A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells |
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Authors: | Nicholas G. James Michelle A. Digman Justin A. Ross Barbara Barylko Lei Wang Jinhui Li Yan Chen Joachim D. Mueller Enrico Gratton Joseph P. Albanesi David M. Jameson |
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Affiliation: | 1. Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, 651 Ilalo Street, Biosciences 222, Honolulu, HI 96813, USA;2. Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA;3. Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, USA;4. School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA |
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Abstract: | BackgroundDynamin 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.MethodsIn 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.ResultsResults 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 significanceThese observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants. |
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Keywords: | Dyn, Dynamin R369W, rat dynamin 2 isoform 2ba construct containing an Arg to Trp mutation at residue 369 Dyn2-EGFP, rat dynamin 2 isoform 2ba with a C- terminal EGFP and terminal hexa-histidine tag MEF, Mouse embryo fibroblasts CNM, centronuclear myopathy FFS, fluorescence fluctuation spectroscopy TIRF, Total Internal Reflection Fluorescence PCH, Photon Counting Histogram PH, Pleckstrin Homology GED, GTPase effector domain wt, wild-type ICS, Image Correlation Spectroscopy N& B, Number and Brightness PM, plasma membrane CME, Clathrin-mediated endocytosis EMCCD, electron multiplying charge-coupled device ACF, autocorrelation function PSF, point spread function |
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