Background Dynamin 2 (Dyn2) is a ~ 100 kDa 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 1 μM. 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.
Bibliographical noteFunding Information:
This work was supported in part by grants RO1GM076665 (DMJ), RO1GM064589 (JDM), and P41-RR03155 , P41 GM103540 and NIH P50-GM076516 (EG and MAD) from the National Institutes of Health . The authors would like to thank Tina Carvalho at the University of Hawaii at Manoa's Biological Electron Microscope Facility for assistance with the collection of TEM images and helpful discussion.
- Centronuclear myopathy
- Dynamin 2
- Fluorescence fluctuation spectroscopy
- R369W mutation