Calpain cleavage and subcellular characterisation of the ferlin family.

Abstract

The ferlins are a family of C2-domain containing proteins. C2 domains regulate vesicle fusion in synaptotagmins, and animal models of ferlin deficiency display pathologies related to Ca2+-dependent vesicle fusion. Dysferlin mutations cause limb-girdle muscular dystrophy due to defective membrane repair. Our group has previously shown that Ca2+-dependent proteases, calpains, cleave dysferlin following membrane injury, releasing mini-dysferlinC72, that we hypothesise mediates membrane repair. Otoferlin mutations cause non-syndromic deafness, while no pathology causing mutations have been identified in other ferlins. My project establishes that dysferlin and myoferlin, type-I ferlins, are present at the plasma membrane and endo-lysosomal pathway while otoferlin and Fer1L6, type-II ferlins, are present at the plasma membrane and recycling trans-Golgi compartments. I also show that dysferlin is cleaved to mini-dysferlinC72 following injury in all cell types by the ubiquitous calpains-1 and -2 in the alternatively spliced exon 40a, indicating dysferlin cleavage is a fundamental response to membrane injury. Exon 40a-containing dysferlin recruits to sites of membrane injury in myotubes, indicating mini-dysferlinC72 may function directly at sites of injury. Finally, I have shown that calpains also cleave otoferlin and myoferlin. Cleavage of other ferlins indicates ferlin cleavage is an evolutionarily conserved event, predating the split between type-I and type-II ferlins.