Vincent GACHE – Myonuclear domain & microtubule network organisation
In the normal course of muscle development or regeneration, nuclei are first found in the center of the myotube before migrating to the periphery of the mature skeletal myofiber. It has long been hypothesized that nuclei are normally positioned to the periphery of the fiber to stay out of the way of the contractile machinery. In fact, nuclei mispositioned in the center of myofibers are a hallmark of a class of muscular diseases called centronuclear myopathies. Additionally, there is growing support for direct connection between regulation of nuclear positioning and myonuclear domains establishment, microtubule architecture maintenance and normal function of muscles. My team goal is to understand how microtubule network is controlled and how nuclei are positioned in developing muscle, and how that relates to diseases.
Aim 1: Elucidate molecular mechanisms regulating myonuclear positioning through MAP7 during muscle development and the progression of centronuclear myopathies.
The microtubule associated protein Ensconsin/MAP7 and the associated kinesin-1 Kif5B are essential regulators of nuclear position in Drosophila and mammalian myotubes. MAP7 functions by interacting with both microtubules and the conventional kinesin-1 Kif5B to maintain particular microtubule architecture. However, nothing is known about how these interactions are regulated during myotube/myofibers development. Two potential regulators are currently investigated to test their implication in myonuclear domains establishment thought MAP7.
Aim 2: Determine mechanisms of microtubule regulation that control myonuclear positioning.
Our previous studies show the importance of the integrity/availability of the microtubule network to position nuclei in myotubes. We are currently screening for microtubule related protein linked to myonuclear positioning using C2C12 cells and Primary cells culture. Few candidates are in the course of investigation.
Aim 3: identification of molecules targeting nuclei spreading in early myotubes.
Previous data demonstrate the importance of precocious nuclei spreading in myotubes to correctly establish myonuclear domains in mature fibers. Our preliminary data show that in a CNM mouse model, myotubes formed from primary myoblasts have a significant reduction in their ability to correctly spaced and aligned nuclei. As this phenotype is an early marker of myofibers in which myonuclear domains establishment will be impaired, we hypothesize that identifying new molecules that restore nuclei spreading in early phase of myotube formaton will help to restore myonuclear domains establishment in mature fibers and will constitute a challenging therapeutic way to treat CMN. The high throughput screening proposed in this project relies on the adaptation of a miniaturized cell-based assay allowing the detection of a restoration of myonuclear domains by automated imaging and image analysis.
Alexandre MÉJAT – Genome folding & chromatin organization
Eukaryotic genomes must accommodate two diametrically opposite requirements: on the one hand, the compaction of ~2 m of genomic DNA into a sphere with a diameter of ~10 µm and, on the other hand, the accessibility of DNA to a wide variety of molecular machines involved in DNA replication, transcription, recombination and repair. Three-dimensional nuclear organization is therefore clearly critical for cellular functions. The nuclear architecture in a differentiated cell results from several factors such as the linear sequence of a gene; long distance interactions between coregulated genes; epigenetic modifications altering DNA (methylation) or histone tails (acetylation, methylation, etc); and interactions between the genome and nuclear envelope components or proteinaceous nuclear sub-compartments. However, the complex interplay between them is not yet elucidated.
The muscle fibre is an ideal model to probe how cellular function is linked to nuclear architecture.
Muscle differentiation is a well-characterized model of cellular differentiation with mononucleated myoblasts irreversibly withdrawing from the cell cycle and differentiating into myocytes that fuse to form multinucleated terminally differentiated myotubes in an ordered series of events. In vivo, each multinucleated fibre is contacted by the axon of a motor neuron initiating the formation of a highly specialized structure dedicated to muscle / nerve communication, called the neuromuscular junction (NMJ). Motor innervation leads to the restriction of expression of synapse-specific genes to a few nuclei located directly under the synapse. Thus, the resulting mature muscle fibre is unique in mammals as it is the only multinucleated cell in which different genetic programs are expressed in different nuclei of the same cell. Finally, many mutations in nuclear envelope components have been linked to several human muscular dystrophies, suggesting that nuclear organization is critical for muscle differentiation/function. However, little is known about how nuclear architecture and dynamics change during muscle differentiation and how nuclear architecture contributes to muscle function.
- Amphiphysin 2 Orchestrates Nucleus Positioning and Shape by Linking the Nuclear Envelope to the Actin and Microtubule Cytoskeleton.
D'Alessandro M, Hnia K, Gache V, Koch C, Gavriilidis C, Rodriguez D, Nicot AS, Romero NB, Schwab Y, Gomes E, Labouesse M, Laporte J. Dev Cell (2015) .
- Moving and positioning the nucleus in skeletal muscle - one step at a time
Cadot B, Gache V, Gomes ER. Nucleus (2015) .
- N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy.
Falcone S, Roman W, Hnia K, Gache V, Didier N, Lainé J, Auradé F, Marty I, Nishino I, Charlet-Berguerand N, Romero NB, Marazzi G, Sassoon D, Laporte J, Gomes ER. EMBO Mol Medicine (2014) .
- Nuclear movement during myotube formation is microtubule and dynein dependent and is regulated by Cdc42, Par6 and Par3.
Gache,V.*, Cadot, B*., Vasyutina, E., Falcone, S., Birchmeier, C., Gomes E. EMBO Reports (2012) .
- MAP and Kinesin dependent nuclear positioning is required for skeletal muscle function.
Gache,V.*, Metzger,T.*, Xu,M., Cadot, B., Folker,E.S., Richardson, B.E., Gomes E., Baylies. M. Nature (2012) .
- Xenopus meiotic microtubule-associated interactome.
Gache, V *., Waridel, P *., Winter, C., Juhem, A., Schroeder, M., Shevchenko, A., A.V. Popov. PLOS One (2010) .
- Drug screening on Hutchinson Gilford progeria pluripotent stem cells reveals aminopyrimidines as new modulators of farnesylation.
Blondel S, Egesipe AL, Picardi P, Jaskowiak AL, Notarnicola M, Ragot J, Tournois J, Le Corf A, Brinon B, Poydenot P, Georges P, Navarro C, Pitrez PR, Ferreira L, Bollot G, Bauvais C, Laustriat D, Méjat A, De Sandre-Giovannoli A, Levy N, Bifulco M, Peschanski M and Nissan X. Cell Death and Dis. (2016) In press.
- PAK1 and CtBP1 regulate the coupling of neuronal activity to muscle chromatin and gene expression.
Thomas JL, Moncollin V, Ravel-Chapuis A, Valente C, Corda D, Méjat A, Schaeffer L. Mol Cell Biol. (2015) Sep 28. pii: MCB.00354-15.
- Histone deactylase 6 is a FoxO transcription factor-dependent effector in skeletal muscle atrophy.
Ratti F, Ramond F, Moncollin V, Simonet T, Milan G, Méjat A, Thomas JL, Streichenberger N, Gilquin B, Khochbin S, Sandri M, Schaeffer L. J Biol Chem. (2015) Feb 13 ; 290(7) : 4215-24.
- Lamin A/C–mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy.
Méjat A, Decostre V, Li J, Renou L, Kesari A, Hantaï D, Stewart CL, Xiao X, Hoffman E, Bonne G, Misteli T. J. Cell Biol. (2009) Jan 12;184(1) :31-44.
- Histone deacetylase 9 couples neuronal activity to muscle chromatin acetylation and gene expression.
Méjat A, Ramond F, Bassel-Duby R, Khochbin S, Olson EN, Schaeffer L. Nat Neuroscience (2005) Mar;8(3):313-21.
The team is currently hosted by the LBMC at the ENS of Lyon
LBMC, UMR5239 ENS Lyon
46 Allée d'Italie
69364 LYON cedex 7