Mapping the determinants of dysferlin dimerization.

<p>(A) Dependence of FRET efficiencies on the protein expression levels of C2A to C2D domains of dysferlin. Hyperbolic fitting showed that the C2B (red), C2C (green) and C2D (purple) domains of dysferlin all mediate the self-interaction. There was no FRET for C2A construct (black). (B) Dependence of FRET efficiencies on the protein expression levels of C2E to C2G domains and the transmembrane (TM) domain of dysferlin. Hyperbolic fitting showed that the C2E (black), C2F (red), C2G (green) and TM (purple) domains of dysferlin also mediate the self-interaction. In A and B, the data were pooled for easy comparison. (C) Independence of dysferlin dimer FRET efficiencies on acceptor concentrations within the range examined. (D) Summary of FRET_max values obtained by fitting (expressed as mean ± S.E.M.). (E) Summary of <i>K_D</i> values obtained by fitting (expressed as mean ± S.E.M.). ND: not determined.</p

Schematic model of dysferlin protein.

<p>(A) We propose dysferlin forms a parallel homodimer through physical interactions of domains C2B to C2G and the transmembrane domains. The domains are color-coded according to measured dissociation constants to indicate the relative contribution of each domain to dimerization. After membrane damage, Ca²⁺ enters the cell and binds to the C2A domains. Ca²⁺-dependent C2A-membrane interactions result in bridging of two membranes: vesicle to plasma membrane (B, D), vesicles to vesicles (C), and two sides of the broken plasma membrane (E), thus promoting the formation of a dysferlin-membrane barrier surrounding membrane pores to accomplish membrane repair.</p

Summary of FCS experiments.

<p>Parameters shown are observed diffusion correlation time (τ_D), diffusion coefficient (D), and apparent hydrodynamic radius (r_h).</p

FRET analyses of adult FDB muscle fibers transfected with CFP-dysferlin and YFP-dysferlin.

<p>(A) CFP-dysferlin and YFP-dysferlin fluorescence images before (Prebleach) and after (Postbleach) YFP-selective photobleaching. Scale bar: 20 µm. (B) Linear relationship between CFP-dysferlin and YFP-dysferlin fluorescence suggests dysferlin forms a dimer in FDB muscle fibers. (C) Independence of dysferlin dimer FRET efficiencies on YFP-dysferlin concentrations within the range examined. (D) Membrane repair defect in dysferlin-deficient muscle fibers was corrected by the CFP-dysferlin, suggesting that the fluorescent protein tag on dysferlin does not compromise the function of dysferlin.</p

Fluorescence correlation spectroscopy and photon counting histogram analyses of dysferlin dimerization.

<p>(A) FCS data obtained from YFP control (black) showed a significantly faster diffusion compared to full length YFP dysferlin in detergent solution. The boxed region is enlarged and reproduced in the next panel. (B) Diffusion of YFP-dysferlin in 1% CHAPS (red) and 0.5% DDM (blue) were well described by a single species diffusion model (<b>Equation 1</b>), with a correlation time of 2.7 ms. This the apparent diffusion time constant was decreased in SDS (green) to 2.1 ms, suggesting a smaller species half the size of YFP-dysferlin in CHAPS or DDM. Triton X-100 solubilization yielded a YFP-dysferlin correlation (gray) that was intermediate between SDS and DDM/CHAPS. These data were best described by a fit to 2 species differing in molecular weight by 2-fold (<b>Equation 3</b>). The data suggest that the dysferlin complex is partially destabilized by Triton X-100, but some dimers remain. (C) PCH analysis yielded the molecular brightness (ε) of diffusing species, normalized to the brightness of control monomeric YFP. The molecular brightness of monomers and dimers are highlighted (dotted red lines). YFP-dysferlin in CHAPS and DDM had a molecular brightness exactly twice the measured brightness of monomeric YFP, suggesting stable dimers in these detergents. YFP-dysferlin in Triton X-100 and individual C2-domains all yielded intermediate molecular brightness, which is compatible with a mixture of monomers and dimers. *** indicates p<0.001, ** p<0.01 and * p<0.05 when compared to the molecular brightness of YFP.</p

Dysferlin self-interaction in living HEK293 cells shown by acceptor-selective photobleaching FRET assay.

<p>(A) Confocal microscopy and total internal reflection fluorescence (TIRF) microscopy images of YFP-dysferlin expressed in HEK293 cells. Scale bars: 10 µm. (B) CFP-dysferlin and YFP-dysferlin fluorescence images before (Prebleach) and after (Postbleach) YFP-selective photobleaching. Scale bar: 5 µm. (C) Quantitative analysis of CFP-dysferlin and YFP-dysferlin fluorescence intensities (F/F₀) during YFP-selective photobleaching. (D) The relationship between the normalized fluorescence of CFP-dysferlin and the normalized fluorescence of YFP-dysferlin during progressive photobleaching was linear, consistent with a homodimeric dysferlin complex.</p