No abstract

Dynamic membrane repair and remodelling is an elemental process that maintains cell integrity and mediates efficient cellular function. Here we report that MG53, a muscle-specific tripartite motif family protein (TRIM72), is a component of the sarcolemmal membrane-repair machinery. MG53 interacts with phosphatidylserine to associate with intracellular vesicles that traffic to and fuse with sarcolemmal membranes. Mice null for MG53 show progressive myopathy and reduced exercise capability, associated with defective membrane-repair capacity. Injury of the sarcolemmal membrane leads to entry of the extracellular oxidative environment and MG53 oligomerization, resulting in recruitment of MG53-containing vesicles to the injury site. After vesicle translocation, entry of extracellular Ca 2+ facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca 2+ -dependent membrane fusion are distinct steps involved in the repair of membrane damage and that MG53 may initiate the assembly of the membrane repair machinery in an oxidation-dependent manner.To maintain cellular homeostasis, eukaryotic cells must conserve the integrity of their plasma membrane through active recycling and repair in response to various sources of damage 1 . Defects in the intrinsic membrane repair response have been linked to numerous disease states, including muscular dystrophy, heart failure and neurodegeneration [2][3][4][5] . Repair of plasma membrane damage requires recruitment of intracellular vesicles to injury sites 6,7 . One protein that has been linked to membrane repair in skeletal muscle is dysferlin [8][9][10] , which is thought to act as a fusogen that participates in restoration of sarcolemmal membrane integrity following muscle injury. Evidence for this role of dysferlin comes, in part, from studies showing that ablation of dysferlin in mice results in muscular dystrophy 8 .Repair of damage to the plasma membrane is an active and dynamic process that requires several steps, including participation of molecular sensor(s) that can detect acute injury to 6 Correspondence should be addressed to J.M. or H.T. (maj2@umdnj.edu; takeshim@pharm.kyoto-u.ac.jp).Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 November 23. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the plasma membrane, nucleation of intracellular vesicles at the injury site and vesicle fusion to enable membrane patch formation. It is well demonstrated that entry of extracellular Ca 2+ is involved in the fusion of intracellular vesicles to reseal the injured plasma membrane 6,11,12 , whereas the molecular machinery involved in sensing the damaged membrane signal and the nucleation process for repair-patch formation have not been fully resolved.We have previously established an immunopr...

Defective membrane repair can contribute to the progression of muscular dystrophy. Although mutations in caveolin-3 (Cav3) and dysferlin are linked to muscular dystrophy in human patients, the molecular mechanism underlying the functional interplay between Cav3 and dysferlin in membrane repair of muscle physiology and disease has not been fully resolved. We recently discovered that mitsugumin 53 (MG53), a muscle-specific TRIM (Tri-partite motif) family protein (TRIM72), contributes to intracellular vesicle trafficking and is an essential component of the membrane repair machinery in striated muscle. Here we show that MG53 interacts with dysferlin and Cav3 to regulate membrane repair in skeletal muscle. MG53 mediates active trafficking of intracellular vesicles to the sarcolemma and is required for movement of dysferlin to sites of cell injury during repair patch formation. Mutations in Cav3 (P104L, R26Q) that cause retention of Cav3 in Golgi apparatus result in aberrant localization of MG53 and dysferlin in a dominant-negative fashion, leading to defective membrane repair. Our data reveal that a molecular complex formed by MG53, dysferlin, and Cav3 is essential for repair of muscle membrane damage and also provide a therapeutic target for treatment of muscular and cardiovascular diseases that are linked to compromised membrane repair.Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis and maintenance of sarcolemma integrity in striated muscle. During the life cycle of striated muscle, membrane repair is a fundamental process in maintaining cellular integrity, as shown by recent studies that link defective membrane repair to the progression of muscular dystrophy (1-3). Repair of the plasma membrane damage requires recruitment of intracellular vesicles to injury sites (4, 5). One protein that has been linked to membrane repair in skeletal muscle is dysferlin (6, 7), which is thought to fuse intracellular vesicles to patch the damaged membrane and restore sarcolemmal integrity following muscle injury. Like dysferlin, caveolin-3 (Cav3) 3 is a muscle-specific protein, and many mutations in Cav3, including P104L, R26Q, and C71W, have been linked to muscular dystrophy (8 -11). Despite extensive research efforts on Cav3 and dysferlin (12)(13)(14), the molecular function of these two proteins in membrane repair in muscle physiology and dystrophy have not been fully defined.Animal model studies reveal that either loss or gain of Cav3 function both result in dystrophic phenotypes in skeletal muscle (15, 16), suggesting that associated cellular components may be involved in the etiology of Cav3-related dystrophy. Although the discovery of dysferlin highlights the importance of membrane repair in the etiology of muscular dystrophy, dysferlin itself does not appear to participate in recruitment of intracellular vesicles because dysferlin Ϫ/Ϫ muscle retains accumulation of vesicles near membrane damage sites (7). This indicates that proteins other than dys...

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.

In skeletal muscle excitation–contraction (E–C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E–C coupling in skeletal muscle.