Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains

Abstract

Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca²⁺-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.

Keywords: C2 domains; actin; annexin A2; dysferlin; dysferlinopathy.

Figure 1

Reduced expression of dysferlin in immortalized myoblasts obtained from dysferlinopathy patients. Dysferlin expression was determined by immunoblotting of total protein extracts from C25 myoblasts obtained from an unaffected donor, or DYSF2, DYSF3, AB320, or ER myoblasts obtained from dysferlinopathy patients. (a) Example of immunoblot detection of dysferlin (upper bands) and β-tubulin (loading control; bottom bands). (b) Relative density (dysferlin/β-tubulin ratio). Data are means ± SEM from 5 independent immunoblots. * p < 0.05 compared to C25 myoblasts (t-test).

Figure 2

G-actin incorporation into filaments in dysferlin-deficient myoblasts. Fluorescently tagged G-actin incorporation into filaments was assayed in myoblasts permeabilized with 20 µM digitonin in the presence of 300 nM Alexa-Fluor-488 actin, 2 mM ATP-Mg²⁺, and 10 µM free Ca²⁺ during 6 min at 37 °C. After permeabilization, cells were fixed and nuclei were stained with DAPI. Confocal images were acquired at the equatorial plane of the cells using identical exposure settings between compared samples. (a–c) Fluorescent actin filaments in control C25 and dysferlinopathy DYSF2, DYSF3, AB320, and ER myoblasts (a) and RCMH myoblasts non-transfected (N-T) or stably transfected with shRNA for dysferlin (c). Scale bar = 20 µm. Insets show digital zooms of the boxed areas; brightness and contrast were increased to appreciate better the pattern of fluorescent actin. (b–d) Data are means ± SEM. Actin fluorescence intensity was measured in a single focal plane at the equator of cells and normalized by the cell area. The number of analyzed cells from five different cultures is indicated in parentheses. * p < 0.05 compared to C25 (b) or N-T RCMH myoblasts (c) (t-test).

Figure 3

Full-length dysferlin-HA restores G-actin incorporation in dysferlinopathy myoblasts. Control C25 or dysferlinopathy DYSF3 myoblasts were transfected with full-length dysferlin-HA (FL-dysferlin-HA), and 24 h fluorescently tagged G-actin incorporation was assayed as previously described. FL-dysferlin-HA expression was assayed by immunofluorescence using a monoclonal antibody against dysferlin and a Cy3-conjugated anti-rabbit secondary antibody. Confocal images were acquired at the equatorial plane of the cells using identical exposure settings between compared samples. (a) C25 and DYSF3 myoblasts with fluorescent actin filaments (green) and FL-dysferlin-HA immunostaining (red). Scale bar = 10 µm. Insets show digital magnification of the boxed areas. (b) Bars represent means ± SEM. Actin fluorescence intensity was measured in a single focal plane at the equator of cells and normalized by the cell area. The number of cells analyzed from four different cultures is indicated in parentheses. No significant differences (NS) were found (t-test).

Figure 4

The N- or C- terminal dysferlin regions are efficient in restoring G-actin incorporation in dysferlinopathy myoblasts. (a) Schematic representation of dysferlin regions included in the N-terminal and C-terminal constructs. Blue arrows indicate positions of dysferlin mutations carried by the cell lines (see Table 1). (b,c) Control C25 or dysferlinopathy DYSF3 myoblasts were transfected with mCherry alone (Cherry), or dysferlin N-terminal (N-term) or C-terminal (C-term) fused to mCherry. Twenty-four hours later, fluorescently tagged G-actin incorporation was assayed as described in Methods, and confocal images were acquired at the equatorial plane of the cells using identical exposure settings between compared samples. (b) Fluorescent actin filaments in C25 or DYSF3 myoblasts expressing Cherry, and DYSF3 myoblasts expressing N-term or C-term. Scale bar = 10 µm. Insets show digital magnification of the boxed areas. (c) Actin fluorescence intensity was measured in a single focal plane and normalized by the cell area. Bars represent means ± SEM. Actin fluorescence intensity was measured in a single focal plane at the equator of cells and normalized by the cell area. The number of analyzed cells from four different cultures is indicated in parentheses. * p < 0.05 compared with C25 myoblasts expressing mCherry, ^& p < 0.05 compared with DYSF3 expressing mCherry (t-test).

Figure 5

Annexin A2 distribution in dysferlinopathy myoblasts. Distribution of annexin A2 was analyzed by immunofluorescence using an anti-annexin A2 antibody and Cy3-conjugated anti-rabbit secondary antibody in C25 and DYSF3 myoblasts permeabilized with 20 µM digitonin in the presence of 300 nM Alexa-Fluor-488 actin, 2 mM ATP-Mg²⁺, and 10 µM free Ca²⁺. Confocal images were captured at the equatorial plane of the cells using identical exposure settings between compared samples; therefore, annexin A2 distribution was measured in a single focal plane. (a) C25 and DYSF3 myoblasts immunostained with annexin A2 (red) and fluorescent G-actin incorporated into filaments (green). Scale bar = 20 µm. Insets show digital magnification of the boxed areas. (b,c) Bars represent means ± SEM of the ratio of the mean fluorescence intensity of the cytosol/whole cell of annexin A2 immunostaining (b) and Pearson correlation coefficient for colocalization of annexin A2 with fluorescent actin filaments (c). The number of analyzed cells from four different cultures is indicated in parentheses. * p < 0.05 (t-test).

Figure 6

Annexin A2 distribution in DYSF3 myoblasts expressing dysferlin constructs. Distribution of annexin A2 was analyzed by immunofluorescence using an anti-annexin A2 antibody and Cy2-conjugated anti-rabbit secondary antibody in C25 and DYSF3 myoblasts expressing full-length dysferlin-HA (FL-dysferlin-HA) (a,b) and mCherry (Cherry), or dysferlin N-terminal (N-term) or C-terminal (C-term) fused to mCherry (c,d). Experiments were performed in digitonin-permeabilized cells in the presence of 10 µM free Ca²⁺. Confocal images were captured at the equatorial plane of the cells using identical exposure settings between compared samples; therefore, annexin A2 distribution was measured in a single focal plane. (a,c) Annexin A2 stained (green) in C25 or DYSF3 myoblasts in a mock condition or expressing FL-dysferlin-HA (a) and N-term or C-term (c). Scale bar = 20 µm. Insets show digital magnification of the boxed areas. (b,d) Data show means ± SEM of the ratio of the mean fluorescence intensity of the cytosol/whole cell of annexin A2 immunostaining. The number of analyzed cells from four different cultures is indicated in parentheses. * p < 0.05 (t-test).