Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair.
Bottom Line: Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a.Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain.Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module.
Affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, Australia.Show MeSH
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Mentions: To determine a potential link between calpain activation, cleavage of dysferlin, and cell survival after an acute membrane injury, we developed a flow cytometry assay to quantify membrane resealing across populations of cells injured by scraping. Primary human myotubes (nascently differentiated for 3 d to activate dysferlin expression) were injured by cell scraping, allowed to reseal for 10 min, and then incubated in cell-impermeable propidium iodide, the “cell death” marker. Flow cytometry was then used to analyze the proportion of cells that failed to reseal and were permeable to propidium iodide. Our results confirm a requirement for calcium for membrane resealing, with ∼30% increase in cell survival when calcium is present (Figure 6A). By titrating levels of extracellular calcium, we show that maximal cell survival is achieved with extracellular calcium concentrations >100–200 μM, remarkably consistent with studies published by Steinhardt et al. (1994).
Affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, Australia.