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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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Mini-dysferlinC72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlinC72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlinC72 from five calcium dose–response curves (EC50 of ∼ 250 μM Ca2+, 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlinC72 is indicated with black arrows.
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Figure 6: Mini-dysferlinC72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlinC72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlinC72 from five calcium dose–response curves (EC50 of ∼ 250 μM Ca2+, 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlinC72 is indicated with black arrows.

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).


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)

Mini-dysferlinC72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlinC72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlinC72 from five calcium dose–response curves (EC50 of ∼ 250 μM Ca2+, 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlinC72 is indicated with black arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Mini-dysferlinC72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlinC72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlinC72 from five calcium dose–response curves (EC50 of ∼ 250 μM Ca2+, 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlinC72 is indicated with black arrows.
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).

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