MG53 Regulates Membrane Budding and Exocytosis in Muscle Cells

Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis. We have identified a tripartite motif (TRIM72) family member protein named MG53 and defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell imaging of green fluorescent protein-MG53 fusion construct in cultured myoblasts showed that although MG53 contains no transmembrane segment it is tightly associated with intracellular vesicles and sarcolemmal membrane. RNA interference-mediated knockdown of MG53 expression impeded myoblast differentiation, whereas overexpression of MG53 enhanced vesicle trafficking to and budding from sarcolemmal membrane. Co-expression studies indicated that MG53 activity is regulated by a functional interaction with caveolin-3. Our data reveal a new function for TRIM family proteins in regulating membrane trafficking and fusion in striated muscles.When myoblasts exit the cell cycle during myogenesis, dramatic changes in membrane organization occur as myoblast fusion allows the formation of multinucleated muscle fibers. In addition to cell fusion events, differentiation of myotubes involves establishment of specialized membrane structures (1, 2). The transverse tubular invagination of sarcolemmal membrane and the intracellular membrane network known as the sarcoplasmic reticulum are two highly organized membrane architectures in cardiac and skeletal muscle. Establishment of these intricate membrane compartments requires extensive remodeling of the immature myoblast membranes. Dynamic membrane remodeling also contributes to many physiologic processes in mature muscle, including Ca²⁺ signaling, trafficking of glucose transporter (GLUT4), and other membrane internalization events involving caveolae structures (3-6). Although defects in membrane integrity have been linked to various forms of muscular dystrophy (7, 8), the molecular machinery regulating these specific membrane recycling and remodeling events in striated muscle is not well defined.The large tripartite motif (TRIM)⁵ family of proteins is involved in numerous cellular functions in a wide variety of cell types. Members of this protein family contain signature motifs that include a RING finger, a zinc binding moiety (B-box), and a coiled coil structure (RBCC), which invariably comprise the amino-terminal domain of TRIM family members (9). The carboxyl-terminal sequence of TRIM proteins is variable; in some cases a subfamily of TRIM proteins contains a SPRY domain, a sequence first observed in the ryanodine receptor Ca²⁺ channel in the sarcoplasmic reticulum membrane of excitable cells (10). Extensive studies have revealed that protein-protein interactions in the cytosol mediate the defined functions of TRIM proteins. For example, the ubiquitin E3 ligase enzymatic activity of several TRIM family members requires the B-box motif (11, 12). Recent studies have also indicated a role for TRIM proteins in defense against events involving membrane penetration, such as protection against infection by various viruses, including human immunodeficiency virus (13-15). Although most of the studies concentrate on the cytosolic action of TRIM, limited reports have investigated the role of TRIM proteins in membrane signaling or recycling.We have previously established an immunoproteomics approach that allows definition of novel components involved in myogenesis, Ca²⁺ signaling, and maintenance of membrane integrity in striated muscle (16). Using this approach, we have shown that junctophilin is a structural protein that establishes functional communication between sarcoplasmic reticulum and transverse tubule membranes at triad and dyad junctions in striated muscle (17-19). Further studies identified mitsugumin 29, a synaptophysin-related protein that is essential for biogenesis of triad membrane structures and Ca²⁺ signaling in skeletal muscle (20, 21). Screening of this immunoproteomics library led to the recent identification of MG53, a muscle-specific TRIM family protein (22). Domain homology analysis revealed that MG53 contains the prototypical RBCC motifs plus a SPRY domain at the carboxyl terminus. Genetic knock-out and functional studies reveal that MG53 nucleates the assembly of the sarcolemmal membrane repair machinery to restore cellular integrity following acute damage to the muscle fiber (22).Here we present evidence illustrating that MG53, in contrast to other known TRIM proteins, can localize to intracellular vesicles and the sarcolemmal membrane. A functional interaction between MG53 and caveolin-3, another muscle-specific protein, plays an essential role in regulating the dynamic process of membrane budding and exocytosis in skeletal muscle.     

Constant rate of p53 tetramerization in response to DNA damage controls the p53 response

The dynamics of the tumor suppressor protein p53 have been previously investigated in single cells using fluorescently tagged p53. Such approach reports on the total abundance of p53 but does not provide a measure for functional p53. We used fluorescent protein-fragment complementation assay (PCA) to quantify in single cells the dynamics of p53 tetramers, the functional units of p53. We found that while total p53 increases proportionally to the input strength, p53 tetramers are formed in cells at a constant rate. This breaks the linear input–output relation and dampens the p53 response. Disruption of the p53-binding protein ARC led to a dose-dependent rate of tetramers formation, resulting in enhanced tetramerization and induction of p53 target genes. Our work suggests that constraining the p53 response in face of variable inputs may protect cells from committing to terminal outcomes and highlights the importance of quantifying the active form of signaling molecules in single cells.Quantification of the dynamics of p53 tetramers in single cells using a fluorescent protein-fragment complementation assay reveals that, while total p53 increases proportionally to the DNA damage strength, p53 tetramers are formed at a constant rate.     

brca2 and tp53 Collaborate in Tumorigenesis in Zebrafish

Germline mutations in the tumor suppressor genes BRCA2 and TP53 significantly influence human cancer risk, and cancers from humans who inherit one mutant allele for BRCA2 or TP53 often display loss of the wildtype allele. In addition, BRCA2-associated cancers often exhibit mutations in TP53. To determine the relationship between germline heterozygous mutation (haploinsufficiency) and somatic loss of heterozygosity (LOH) for BRCA2 and TP53 in carcinogenesis, we analyzed zebrafish with heritable mutations in these two genes. Tumor-bearing zebrafish were examined by histology, and normal and neoplastic tissues were collected by laser-capture microdissection for LOH analyses. Zebrafish on a heterozygous tp53^M214K background had a high incidence of malignant tumors. The brca2^Q658X mutation status determined both the incidence of LOH and the malignant tumor phenotype. LOH for tp53 occurred in the majority of malignant tumors from brca2 wildtype and heterozygous mutant zebrafish, and most of these were malignant peripheral nerve sheath tumors. Malignant tumors in zebrafish with heterozygous mutations in both brca2 and tp53 frequently displayed LOH for both genes. In contrast, LOH for tp53 was uncommon in malignant tumors from brca2 homozygotes, and these tumors were primarily undifferentiated sarcomas. Thus, carcinogenesis in zebrafish with combined mutations in tp53 and brca2 typically requires biallelic mutation or loss of at least one of these genes, and the specific combination of inherited mutations influences the development of LOH and the tumor phenotype. These results provide insight into cancer development associated with heritable BRCA2 and TP53 mutations.