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Proteomics of skeletal muscle triads

Objectifs

The project aims at identifying proteins localized at skeletal muscle triads, which are essential structures for excitation-contraction coupling and are defective in several muscle genetic diseases. This will allow the identification of pathways potentially involved in the formation and maintenance of triads.

Résumé

In skeletal muscle, triads are composed of an invagination of the plasma membrane, the transverse tubule, flanked by two terminal cisternae of the sarcoplasmic reticulum (SR) which are calcium storage sites. Action potentials transmitted by nerves activate channels at triads, leading to intracellular SR calcium release and sarcomere contraction. The mechanisms governing the dynamics of triads remain mysterious. To elucidate those mechanisms, the master student will identify proteins localized at triads using the recently developed technique of proximity-dependent biotinylation identification (BioID). He/she will learn the technics and test proteins already known to be localized at triads as anchors for BioID biotin ligase. The optimal fusion BioID anchor protein will be expressed in cultured muscle cells (1) at different time points of differentiation, (2) in differentiated muscle cells stimulated or not to mimic nerve stimulation. Proteins biotinylated by the proximity of the BioID anchor will then be identified by mass spectrometry.

Méthodes

Methods used will include molecular biology, biochemistry, cell culture and microscopy in cell lines and primary cultures of mouse and human muscle cells.

Références

Sébastien et al. (2020) Dynamics of triadin, a muscle-specific triad protein, within sarcoplasmic reticulum subdomains. Mol Biol Cell. 31(4):261-272.

Osseni et al. (2016) Triadin and CLIMP-63 form a link between triads and microtubules in muscle cells. J Cell Sci. 2016. 129(20):3744-3755.

Domaines d'expertise requis

molecular biology, biochemistry, cell biology, imaging

Contacts

Dr Anne-Sophie Nicot, MCU
Email : nicotan@univ-grenoble-alpes.fr
Phone : 04 56 52 05 70

Téléchargement(s)

GIN_offre-stage-M2_2022-2023_ASNicot_20220707.pdf (PDF, 136983 Ko)

Mise à jour le 8 juillet 2022

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