Subcellular distribution of cell surface-biotinylated β-dystroglycan in C2C12 myoblasts overexpressing active ezrin.

<p><b>A.</b> Cells were transfected to transitory express either ET567D-GFP, Ez-T567A-GFP or GFP alone and incubated with biotin 24 h post-transfection to label cell surface proteins, as described in Material and Methods. Cytosolic and nuclear fractions isolated from biotinylated cells were pulled-down using streptavidin-agarose beads and precipitated proteins were subjected to SDS-PAGE/Western analysis using an anti-β-DG antibody (7D11). Input, biotinylated cytosolic and nuclear extracts subjected to SDS-PAGE/Western analysis without previous streptavidin-mediated precipitation. Membranes were stripped and reprobed for calnexin (Clnx) and Sp3, loading controls for cytoplasmic and nuclear lysates respectively. <b>B.</b> Nuclear to cytoplasmic levels (n/c) of β-DG were quantified as per the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a> and results plotted represent the mean +/- SD from a series of three separate experiments, with significant differences determined by Student t-test.</p

β-DG interacts with ezrin in C2C12 myoblasts.

<p><b>A.</b> Schematic of the domain structure of β-DG. EC, extracellular domain; TM, transmembrane domain; NLS, nuclear localization signal; CYT, cytoplasmic tail. The NLS of β-DG serves as binding site for both importin α2/β1 and ezrin. <b>B.</b> C2C12 cells grown on glass coverslips were fixed and double stained with anti-ezrin (green) and anti- β-DG antibodies prior to be analyzed by confocal laser scanning microscopy (CLSM). Merged images show colocalization between ezrin and β-DG (yellow), specifically at plasma membrane projections (Inset); scale bar is 10 µm. <b>C.</b> C2C12 total extracts were immunoprecipitated (IP) using an anti-β-DG antibody or control antibody (IgG0), and precipitated proteins subjected to SDS-PAGE/Western analysis employing anti-β-DG or anti-ezrin antibodies. <b>D.</b> The IMPα2/β1 heterodimer interacts with the NLS of β-DG with higher affinity than ezrin. Increasing concentrations (0-60 nm) of GST-tagged ezrin, GST-tagged predimerized IMPα2/β1 or GST alone as a control were incubated with a GFP-β-DGNLS fusion protein and an ALPHA-Screen assay performed as described in Materials and Methods. Sigmoidal curves were fitted using the SigmaPlot software to determine the apparent dissociation constants (Kd) as indicated. Each data point represents the average of three measurements from a single typical experiment from a series of three separate experiments.</p

Nuclear translocation of endogenous β-DG induced by active ezrin is dependent on IMPβ1.

<p>C2C12 myoblasts stably transfected with vector expressing either the control or importin β1 (IMPβ1) RNAi were cultured on glass coverslips, fixed and immunostained for IMPβ1 <b>A</b> or β-DG <b>B</b>, using FITC-conjugated secondary antibody (green), with nuclei stained using DAPI (blue). Cells were imaged by CLSM, with typical single Z-sections shown (scale bar is 10 µm). <b>B.</b> Quantitative analysis to determine the nuclear to cytoplasmic ratio (Fn/c) of β-DG was performed in control- and RNAi IMPβ1-transfected cells (bottom panel), as per the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/– SD (n &gt; 50 cells) from a series of three separate experiments, with significant differences between cells expressing the control or IMPβ1 RNAi determined by Student t-test. <b>C.</b> Cytoplasmic and nuclear fractions obtained from cells stably expressing either the control or IMPβ1 RNAi and transiently expressing GFP or Ez-T567D-GFP fusion proteins were analyzed by SDS-PAGE/Western using an anti-β-DG antibody (upper panels). Membranes were stripped and reprobed with antibodies against calnexin (Clnx) and Sp3, loading controls for cytoplasmic and nuclear lysates respectively. Nuclear to cytoplasmic ratio (n/c) of β-DG levels were quantified and plotted (bottom panel), as per the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/– SD from a series of three separate experiments, with significant differences between cells expressing the control or IMPβ1 RNAi determined by Student t-test.</p

Nuclear Import of β-Dystroglycan Is Facilitated by Ezrin-Mediated Cytoskeleton Reorganization

<div><p>The β-dystroglycan (β-DG) protein has the ability to target to multiple sites in eukaryotic cells, being a member of diverse protein assemblies including the transmembranal dystrophin-associated complex, and a nuclear envelope-localised complex that contains emerin and lamins A/C and B1. We noted that the importin α2/β1-recognised nuclear localization signal (NLS) of β-DG is also a binding site for the cytoskeletal-interacting protein ezrin, and set out to determine whether ezrin binding might modulate β-DG nuclear translocation for the first time. Unexpectedly, we found that ezrin enhances rather than inhibits β-DG nuclear translocation in C2C12 myoblasts. Both overexpression of a phosphomimetic activated ezrin variant (Ez-T567D) and activation of endogenous ezrin through stimulation of the Rho pathway resulted in both formation of actin-rich surface protrusions and significantly increased nuclear translocation of β-DG as shown by quantitative microscopy and subcellular fractionation/Western analysis. In contrast, overexpression of a nonphosphorylatable inactive ezrin variant (Ez-T567A) or inhibition of Rho signaling, decreased nuclear translocation of β-DG concomitant with a lack of cell surface protrusions. Further, a role for the actin cytoskeleton in ezrin enhancement of β-DG nuclear translocation was implicated by the observation that an ezrin variant lacking its actin-binding domain failed to enhance nuclear translocation of β-DG, while disruption of the actin cytoskeleton led to a reduction in β-DG nuclear localization. Finally, we show that ezrin-mediated cytoskeletal reorganization enhances nuclear translocation of the cytoplasmic but not the transmembranal fraction of β-DG. This is the first study showing that cytoskeleton reorganization can modulate nuclear translocation of β-DG, with the implication that β-DG can respond to cytoskeleton-driven changes in cell morphology by translocating from the cytoplasm to the nucleus to orchestrate nuclear processes in response to the functional requirements of the cell.</p></div

Ezrin-mediated cytoskeleton reorganization facilitates nuclear translocation of β-DG.

<p><b>A.</b> C2C12 myoblasts grown on coverslips were transfected to express GFP-tagged-EzT567D (active ezrin), -EzT567ΔNLS (active ezrin carrying a deletion of the NLS), or -EzΔABD (ezrin variant with a deletion of the actin-binding domain), or GFP alone. Transfected cells were immunostained with anti-β-DG primary and TRITC-conjugated secondary antibodies and counterstained with DAPI (nuclei), prior to CLSM, with typical single Z-sections shown (scale bar is 10 µm). Images were analysed to determine the Fn/c for β-DG (bottom), as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/– SD (n &gt; 50) from three separate experiments, with significant differences in the Fn/c of β-DG between cells expressing GFP alone and those expressing GFP-Ez-T567D, and between cells expressing GFP-Ez-T567D- and GFP-EzΔABD, as denoted by the p values. <b>B.</b> Cytoplasmic and nuclear extracts from transfected cells with the above constructs were analyzed by SDS/Western blotting using anti-β-DG antibodies. Sp3 and calnexin (Clnx) were immunodetected as loading controls for nuclear and cytoplasmic fractions respectively. Densitometric analysis was performed to obtain the n/c ratio of β-DG for the different transfected cultures (right panel), as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/- SD for 3 separate experiments, with significant differences between cells expressing GFP alone and those expressing GFP-Ez-T567D, as well as between cell expressing GFP-Ez-T567D and those expressing GFP-EzΔABD, as denoted by the p values. <b>C.</b> C2C12 myoblasts seeded in coverslips were treated without (control) or with 6 µM cytochalasin B (Cyt B) for 1 h. Treated cells were fixed and double-stained with anti-β-DG primary antibody and fluorescein-conjugated secondary antibody, and with TRITC-phalloidin to visualize effects on the actin-based cytoskeleton network. Nuclei were counterstained with DAPI. Samples were imaged and subjected to image analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/- SD for three separate experiments (n&gt; 50), with p values determined by Student t-test denoting significant differences between control and Cyt B-treated cells (bottom panel).</p

Rho signaling-mediated activation of ezrin facilitates nuclear accumulation of β-DG.

<p><b>A.</b> Treatment of C2C12 cells with the toxin C3, a specific Rho inhibitor, decreased β-DG nuclear accumulation. Lysates from cells treated or not with C3 were analyzed by SDS/Western blotting using an antibody that recognizes phosphorylated ezrin, raxidin and moesin (p-ERM). The membranes were reprobed with antibody for total ezrin (upper panel). Cells grown on coverslips were treated without or with C3, fixed and immunolabeled using anti-β-DG primary and a fluorescein-conjugated secondary antibodies and nuclei stained with DAPI. Cells were imaged and quantitative analysis for Fn/c of β-DG was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a> (bottom right panel) showing significant differences between cells treated or not with C3. <b>B.</b> Nuclear and cytoplasmic fractions from control and C3-treated cells were analyzed by SDS/Western blotting using anti- β-DG antibodies (upper panels). Immunodetection of Sp3 and Calnexin (Clnx) was used as loading control for nuclear and cytoplasmic fractions respectively. The n/c ratio for β-DG was quantified and plotted as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a> (bottom panel) with significant differences determined by <i>p</i> value. <b>C.</b> C2C12 cells treated with LPA (inductor of the Rho pathway) show increased β-DG nuclear accumulation. Lysates from cells pretreated with LPA or PBS for 0–30 min were analyzed by SDS/Western blotting using anti-p-ERM or anti-ezrin antibodies (upper left panel). Control and LPA-treated cells were immunolabeled for β-DG and imaged as before (left panel) with significant differences between control and treated cells. <b>D.</b> Cytoplasmic and nuclear extracts of untreated and LPA-treated cells were analyzed by SDS/Western blotting using anti-β-DG antibodies. Sp3 and Calnexin were employed as loading controls (upper panels). The n/c ratio for β-DG shows significant differences between control and treated cells. All results representing the mean +/- SD from three separate experiments were analyzed by Student t-test.</p

Overexpression of active ezrin facilitates nuclear translocation of β-DG.

<p><b>A.</b> C2C12 myoblasts cultured on glass coverslips were transfected to express ezrin-GFP (Ez) fusion proteins (either wild type, WT, or the mutated variants T567D and Ez-T567A) or GFP alone. Cells were fixed and stained 24 h post-transfection with a polyclonal anti-β-DG antibody (JAF) and TRITC-conjugated secondary antibody, with nuclei stained using DAPI (blue). Cells were imaged by CLSM, with typical single Z-sections shown (scale bar is 10 µm). Quantitative analysis for the nuclear to cytoplasmic ratio (Fn/c) of β-DG was performed (bottom panel) using the Image J software, as described in Material and Methods. Results represent the mean +/– SD (n &gt; 50 cells) from a series of three separate experiments, with significant differences between cells expressing GFP alone and cells expressing the different GFP-tagged ezrin variants determined by Student t-test. <b>B.</b> Cytoplasmic and nuclear extracts obtained from cells transfected to express the above constructs were separated by SDS-PAGE and subjected to Western analysis for β-DG. Membranes were stripped and reprobed for Sp3 and calnexin (Clnx); loading controls for nuclear and cytoplasmic extracts respectively. Densitometric analysis of autoradiograms was performed, and the nuclear/cytoplasmic ratio (n/c) for β-DG obtained by dividing the relative levels of β-DG in the nuclear extracts with those obtained in the corresponding cytoplasm extracts (bottom panel). Results represent the mean +/– SD for 3 separate experiments, with significant differences between cells expressing GFP alone and those expressing the different GFP-tagged ezrin variants determined by Student t-test.</p