Limits...
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.

Show MeSH

Related in: MedlinePlus

Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlinC72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca2+ or the presence of Ca2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlinC72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlinC72 occurs independently of MG53.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230592&req=5

Figure 4: Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlinC72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca2+ or the presence of Ca2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlinC72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlinC72 occurs independently of MG53.

Mentions: Although skeletal muscle is believed to be particularly prone to membrane injury due to its contractile functions under load, membrane injury is also a feature of ischemic injury to heart and brain and shear stress injury of blood vessel endothelia. Because dysferlin is ubiquitously expressed and readily detected in endothelia and brain, we studied whether the calcium-activated calpain cleavage of dysferlin represents a ubiquitous response to acute membrane injury or is a specific adaptation of skeletal muscle. In primary cultures of human myotubes, human umbilical vein endothelial cells (HUVECs), mouse astrocytes, and microglia, as well as secondary human oligodendrocytes (MO3.13), each displayed calcium-dependent, calpeptin-sensitive formation of mini-dysferlinC72 after scrape damage (Figure 4A). Primary mouse astrocytes and HUVECs do not express detectable levels of MG53 but are still capable of forming mini-dysferlinC72, indicating that MG53 is not required for calpain cleavage of dysferlin (Figure 4B).


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 is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlinC72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca2+ or the presence of Ca2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlinC72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlinC72 occurs independently of MG53.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230592&req=5

Figure 4: Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlinC72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca2+ or the presence of Ca2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlinC72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlinC72 occurs independently of MG53.
Mentions: Although skeletal muscle is believed to be particularly prone to membrane injury due to its contractile functions under load, membrane injury is also a feature of ischemic injury to heart and brain and shear stress injury of blood vessel endothelia. Because dysferlin is ubiquitously expressed and readily detected in endothelia and brain, we studied whether the calcium-activated calpain cleavage of dysferlin represents a ubiquitous response to acute membrane injury or is a specific adaptation of skeletal muscle. In primary cultures of human myotubes, human umbilical vein endothelial cells (HUVECs), mouse astrocytes, and microglia, as well as secondary human oligodendrocytes (MO3.13), each displayed calcium-dependent, calpeptin-sensitive formation of mini-dysferlinC72 after scrape damage (Figure 4A). Primary mouse astrocytes and HUVECs do not express detectable levels of MG53 but are still capable of forming mini-dysferlinC72, indicating that MG53 is not required for calpain cleavage of dysferlin (Figure 4B).

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