Dysferlin Interacts with Annexins A1 and A2 and Mediates Sarcolemmal Wound-healing

Mutations in the dysferlin gene cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. We report here the results of expression profile analyses and in vitro investigations that point to an interaction between dysferlin and the Ca2+ and lipid-binding proteins, annexins A1 and A2, and define a role for dysferlin in Ca2+-dependent repair of sarcolemmal injury through a process of vesicle fusion. Expression profiling identified a network of genes that are co-regulated in dysferlinopathic mice. Co-immunofluorescence, co-immunoprecipitation, and fluorescence lifetime imaging microscopy revealed that dysferlin normally associates with both annexins A1 and A2 in a Ca2+ and membrane injury-dependent manner. The distribution of the annexins and the efficiency of sarcolemmal wound-healing are significantly disrupted in dysferlin-deficient muscle. We propose a model of muscle membrane healing mediated by dysferlin that is relevant to both normal and dystrophic muscle and defines the annexins as potential muscular dystrophy genes. Mutations in the dysferlin gene cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. We report here the results of expression profile analyses and in vitro investigations that point to an interaction between dysferlin and the Ca2+ and lipid-binding proteins, annexins A1 and A2, and define a role for dysferlin in Ca2+-dependent repair of sarcolemmal injury through a process of vesicle fusion. Expression profiling identified a network of genes that are co-regulated in dysferlinopathic mice. Co-immunofluorescence, co-immunoprecipitation, and fluorescence lifetime imaging microscopy revealed that dysferlin normally associates with both annexins A1 and A2 in a Ca2+ and membrane injury-dependent manner. The distribution of the annexins and the efficiency of sarcolemmal wound-healing are significantly disrupted in dysferlin-deficient muscle. We propose a model of muscle membrane healing mediated by dysferlin that is relevant to both normal and dystrophic muscle and defines the annexins as potential muscular dystrophy genes. Mutations in the dysferlin gene DYSF cause limb girdle muscular dystrophy (LGMD) 1The abbreviations used are: LGMDlimb girdle muscular dystrophyPBSphosphate-buffered salineFLIMfluorescence lifetime imaging microscopyDAPI4′,6-diamidino-2-phenylindole.1The abbreviations used are: LGMDlimb girdle muscular dystrophyPBSphosphate-buffered salineFLIMfluorescence lifetime imaging microscopyDAPI4′,6-diamidino-2-phenylindole. type 2B and Miyoshi myopathy (1.Liu is a muscle membrane that to the to of to the membrane a and the of the Ca2+-dependent of the of dysferlin to a that muscular dystrophy muscle an of the membrane We a interaction between dysferlin and a and muscle that and of the of gene expression in normal and dysferlin-deficient and in muscle We a that gene expression in The identified a of genes expression are in dysferlinopathic of network to the of A1 and A2 in the are and that are in membrane of A2 and The of of the annexins are annexins A1 and A2 to and in a Ca2+-dependent the of in A1 by a with and A2 to a with the for a Ca2+-dependent interaction between dysferlin and annexins A1 and a membrane is of dysferlin to Ca2+-dependent vesicle and with the We that membrane repair process is in dysferlinopathic the disrupted membrane healing in dysferlin and that by for We propose a role for dysferlin in that a healing model in muscle the and of and of of of and in the and a for to the as in The for and used to the expression of gene the of between and to a gene expression as and muscle the used for of in the normal dysferlin-deficient by the by of with the and of and and to the The gene with in a and of genes. with a of and and dysferlin-deficient with a of in with the the dysferlin the and and as in and in in to and by to in and for with and for with to with The in as with an and for in by a the of the in the of in for and in for to with in for for as and by the dysferlin in the with a to a a with a with and a to the interaction of proteins, the with both the 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repair of through interaction with the in dysferlin is to the with annexins A1 and A2 associates with and in A1 to The annexins as both and with and a of the sarcolemmal membrane results in the of Ca2+ that the annexins A1 and A2 to the of injury through an interaction with with the sarcolemmal and to the model in for the the of both annexins A1 and A2 with dysferlin the of normal muscle and the of in dysferlinopathic muscle the of dysferlin to both annexins in The Ca2+ and injury of is as a to to a interaction between dysferlin and A1 in Ca2+ in A2 with dysferlin in a in both and as by and by the of an of the as that with vesicle process is disrupted in is by the of expression in the of is a significantly of expression in both normal and dysferlin-deficient that is an of membrane repair that is of Ca2+ and dysferlin analyses of Ca2+ a of membrane healing in in the of the model the that membrane repair in a dysferlin that normal membrane healing that are by that microscopy of dysferlin-deficient muscle membrane and vesicle and in dysferlin-deficient are of (1.Liu of model are in repair as annexins A1 and A2 are to by and in the gene with muscular dystrophy type The cause muscular dystrophy to of annexins A1 and A2 for membrane The of model is the of Mutations in cause muscular dystrophy by an interaction between dysferlin and A2 is to of A2 and the repair model for the normal of of the in muscle are and and healing are to are in in and dysferlin in and membrane annexins A1 and A2, to and in the for to limb girdle in with in DYSF Mutations in the dysferlin gene DYSF cause limb girdle muscular dystrophy (LGMD) 1The abbreviations used are: LGMDlimb girdle muscular dystrophyPBSphosphate-buffered salineFLIMfluorescence lifetime imaging microscopyDAPI4′,6-diamidino-2-phenylindole.1The abbreviations used are: LGMDlimb girdle muscular dystrophyPBSphosphate-buffered salineFLIMfluorescence lifetime imaging microscopyDAPI4′,6-diamidino-2-phenylindole. type 2B and 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process in with of in with is significantly that of normal in the of a of dysferlin expression an the efficiency of sarcolemmal and with the Ca2+ and in the disrupted with a of a and and used to a of in to membrane that to normal a to normal Ca2+ significantly in in that of the Ca2+ for the of the that to and of injury for to the in between the for the in and to to a of a of is significantly normal the of injury normal and in as the for is report the of gene expression between normal and dysferlin-deficient muscle and the of a of co-regulated genes in dysferlinopathic of to the of of A1 and A2, with for the normal of in the expression of genes in both and of muscular dystrophy the expression of normal dysferlin-deficient The of gene here with muscular dystrophy as as the in relevant gene expression of genes a of interaction The is that of genes that are in of expression are to co-regulated in in a between genes that and the of a in genes muscle of muscle that a network of genes is to the dysferlinopathic of network for the of muscle in and Miyoshi myopathy. the genes in network are of and of the as as the annexins A1 and a Ca2+-dependent interaction between dysferlin and annexins A1 and A2 that a role in the and of in to membrane injury model is to the of membrane healing for dysferlin in the the annexins A1 and A2, in are to the with the dysferlin injury the muscle the of Ca2+ a The Ca2+ in a of that results in the of as to a to and with the to to the is to as a Ca2+-dependent that the of the repair with the model in for the the of both annexins A1 and A2 with dysferlin the of normal muscle and the of in dysferlinopathic muscle the of dysferlin to both annexins in The Ca2+ and injury of is as a to to a interaction between dysferlin and A1 in Ca2+ in A2 with dysferlin in a in both and as by and by the of an of the as that with vesicle process is disrupted in is by the of expression in the of is a significantly of expression in both normal and dysferlin-deficient that is an of membrane repair that is of Ca2+ and dysferlin analyses of Ca2+ a of membrane healing in in the of the model the that membrane repair in a dysferlin that normal membrane healing that are by that microscopy of dysferlin-deficient muscle membrane and vesicle and in dysferlin-deficient are of (1.Liu of model are in repair as annexins A1 and A2 are to by and in the gene with muscular dystrophy type The cause muscular dystrophy to of annexins A1 and A2 for membrane The of model is the of Mutations in cause muscular dystrophy by an interaction between dysferlin and A2 is to of A2 and the repair model for the normal of of the in muscle are and and healing are to are in in and dysferlin in and membrane annexins A1 and A2, to and in the for to limb girdle in with in DYSF report the of gene expression between normal and dysferlin-deficient muscle and the of a of co-regulated genes in dysferlinopathic of to the of of A1 and A2, with for the normal of in muscle. the expression of genes in both and of muscular dystrophy the expression of normal dysferlin-deficient The of gene here with muscular dystrophy as as the in relevant gene expression of genes a of interaction The is that of genes that are in of expression are to co-regulated in in a between genes that and the of a in genes muscle of muscle that a network of genes is to the dysferlinopathic of network for the of muscle in and Miyoshi myopathy. the genes in network are of and of the as as the annexins A1 and a Ca2+-dependent interaction between dysferlin and annexins A1 and A2 that a role in the and of in to membrane injury model is to the of membrane healing for dysferlin in the the annexins A1 and A2, in are to the with the dysferlin injury the muscle the of Ca2+ a The Ca2+ in a of that results in the of as to a to and with the to to the is to as a Ca2+-dependent that the of the repair with the model in for the the of both annexins A1 and A2 with dysferlin the of normal muscle and the of in dysferlinopathic muscle the of dysferlin to both annexins in The Ca2+ and injury of is as a to to a interaction between dysferlin and A1 in Ca2+ in A2 with dysferlin in a in both and as by and by the of an of the as that with vesicle process is disrupted in is by the of expression in the of is a significantly of expression in both normal and dysferlin-deficient that is an of membrane repair that is of Ca2+ and dysferlin analyses of Ca2+ a of membrane healing in in the of the model the that membrane repair in a dysferlin that normal membrane healing that are by that microscopy of dysferlin-deficient muscle membrane and vesicle and in dysferlin-deficient are of (1.Liu of model are in repair as annexins A1 and A2 are to by and in the gene with muscular dystrophy type The cause muscular dystrophy to of annexins A1 and A2 for membrane The of model is the of Mutations in cause muscular dystrophy by an interaction between dysferlin and A2 is to of A2 and the repair model for the normal of of the in muscle are and and healing are to are in in and dysferlin in and membrane annexins A1 and A2, to and in the for to limb girdle in with in DYSF We and of for with the We for the gene expression that in the