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

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

Summary of FCS experiments.

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

Anoctamin 6 Regulates C2C12 Myoblast Proliferation

<div><p>Anoctamin 6 (<i>Ano6</i>) belongs to a conserved gene family (TMEM16) predicted to code for eight transmembrane proteins with putative Ca²⁺-activated chloride channel (CaCC) activity. Recent work revealed that disruption of <i>ANO6</i> leads to a blood coagulation defect and impaired skeletal development. However, its function in skeletal muscle cells remains to be determined. By using a RNA interference mediated (RNAi) loss-of-function approach, we show that <i>Ano6</i> regulates C2C12 myoblast proliferation. <i>Ano6</i> is highly expressed in C2C12 myoblasts and its expression decreases upon differentiation. Knocking down <i>Ano6</i> significantly reduces C2C12 myoblast proliferation but has minimal effect on differentiation. <i>Ano6</i> deficiency significantly reduces ERK/AKT phosphorylation, which has been shown to be involved in regulation of cancer cell proliferation by another Anoctamin member. Taken together, our data demonstrate for the first time that <i>Ano6</i> plays an essential role in C2C12 myoblast proliferation, likely via regulating the ERK/AKT signaling pathway.</p></div

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

Altered ERK/AKT signaling in <i>Ano6</i>-KD C2C12 myoblasts.

<p>(A) Western blotting analysis of ERK phosphorylation (pERK) and total ERK expression (ERK) in C2C12 myoblasts of different stable lines. Three independent experiments per cell line were loaded on the gel. (B) Normalized expression levels of ERK phosphorylation and total ERK by membrane densitometry. (C) Western blotting analysis of AKT phosphorylation (pAKT) and total AKT expression (AKT) in C2C12 myoblasts of different stable lines. Three independent experiments per cell line were loaded on the gel. (D) Normalized expression levels of AKT phosphorylation and total AKT by membrane densitometry. (E) Western blotting analysis of cyclin D1 in C2C12 myoblasts of different stable lines. (F) Normalized expression levels of cyclin D1 by membrane densitometry. Three independent experiments per cell line were loaded on the gel. GAPDH was used as a loading control. *p<0.05.</p

Expression of Ano6 in C2C12 muscle cells and mouse skeletal muscle.

<p>(A) Semi-quantitative RT-PCR analysis of Ano6 and GAPDH expression in C2C12 cells during differentiation. (B) Relative expression of Ano6 (normalized to GAPDH) examined by qRT-PCR in C2C12 cells during differentiation. (C) Relative expression of Ano6 (normalized to GAPDH) examined by qRT-PCR in the quadriceps muscles of mice at 6 days, 6 weeks and 6 months of age. **p<0.01; ***p<0.001.</p

Effects of <i>Ano6</i>-KD on the proliferation of C2C12 myoblasts.

<p>(A) Relative expression of Ano6 (normalized to GAPDH) examined by quantitative RT-PCR in C2C12 stable cells lines (Scramble [shSCR], <i>Ano6</i>-KD). (B) Representative photographs of stable C2C12 cell lines expressing either a Scramble shRNA or the shRNA targeting <i>Ano6</i>-KD 48 hours post plating. (C) Quantitative analysis of C2C12 myoblast proliferation using the MTT assay. Scale bar  =  150 μm. ***p<0.001.</p

Effect of the ERK inhibitor UO126 on proliferation of control and <i>Ano6</i>-KD C2C12 myoblasts.

<p>(A) Proliferation analysis of control C2C12 cells treated by UO126 (10 μM) or the vehicle alone (DMSO) measured by MTT assay. (B) Proliferation analysis of <i>Ano6</i>-KD C2C12 cells treated by UO126 or the vehicle alone (DMSO) measured by MTT assay. Note that the dashed lines were re-plotted from panel A.</p