-crystalline pattern of myosin motors (thick filaments), actin tracks (thin filaments) linked by gigantic titin molecules (C-filament) is built in every muscle. Using in vivo imaging we found that myofibrillar pattern organises simultaneously across the entire muscle fiber after tension has been built up. We hypothesize that tension is used as a molecular compass to direct myofibril assembly, confirming that each myofibril spans across the entire muscle fiber. This guarantees effective muscle contractions in the mature muscle. We are testing this hypothesis by applying molecular force sensors, which enable us to quantify tension across individual molecules within developing myofibrils. We are also applying novel high-resolution microscopy techniques to monitor myofibril assembly at the nanoscale.

To walk or to fly

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.

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. Note the transformation of flight muscle to leg muscle morphology after loss of spalt.

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 (Schönbauer et al. 2011). We found that downstream of Spalt the muscle specific splicing program is regulated by the RNA binding protein Arrest (Bruno) (Spletter et al. 2015). 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 specific alternative splicing as well as the individual impact of the regulated components on myofibril assembly and muscle biomechanics.

CRISPRing the fly

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.

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.

We have developed an efficient two-step genome engineering protocol to manipulate any Drosophila gene of interest at its endogenous locus (Zhang et al. 2014). 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 two attP sites. In step 2, we replace the inserted marker with any DNA of choice using ΦC31-mediate 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

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

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 the in vivo dynamics and subcellular localisation of the tagged protein. 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.


Main publications

PUBLICATION

Mechanical forces during muscle development

Lemke SB, Schnorrer F.
Mech Dev. 2017 Apr;144(Pt A):92-101. PMID: 27913119

PUBLICATION

Mechanical tension and spontaneous muscle twitching precede the formation of cross-striated muscle in vivo.

Weitkunat M, Brasse M, Bausch AR, Schnorrer F.
Development. 2017 Apr 1;144(7):1261-1272. http://www.pubmed.com/28174246/

PUBLICATION

A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila.

Kaya-Çopur A, Schnorrer F.
Methods Mol Biol. 2016;1478:117-143. http://www.pubmed.com/27730578

PUBLICATION

A genome-wide resource for the analysis of protein localisation in Drosophila.

Sarov M, Barz C, Jambor H, Hein MY, Schmied C, Suchold D, Stender B, Janosch S, Kj VV, Krishnan RT, Krishnamoorthy A, Ferreira IR, Ejsmont RK, Finkl K, Hasse S, Kämpfer P, Plewka N, Vinis E, Schloissnig S, Knust E, Hartenstein V, Mann M, Ramaswami M, VijayRaghavan K, Tomancak P, Schnorrer F.
Elife. 2016 Feb 20;5. pii: e12068. PMID: 26896675

PUBLICATION

The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle.

Spletter ML, Barz C, Yeroslaviz A, Schönbauer C, Ferreira IR, Sarov M, Gerlach D, Stark A, Habermann BH, Schnorrer F.
EMBO Rep. 2015 Feb;16(2):178-91. PMID: 25532219

PUBLICATION

Tension and force-resistant attachment are essential for myofibrillogenesis in Drosophila flight muscle.

Weitkunat M, Kaya-Çopur A, Grill SW, Schnorrer F.
Curr Biol. 2014 Mar 31;24(7):705-16. PMID: 24631244

PUBLICATION

Spalt mediates an evolutionary conserved switch to fibrillar muscle fate in insects

Schönbauer C, Distler J, Jährling N, Radolf M, Dodt HU, Frasch M, Schnorrer F.
Nature. 2011 Nov 16;479(7373):406-9. PMID: 22094701

PUBLICATION

Systematic genetic analysis of muscle morphogenesis and function in Drosophila

Schnorrer F, Schönbauer C, Langer CC, Dietzl G, Novatchkova M, Schernhuber K, Fellner M, Azaryan A, Radolf M, Stark A, Keleman K, Dickson BJ.
Nature. 2010 Mar 11;464(7286):287-91. PMID: 20220848

PUBLICATION

A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.

Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M, Gasser B, Kinsey K, Oppel S, Scheiblauer S, Couto A, Marra V, Keleman K, Dickson BJ.
Nature. 2007 Jul 12;448(7150):151-6. PMID: 17625558

PUBLICATION

The transmembrane protein Kon-tiki couples to Dgrip to mediate myotube targeting in Drosophila

Schnorrer F, Kalchhauser I, Dickson BJ.
Dev Cell. 2007 May;12(5):751-66. PMID: 17488626
Other publications

PUBLICATION

AIDing-targeted protein degradation in Drosophila.

Zhang X, Schnorrer F.
FEBS J. 2017 Apr;284(8):1178-1181. PMID: 28382696

PUBLICATION

Slit cleavage is essential for producing an active, stable, non-diffusible short-range signal that guides muscle migration.

Ordan E, Brankatschk M, Dickson B, Schnorrer F, Volk T.
Development. 2015 Apr 15;142(8):1431-6. PMID: 25813540

PUBLICATION

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

Klein P, Müller-Rischart AK, Motori E, Schönbauer C, Schnorrer F, Winklhofer KF, Klein R.
EMBO J. 2014 Feb 18;33(4):341-55. PMID: 24473149

PUBLICATION

Transcriptional regulation and alternative splicing cooperate in muscle fiber-type specification in flies and mammals

Spletter ML, Schnorrer F.
Exp Cell Res. 2014 Feb 1;321(1):90-8. PMID: 24145055

PUBLICATION

A guide to study Drosophila muscle biology.

Weitkunat M, Schnorrer F.
Methods. 2014 Jun 15;68(1):2-14. PMID: 24625467

PUBLICATION

A simple protocol to efficiently engineer the Drosophila genome by TALENs

Zhang X., Ferreira I., and Schnorrer F
Methods, 69, 32-7 (2014)

PUBLICATION

The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures.

Hatan M, Shinder V, Israeli D, Schnorrer F, Volk T.
J Cell Biol. 2011 Jan 24;192(2):307-19. PMID: 21242289

PUBLICATION

Three-dimensional reconstruction and segmentation of intact Drosophila by ultramicroscopy.

Jährling N, Becker K, Schönbauer C, Schnorrer F, Dodt HU.
Front Syst Neurosci. 2010 Feb 8;4:1. PMID: 20204156

PUBLICATION

In vivo RNAi rescue in Drosophila melanogaster with genomic transgenes from Drosophila pseudoobscura

Langer CC, Ejsmont RK, Schönbauer C, Schnorrer F, Tomancak P.
PLoS One. 2010 Jan 28;5(1):e8928. PMID: 20126626

PUBLICATION

High-resolution, high-throughput SNP mapping in Drosophila melanogaster

Chen D, Ahlford A, Schnorrer F, Kalchhauser I, Fellner M, Viràgh E, Kiss I, Syvänen AC, Dickson BJ
Nat Methods. 2008 Apr;5(4):323-9. PMID: 18327265

PUBLICATION

Ultramicroscopy: 3D reconstruction of large microscopical specimens

Becker K, Jährling N, Kramer ER, Schnorrer F, Dodt HU.
J Biophotonics. 2008 Mar;1(1):36-42. PMID: 19343633

PUBLICATION

Positional cloning by fast-track SNP-mapping in Drosophila melanogaster

Schnorrer F, Ahlford A, Chen D, Milani L, Syvänen AC.
Nat Protoc. 2008;3(11):1751-65. PMID: 18948975

PUBLICATION

The gammaTuRC components Grip75 and Grip128 have an essential microtubule-anchoring function in the Drosophila germline

Vogt N, Koch I, Schwarz H, Schnorrer F, Nüsslein-Volhard C
Development. 2006 Oct;133(20):3963-72. PMID: 16971473

PUBLICATION

RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation

Grosshans J, Wenzl C, Herz HM, Bartoszewski S, Schnorrer F, Vogt N, Schwarz H, Müller HA
Development. 2005 Mar;132(5):1009-20. PMID: 15689371

PUBLICATION

Muscle building; mechanisms of myotube guidance and attachment site selection

Schnorrer F, Dickson BJ
Dev Cell. 2004 Jul;7(1):9-20. PMID: 15239950

PUBLICATION

Axon guidance: morphogens show the way

Schnorrer F, Dickson BJ
Curr Biol. 2004 Jan 6;14(1):R19-21. PMID: 14711429

PUBLICATION

Gamma-tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during drosophila oogenesis

Schnorrer F, Luschnig S, Koch I, Nüsslein-Volhard C.
Dev Cell. 2002 Nov;3(5):685-96. PMID: 12431375

PUBLICATION

The molecular motor dynein is involved in targeting swallow and bicoid RNA to the anterior pole of Drosophila oocytes.

Schnorrer F, Bohmann K, Nüsslein-Volhard C.
Nat Cell Biol. 2000 Apr;2(4):185-90. PMID: 10783235

PUBLICATION

Oligomerisation of Tube and Pelle leads to nuclear localisation of dorsal

Grosshans J, Schnorrer F, Nüsslein-Volhard C.
Mech Dev. 1999 Mar;81(1-2):127-38.

PUBLICATION

The cellular localization of the murine serine/arginine-rich protein kinase CLK2 is regulated by serine 141 autophosphorylation

Nayler O, Schnorrer F, Stamm S, Ullrich A.
J Biol Chem. 1998 Dec 18;273(51):34341-8. PMID: 9852100

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  Celine Guichard Vincent Loreau Nuno Luis  
Frank Schnorrer
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Frank Schnorrer

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Jérôme Avellaneda

MSc student

Celine Guichard
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Celine Guichard

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Vincent Loreau
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Vincent Loreau

PhD student

Nuno Luis
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Nuno Luis

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christophe Pitaval

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