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Elife. 2015 Oct 13;4. doi: 10.7554/eLife.06541.

In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis.

Author information

1
Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
2
CNRS and Université Paul Sabatier, Toulouse, France.
3
Department of Physics, University of Surrey, Guildford, United Kingdom.
4
Université Versailles Saint-Quentin, Montigny-le-Bretonneux, France.
5
Laboratoire International Associé-Biologie appliquée aux handicaps neuromusculaires, Centre Scientifique de Monaco, Monaco, Monaco.

Abstract

Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmd(ta222a/ta222a) zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)(ct90a) that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics.

KEYWORDS:

Dystrophin; FRAP; binding dynamics; cell biology; developmental biology; diffusion; muscle; software; stem cells; zebrafish

PMID:
26459831
PMCID:
PMC4601390
DOI:
10.7554/eLife.06541
[Indexed for MEDLINE]
Free PMC Article

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