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

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.

DOI: http://dx.doi.org/10.7554/eLife.06541.001

eLife digest

A protein called Dystrophin plays a key role in maintaining the structural integrity of muscle cells as they contract and relax. Mutations in the gene that encodes Dystrophin can cause several different types of muscular dystrophy, a group of diseases in which muscle progressively weakens. Some mutations in Dystrophin can lead to mild symptoms that may affect the quality of life but are not life threatening. However, in more serious cases, patients lose the ability to walk in childhood and have shortened life expectancy. There is no cure for these diseases, and there are still big gaps in our understanding of how Dystrophin works, which makes it more difficult to develop efficient therapies.

The zebrafish is often used as a model to study muscular dystrophies. In this study, Bajanca et al. introduced human Dystrophin into zebrafish muscle cells and analysed its behaviour using a combination of mathematical modelling and a method known as ‘fluorescence recovery after photobleaching’. In these experiments, the human Dystrophin was attached to a tag that fluoresces green under a microscope, which allowed it to be easily seen and be followed in real time inside the cells of live animals.

Bajanca et al. observed that Dystrophin could either remain firmly associated with the membrane that surrounds the cell over long periods of time or interact briefly with the membrane. Bajanca et al. carried out further experiments with the Dystrophin protein naturally found in zebrafish and observed that it behaved in a similar manner to the human protein, suggesting this behaviour is likely to be important for the ability of the protein to work.

Bajanca et al.'s findings reveal that Dystrophin displays complex behaviour in living muscle cells. The fact that some Dystrophin molecules are firmly attached to the membrane support previous findings that this protein provides mechanical stability to the cells. However, the discovery that there is a group of more mobile Dystrophin molecules within muscle cells suggests that this protein may also play other roles. Therefore, these findings open a new avenue for research that may contribute to the development of new therapy approaches in future.

DOI: http://dx.doi.org/10.7554/eLife.06541.002