Dynamin 2 Mutants Linked to Centronuclear Myopathies Form Abnormally Stable Polymers

Abstract

Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM). Dynamin 2 is a ubiquitously expressed ϳ100-kDa GTPase that assembles around the necks of vesiculating membranes and promotes their constriction and scission. It has also been implicated in regulation of the actin and microtubule cytoskeletons. At present, the cellular functions of dynamin 2 that are affected by CNM-linked mutations are not well defined, and the effects of these mutations on the physical and enzymatic properties of dynamin have been not examined. Here, we report the expression, purification, and characterization of four CNMassociated dynamin mutants. All four mutants display higher than wild-type GTPase activities, and more importantly, the mutants form high order oligomers that are significantly more resistant than wild-type dynamin 2 to disassembly by guanine nucleotides or high ionic strength. These observations suggest that the corresponding wild-type residues serve to prevent excessive or prolonged dynamin assembly on cellular membranes or inappropriate self-assembly in the cytoplasm. To our knowledge, this report contains the first identification of point mutations that enhance the stability of dynamin polymers without impairing their ability to bind and/or hydrolyze GTP. We envision that the formation of abnormally large and stable complexes of these dynamin mutants in vivo contributes to their role in CNM pathogenesis. Autosomal dominant CNM2 is a congenital disorder that commonly results in muscle weakness and wasting, ptosis, and ophthalmoplegia (reviewed in Ref. 1). As the name implies, the most evident histopathological feature is the presence of a large number of muscle fibers of centrally (rather than peripherally) located nuclei. Other characteristics include a relative increase in the number of type I fibers, hypertrophy of these fibers, and the presence of sarcoplasmic strands distributed radially around the central nuclei. In 2005, Bitoun et al. (2) reported the identification of four mutations in the DNM2 (dynamin 2) gene in patients with autosomal dominant CNM, and an additional seven mutations have since been identified (3, 4). DNM2 is a ubiquitously expressed ϳ100-kDa GTPase that assembles into helical polymers around the necks of vesiculating membranes, thereby providing force for their constriction and scission (reviewed in Refs. 5-8). In addition to its well characterized roles in endocytosis and Golgi budding, DNM2 is also implicated in regulation of the actin (9, 10) and microtubule (11, 12) cytoskeletons. The DNM2 molecule consists of five functional domains: an N-terminal catalytic domain; a so-called "middle domain" implicated in dynamin-dynamin interactions; a PH domain involved in phosphoinositide binding; a GTPase effector domain, which interacts with the catalytic domain and stimulates its GTPase activity; and a C-terminal proline/arginine-rich domain, which mediates interactions of dynamin with other proteins. Most of the currently kn...

Reducing dynamin 2 expression rescues X-linked centronuclear myopathy

“…Recent biochemical studies have indicated that some CNMcausing DNM2 mutations increase dynamin oligomer stability and GTPase activity (26,27). This was supported in vivo by either knockin or overexpression of the most common CNM-DNM2 patient mutation in mice, which induced CNM-like features at adulthood (28,29), indicating that the disease is not due to haploinsufficiency.…”

“…Loss-of-function mutations in MTM1 and dominant mutations in DNM2 lead to CNM in human and CNM phenotypes in mice models (1,9). Moreover, biochemical analysis suggests several DNM2 CNM mutants increase DNM2 GTPase activity and oligomerization (26,27). Furthermore, overexpression of WT DNM2 by AAV or by miR-133a inhibition resulting in increased DNM2 expression promoted muscle defects and centralization of nuclei in mouse muscle (28,30).…”

Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation

“…Recessive BIN1-CNM is due to BIN1 loss of function and dominant DNM2-CNM is probably due to DNM2 gain of function (5,7,(15)(16)(17). We hypothesize that these 2 proteins are working antagonistically in the same pathway for muscle maturation and that reducing DNM2 expression might rescue the neonatal lethality observed in …”

“…DNM2 is a large GTPase involved in membrane fission and endocytosis (13,14). Biochemical studies indicated that several DNM2 mutations causing CNMs increase dynamin oligomer stability and GTPase activity (15)(16)(17). Moreover, overexpression of WT or a DNM2-CNM mutation leads to CNM-like features in mice and perturbation of muscle and T-tubules in Drosophila (17)(18)(19).…”

Mice lacking microRNA 133a develop dynamin 2–dependent centronuclear myopathy

“…However, a recent study showed that specific CNM-related DNM2 mutations cause increased GTPase activity and promote dynamin oligomerization without altering lipid binding (32). Another study also showed that CNM-related DNM2 mutants enhance the stability of dynamin polymers without impairing their ability to bind and/or hydrolyze GTP (33).…”