Dynamin 2 Mutants Linked to Centronuclear Myopathies Form Abnormally Stable Polymers

Lei Wang,Barbara Barylko,Christopher Byers,Justin A Ross,David M Jameson,Joseph P Albanesi

doi:10.1074/jbc.c110.130013

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 approximately 100-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 CNM-associated 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.

Highlights

Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM)
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
We found that all four mutants have a greater propensity to self-assemble than wild-type DNM2 and that the resulting structures are more resistant to depolymerization by guanine nucleotides or high ionic strength

Summary

Introduction

Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM). 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. We found that all four mutants have a greater propensity to self-assemble than wild-type DNM2 and that the resulting structures are more resistant to depolymerization by guanine nucleotides or high ionic strength The stability of these higher order oligomers in the presence of GTP is not due to defects in GTP binding and/or hydrolysis, as the mutants express higher than wild-type catalytic activities

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