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.
Highlights
Muscle Dystrophin establishes a link between Dystroglycan complexes at the cell membrane and actin in the cortical cytoskeleton (Ibraghimov-Beskrovnaya et al, 1992; Levine et al, 1992; Ervasti and Campbell, 1993; Rybakova et al, 1996, 2000)
It is important to understand the dynamics of Dystrophin in vivo and how this could vary within cellular context, influencing the phenotype of Becker muscular dystrophy (BMD) and gene therapy planning for patients with Duchenne muscular dystrophy (DMD)
Expression of human Dystrophin (huDys) or GFP control in zebrafish embryos was achieved through the injection of the DNA constructs into newly fertilized embryos at the early 1 cell stage, aiming to obtain mosaic expression to facilitate single cell analysis (Figure 1B,C)
Summary
Muscle Dystrophin establishes a link between Dystroglycan complexes at the cell membrane and actin in the cortical cytoskeleton (Ibraghimov-Beskrovnaya et al, 1992; Levine et al, 1992; Ervasti and Campbell, 1993; Rybakova et al, 1996, 2000). Mutations in the Dystrophin gene often lead to a non-functional protein and Duchenne muscular dystrophy (DMD), characterised by severe muscle degeneration from early childhood. In-frame deletions within the Dystrophin sequence can result in a shortened but partially functional protein that causes Becker muscular dystrophy (BMD) (Koenig et al, 1989). There are still big gaps on our understanding of how Dystrophin works within cells. It is important to understand the dynamics of Dystrophin in vivo and how this could vary within cellular context, influencing the phenotype of BMD and gene therapy planning for patients with DMD. Many current approaches for gene therapy in DMD aim to restore ‘short’ Dystrophins, known to be partially functional from studies of patients with BMD and murine transgenic models (Konieczny et al, 2013). If some short Dystrophin forms bind more efficiently and stably than
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