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Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair.

Redpath GM, Woolger N, Piper AK, Lemckert FA, Lek A, Greer PA, North KN, Cooper ST - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

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.

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Dysferlin exon 40a and calpain recruit to sites of membrane injury. Cultured MO3.13 secondary oligodendrocytes (row 1) and primary human myotubes (row 2) were shot with 4-μm silica beads using a Bio-Rad Helios Gene Gun, fixed at 10 s postinjury in cold 3% paraformaldehyde, and then permeabilized and immunolabeled (see Materials and Methods). Romeo was applied for 2 h before Hamlet-1 to bias the detection of the N-terminal dysferlin epitope. Dysferlin was detectable only at sites of membrane injury with Hamlet-1 (rows 1 and 2). Staining with an antibody raised to dysferlin exon 40a revealed exon 40a–containing dysferlin recruits to sites of injury within 10 s (row 3). Calpain-2 was detectable at sites of membrane injury at 2 s (T2, row 4) and 10 s postdamage (T10, row 5). Large-injury sites often showed a void of negative labeling for calpain-2 (T10, row 6), suggesting that calpain might be extracted or escape from large injuries. Scale bar, 5 μm.
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Figure 5: Dysferlin exon 40a and calpain recruit to sites of membrane injury. Cultured MO3.13 secondary oligodendrocytes (row 1) and primary human myotubes (row 2) were shot with 4-μm silica beads using a Bio-Rad Helios Gene Gun, fixed at 10 s postinjury in cold 3% paraformaldehyde, and then permeabilized and immunolabeled (see Materials and Methods). Romeo was applied for 2 h before Hamlet-1 to bias the detection of the N-terminal dysferlin epitope. Dysferlin was detectable only at sites of membrane injury with Hamlet-1 (rows 1 and 2). Staining with an antibody raised to dysferlin exon 40a revealed exon 40a–containing dysferlin recruits to sites of injury within 10 s (row 3). Calpain-2 was detectable at sites of membrane injury at 2 s (T2, row 4) and 10 s postdamage (T10, row 5). Large-injury sites often showed a void of negative labeling for calpain-2 (T10, row 6), suggesting that calpain might be extracted or escape from large injuries. Scale bar, 5 μm.

Mentions: Immunolabeling experiments of differentiated MO3.13 oligodendrocytes (differentiation increases dysferlin expression) subjected to ballistics injury with silica microparticles (4-μm diameter) revealed robust labeling for dysferlin at sites of membrane injury within 10 s of injury (Figure 5, top, left; Hamlet-1, green), concordant with our previous study in cultured human myotubes (Lek et al., 2013). Similarly, injury-induced dysferlin accumulation could be detected only with the C-terminal antibody Hamlet-1 and not the N-terminal antibody Romeo (Figure 5, top, middle; Romeo, red). Staining of primary human myotubes, biasing antibody binding to the dysferlin N-terminus by incubating with the N-terminal Romeo antibody before the C-terminal antibody Hamlet-1, similarly highlights specific recognition of the dysferlin C-terminus but not the dysferlin N-terminus at injury sites (Figure 5, second row).


Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair.

Redpath GM, Woolger N, Piper AK, Lemckert FA, Lek A, Greer PA, North KN, Cooper ST - Mol. Biol. Cell (2014)

Dysferlin exon 40a and calpain recruit to sites of membrane injury. Cultured MO3.13 secondary oligodendrocytes (row 1) and primary human myotubes (row 2) were shot with 4-μm silica beads using a Bio-Rad Helios Gene Gun, fixed at 10 s postinjury in cold 3% paraformaldehyde, and then permeabilized and immunolabeled (see Materials and Methods). Romeo was applied for 2 h before Hamlet-1 to bias the detection of the N-terminal dysferlin epitope. Dysferlin was detectable only at sites of membrane injury with Hamlet-1 (rows 1 and 2). Staining with an antibody raised to dysferlin exon 40a revealed exon 40a–containing dysferlin recruits to sites of injury within 10 s (row 3). Calpain-2 was detectable at sites of membrane injury at 2 s (T2, row 4) and 10 s postdamage (T10, row 5). Large-injury sites often showed a void of negative labeling for calpain-2 (T10, row 6), suggesting that calpain might be extracted or escape from large injuries. Scale bar, 5 μm.
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Related In: Results  -  Collection

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Figure 5: Dysferlin exon 40a and calpain recruit to sites of membrane injury. Cultured MO3.13 secondary oligodendrocytes (row 1) and primary human myotubes (row 2) were shot with 4-μm silica beads using a Bio-Rad Helios Gene Gun, fixed at 10 s postinjury in cold 3% paraformaldehyde, and then permeabilized and immunolabeled (see Materials and Methods). Romeo was applied for 2 h before Hamlet-1 to bias the detection of the N-terminal dysferlin epitope. Dysferlin was detectable only at sites of membrane injury with Hamlet-1 (rows 1 and 2). Staining with an antibody raised to dysferlin exon 40a revealed exon 40a–containing dysferlin recruits to sites of injury within 10 s (row 3). Calpain-2 was detectable at sites of membrane injury at 2 s (T2, row 4) and 10 s postdamage (T10, row 5). Large-injury sites often showed a void of negative labeling for calpain-2 (T10, row 6), suggesting that calpain might be extracted or escape from large injuries. Scale bar, 5 μm.
Mentions: Immunolabeling experiments of differentiated MO3.13 oligodendrocytes (differentiation increases dysferlin expression) subjected to ballistics injury with silica microparticles (4-μm diameter) revealed robust labeling for dysferlin at sites of membrane injury within 10 s of injury (Figure 5, top, left; Hamlet-1, green), concordant with our previous study in cultured human myotubes (Lek et al., 2013). Similarly, injury-induced dysferlin accumulation could be detected only with the C-terminal antibody Hamlet-1 and not the N-terminal antibody Romeo (Figure 5, top, middle; Romeo, red). Staining of primary human myotubes, biasing antibody binding to the dysferlin N-terminus by incubating with the N-terminal Romeo antibody before the C-terminal antibody Hamlet-1, similarly highlights specific recognition of the dysferlin C-terminus but not the dysferlin N-terminus at injury sites (Figure 5, second row).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus