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.
Keywords: ACF; CME; CNM; Centronuclear myopathy; Clathrin-mediated endocytosis; Dyn; Dyn2-EGFP; Dynamin; Dynamin 2; EGFP; EMCCD; FFS; Fluorescence fluctuation spectroscopy; GED; GTPase effector domain; ICS; Image Correlation Spectroscopy; MEF; Mouse embryo fibroblasts; N&B; Number and Brightness; PCH; PH; PM; PSF; Photon Counting Histogram; Pleckstrin Homology; R369W; R369W mutation; TIRF; Total Internal Reflection Fluorescence; autocorrelation function; centronuclear myopathy; electron multiplying charge-coupled device; fluorescence fluctuation spectroscopy; plasma membrane; point spread function; rat dynamin 2 isoform 2ba construct containing an Arg to Trp mutation at residue 369; rat dynamin 2 isoform 2ba with a C- terminal EGFP and terminal hexa-histidine tag; wild-type; wt.