A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells

• Published in: Biochimica et biophysica acta Vol. 1840; no. 1; pp. 315 - 321
• Main Authors: James, Nicholas G., Digman, Michelle A., Ross, Justin A., Barylko, Barbara, Wang, Lei, Li, Jinhui, Chen, Yan, Mueller, Joachim D., Gratton, Enrico, Albanesi, Joseph P., Jameson, David M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2014
• Subjects: Animals; Bone Neoplasms - genetics; Bone Neoplasms - metabolism; Bone Neoplasms - pathology; Cell Membrane - metabolism; Cells, Cultured; Centronuclear myopathy; Clathrin - metabolism; cytosol; Cytosol - metabolism; Dynamin 2; Dynamin II - genetics; Dynamin II - metabolism; dynamins; EGFP; Embryo, Mammalian - cytology; Embryo, Mammalian - metabolism; Endocytosis; Fibroblasts - cytology; Fibroblasts - metabolism; fluorescence; Fluorescence fluctuation spectroscopy; Golgi Apparatus - metabolism; Humans; Image Processing, Computer-Assisted; Mice; Microscopy, Fluorescence; muscular diseases; mutants; mutation; Mutation - genetics; Myopathies, Structural, Congenital - genetics; Myopathies, Structural, Congenital - metabolism; Myopathies, Structural, Congenital - pathology; Osteosarcoma - genetics; Osteosarcoma - metabolism; Osteosarcoma - pathology; patients; physiological transport; plasma membrane; polymers; Protein Multimerization - genetics; Protein Transport; R369W mutation; spectroscopy; TIRF; PSF; CNM; ACF; Fluorescence fluctuation spectroscopy; GED; R369W; N&B; EMCCD; EGFP; TIRF; MEF; FFS; R369W mutation; PCH; Dyn2-EGFP; Dyn; PH; ICS; CME; Centronuclear myopathy; Dynamin 2; wt; PM; Mouse embryo fibroblasts; rat dynamin 2 isoform 2ba with a C- terminal EGFP and terminal hexa-histidine tag; point spread function; Number and Brightness; autocorrelation function; Pleckstrin Homology; Image Correlation Spectroscopy; Clathrin-mediated endocytosis; plasma membrane; GTPase effector domain; Photon Counting Histogram; Total Internal Reflection Fluorescence; rat dynamin 2 isoform 2ba construct containing an Arg to Trp mutation at residue 369; electron multiplying charge-coupled device; Dynamin; wild-type
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.09.001

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. 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 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. These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants. •The properties of a CNM-associated dynamin mutant in living cells are studied using fluorescence.•The R369W dynamin mutant forms larger oligomers in the cytosol than does wild-type dynamin.•The R369W dynamin assembles into large polymers on clathrin-coated pits in the plasma membrane.•These observations provide insight into the molecular defects underlying dynamin-dependent CNM.