BACKGROUND: Dynamin 2 (Dyn2) is a ~100kDa GTPase that assembles around the necks 
of nascent endocytic and Golgi vesicles and catalyzes membrane scission. 
Mutations in Dyn2 that cause centronuclear myopathy (CNM) have been shown to 
stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely 
timed regulation of assembly and disassembly is believed to be critical for Dyn2 
function in membrane vesiculation, and the CNM mutations interfere with this 
regulation by shifting the equilibrium toward the assembled state.
METHODS: In this study we use two fluorescence fluctuation spectroscopy (FFS) 
approaches to show that a CNM mutant form of Dyn2 also has a greater propensity 
to self-assemble in the cytosol and on the plasma membrane of living cells.
RESULTS: Results obtained using brightness analysis indicate that unassembled 
wild-type Dyn2 is predominantly tetrameric in the cytosol, although different 
oligomeric species are observed, depending on the concentration of expressed 
protein. In contrast, an R369W mutant identified in CNM patients forms 
higher-order oligomers at concentrations above 1μM. Investigation of Dyn2-R369W 
by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant 
forms larger and more stable clathrin-containing structures on the plasma 
membrane than wild-type Dyn2.
CONCLUSIONS AND GENERAL SIGNIFICANCE: These observations may explain defects in 
membrane trafficking reported in CNM patient cells and in heterologous systems 
expressing CNM-associated Dyn2 mutants.