Mutations Associated with Centronuclear Myopathy (CNM) Enhance the Size and Stability of Dynamin 2 Clusters in Cells

Dynamin 2 (Dyn2), a ∼ 100 kDa, GTPase that self-assembles into rings around the necks of budding vesicles, has been implicated in generating force for endocytic vesicle scission from the plasma membrane. Self-association of Dyn2 at the site of membrane invagination, and subsequent catalysis of membrane scission, is tightly coupled to GTP binding and hydrolysis. Mutations that affect the stability of Dyn2 polymers have been linked to autosomal dominant forms of CNM, a congenital disorder characterized by muscle weakness and wasting. In vitro biochemical analysis revealed that CNM-causing Dyn2 mutants express enhanced GTPase activity relative to wild-type Dyn2, and form polymers that are more resistant to GTPase-dependent disassembly. We compared the physical properties of assembled wild-type and CNM-linked mutant forms of Dyn2 in the cytosol and plasma membrane of living cells using fluorescence fluctuation spectroscopy (FFS). Our data indicate that unassembled wild-type Dyn2 is a tetramer in the cytosol, whereas CNM-associated R369W and A618T mutations induce the formation of higher order cytosolic oligomers (10- and 22-mers, respectively). FFS coupled with total internal reflection microscopy established that mutant forms of Dyn2 assemble into larger, more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2. Based on these observations, a model to explain the defects in membrane trafficking in CNM muscle cells was developed.