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First published online 3 March 2009
doi: 10.1242/jcs.040121

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TC10 controls human myofibril organization and is activated by the sarcomeric RhoGEF obscurin

¹ INSERM, ERI 25, `Muscle and Pathologies', F-34295 Montpellier, France
² Université Montpellier I, EA4202, F-34295 Montpellier, France
³ IGMM, CNRS-UMR5535, 1919 route de Mende, F-34293 Montpellier, France
⁴ Universités Montpellier II et I, Centre de Recherche en Biologie Macromoléculaire, F-34293, Montpellier, France
⁵ CNRS, UMR5237, 1919 Route de Mende, F-34293 Montpellier, France

View larger version (36K): [in this window] [in a new window]   Fig. 1. Expression and activity of TC10 during differentiation of human primary myoblasts. (A) Lysates from human primary myoblasts, collected at different times of differentiation, were separated by SDS-PAGE and western blotted with the indicated antibodies (supplementary material Fig. S1). The results shown are typical of four independent experiments. (B) The level of active TC10 in lysates from proliferating (proliferation) and differentiated (differentiation) myoblasts was measured by binding to GST-PBD (IP). Proteins were separated by SDS-PAGE and visualized by immunoblotting (IB) with the indicated antibodies. The results shown are typical of three independent experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Interaction between obscurin and TC10 proteins overexpressed in HEK293T cells. (A) Schematic representation of the obscurin C-terminus and the various expression vectors used. (B) HEK293T cells were transfected with expression vectors for Myc-tagged DH domain (Myc-ObsDH) or C-terminal region (Myc-ObsCT) of obscurin, along with HA-TC10 or HA-Elmo, as indicated. The latter were immunoprecipitated (IP) from total cell lysates with an anti-HA monoclonal antibody (HA), and the binding of obscurin was detected by immunoblotting (IB) with a polyclonal antibody to the Myc epitope. The results shown are typical of four independent experiments. (C) The same experiment as B, except that the immunoprecipitation (IP) was performed with anti-obscurin polyclonal antiserum (Obs) or non-specific rabbit IgG (IgG). Binding of TC10 to the immunoprecipitated protein was detected by immunoblotting (IB) with an anti-HA monoclonal antibody. The lanes marked Lysate were loaded with 10% of the total cell lysates. The results shown are typical of four independent experiments. (D) HEK293T cells were transfected with expression vectors for either Myc-tagged C-terminal region (Myc-ObsCT) of obscurin or same vector lacking the DH-PH domain [Myc-ObsCT(DH-PH)], along with either HA-TC10CA (TC10/Q75L) (Chiang et al., 2001) or HA-TC10DN (TC10/T31N) (Chiang et al., 2001) as indicated. The immunoprecipitation was performed with anti-obscurin polyclonal antiserum (IP Obs). Binding of TC10 mutants to the immunoprecipitated protein was detected by immunoblotting (IB) with an anti-HA monoclonal antibody. The lanes marked Lysate were loaded with 10% of the total cell lysates. The results shown are typical of three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Obscurin interacts with TC10 in myotubes. (A) Differentiated human myotubes were transduced with empty lentiviral vector or vector expressing HA-TC10 as indicated. Endogenous obscurin was immunoprecipitated (IP) from total cell lysates with anti-obscurin polyclonal antiserum (Obs) or non-specific rabbit IgG (IgG). Binding of TC10 to the obscurin immunoprecipitates was detected by immunoblotting (IB) with an anti-HA monoclonal antibody (HA). Lysate represents 5% of the total cell lysates. The results shown are typical of three independent experiments. (B) Purified recombinant GST-obscurin DH domain (GST-ObsDH) or GST alone were incubated with immobilized MBP-TC10, and the washed complexes were separated by SDS-PAGE. Binding of the GST fusion was detected by immunoblotting (IB) with an anti-GST polyclonal antiserum (GST). 5% of the recombinant protein input was loaded in the lane marked Lysate. The results shown are typical of three independent experiments.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Obscurin activates TC10. (A) HEK293T cells were transfected with expression vectors for Myc-tagged C-terminal region of obscurin (Myc-ObsCT), HA-tagged wild-type (HA-TC10 wt) or constitutively active TC10 (HA-TC10 CA), and the PBD of PAK1 fused to GFP (GFP-PBD), as indicated. Expression levels of the different proteins were confirmed by immunoblotting after SDS-PAGE of 10% of each cell lysate (IB). Active TC10 was isolated by immunoprecipitating its complexes with GFP-PBD with an anti-GFP polyclonal antiserum (IP PBD) and was visualized by immunoblotting with the HA monoclonal antibody. The histogram represents the level of active HA-TC10 normalized for the amount of total HA-TC10 expressed in cells. Numbers on x-axis correspond to the lanes in blots above. Data are the mean ± s.e.m. of four experiments (n=4) (**P<0.001). (B) Human primary myotubes were transfected with Myc-tagged C-terminal region of obscurin (Myc-ObsCT) or the same vector lacking the DH-PH domain [Myc-ObsCT(DH-PH)] as indicated (+). The endogenous Rho GTPases TC10, Cdc42 and Rac in their active state were isolated via their binding to GST-PBD (IP PBD). Cell lysates were separated by SDS-PAGE and proteins visualized by immunoblotting (IB) with the indicated antibodies. Lysate represents 5% of the total cell lysates. The histogram represents the level of active Rho GTPase normalized for the amount of total Rho GTPase expressed in cells as indicated. Data are the means ± s.e.m. of three independent experiments (n=3) (**P<0.001).

View larger version (43K): [in this window] [in a new window]   Fig. 5. Activation of TC10 by expression of the C-terminal region of obscurin induces PAK autophosphorylation in human primary myotubes. (A) Lysates from human primary myoblasts, collected at different times of differentiation, were separated by SDS-PAGE and western blots were incubated with the indicated antibodies. The histogram represents the endogenous level of phosphorylated PAK normalized for the amount of total PAK expressed in cells. Numbers on x-axis correspond to the lanes in blots above. Data are the means ± s.e.m. of three independent experiments (n=3). (B) Human myoblasts were transduced with lentiviral vectors expressing either a control (C shRNA, lane 1) or a TC10-specific shRNA (TC10 shRNA, lane 2) and maintained in culture until terminal differentiation. Cell lysates were separated by SDS PAGE and proteins visualized by immunoblotting (IB) with the indicated antibodies. The histogram shows the quantification of phosphorylated PAK normalized to the amount of PAK in cells. Data are the means ± s.e.m. of five independent experiments (n=5) (***P<0.0001). (C) Human primary myotubes were transfected with expression vectors for Myc-tagged C-terminal region of obscurin (Myc-ObsCT) and/or HA-tagged wild-type (HA-TC10 wt) or constitutively active TC10 (HA-TC10 CA), as indicated. Cell lysates were separated by SDS-PAGE and proteins visualized by immunoblotting (IB) with the indicated antibodies. The histogram shows the quantification of phosphorylated PAK (PPAK) normalized for the amount of PAK in cells. Data are the means ± s.e.m. of four independent experiments (n=4) (*P<0.05).

View larger version (47K): [in this window] [in a new window]   Fig. 6. Effect of TC10 on sarcomeric protein expression. (A) Human myoblasts were transduced with lentiviral vectors expressing either a control (C shRNA, lane 1) or a TC10-specific shRNA (TC10 shRNA, lane 2) and maintained in culture until terminal differentiation. Cell lysates were separated by SDS-PAGE and proteins analyzed by immunoblotting with the indicated antibodies. The results shown are typical of six independent experiments. (B) Human myoblasts were transduced with empty lentiviral vector (C, lane 1) or vectors expressing either HA-tagged wild-type (HA-TC10 wt, lane 2) or constitutively active TC10 (HA-TC10 CA, lane 3), as indicated. As above, cells were maintained in culture until terminal differentiation. Cell lysates were separated by SDS-PAGE and proteins visualized by immunoblotting with the indicated antibodies. The results shown are typical of six independent experiments. (C) Quantification of the expression level of the proteins obtained in A and B. Data are the means ± s.e.m. of six independent experiments (n=6) (***P<0.0001).

View larger version (56K): [in this window] [in a new window]   Fig. 7. Inhibition of TC10 expression or activity blocks myofibrillogenesis. (A) Human myotubes were transfected with expression vectors for GFP or either HA-tagged wild-type (HA-TC10 wt) or a dominant-negative TC10 (HA-TC10 DN, TC10/T31N). Cells were fixed and incubated with antibodies to the HA epitope and sarcomeric -actinin as indicated, followed by Fluorescently labeled secondary antibodies. DNA was stained with DAPI. Proteins and DNA were detected by fluorescence microscopy: GFP or HA-tagged TC10 proteins (green), sarcomeric -actinin (red) and nuclei (blue). The upper panels show the merged images and the lower panels show -actinin staining. The results shown are typical of three independent experiments. Scale bars: 10 µm. (B) Human myoblasts were transduced with lentiviral vectors expressing GFP and either a control shRNA (shCTL) or TC10 shRNA (shTC10). The cells were maintained in culture until terminal differentiation. Myotubes were fixed and immunostained for sarcomeric -actinin (red) and with DAPI to visualize the nuclei (blue). The upper panels show the merge of GFP, -actinin and DAPI fluorescence. The lower panels are a higher magnification of the boxed areas in the upper panel and show -actinin staining. The results shown are typical of three independent experiments. Scale bars: 10 µm. (C) Human myotubes described in B were analyzed for their myofibril content by -actinin immunostaining. This revealed punctate filaments (premyofibrils), filaments that have started to align (nascent myofibrils) and cross-striated myofibrils (mature myofibrils). Myotubes containing cross-striated myofibrils were counted as mature myofibrils. Myotubes containing aligned but not cross-striated myofibrils were counted as nascent myofibrils and other myotubes, containing only punctuated stain, were counted as pre-myofibrils. The percentage of myotubes displaying myofibrils at different stages of maturation (pre-, nascent or mature) was determined by immunostaining for sarcomeric -actinin in three independent experiments.

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