N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy

Abstract

Mutations in amphiphysin-2/BIN1, dynamin 2, and myotubularin are associated with centronuclear myopathy (CNM), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis-splicing of amphiphysin-2/BIN1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM. How amphiphysin-2 orchestrates nuclear positioning and triad organization and how CNM-associated mutations lead to muscle dysfunction remains elusive. We find that N-WASP interacts with amphiphysin-2 in myofibers and that this interaction and N-WASP distribution are disrupted by amphiphysin-2 CNM mutations. We establish that N-WASP functions downstream of amphiphysin-2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b-dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N-WASP and amphiphysin-2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N-WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N-WASP in amphiphysin-2-dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology.

Keywords: centronuclear myopathy; cytoskeleton; nuclear movement; triad formation.

Figure 1. Peripheral localization of nuclei and organization of transversal T-tubules paired with sarcoplasmic reticulum (SR) in in vitro myofibers

1. Images from a representative time-lapse dual color phase-contrast movie of H2B-GFP myotubes (Supplementary Movie S1), recorded from day 1 (after agrin addition) until day 10, showing nuclear positioning to the periphery during myofiber maturation. Arrowhead indicates an example of a nucleus going to the periphery. Bar, 15 µm.

2. Quantification of peripheral nuclei in H2B-GFP and WT myofibers, treated or untreated with agrin and differentiated for 5 or 10 days. Error bars, s.e.m., n = 3. P-values from t-test (untreated versus treated condition).

3. Representative immunofluorescence images of WT myofibers treated with agrin for 5 days immunostained for DHPR (red), a T-tubule marker, triadin (green), a junctional SR marker, and DAPI (blue). On the right are 2× magnifications of rectangles. Bar, 15 μm.

4. Representative immunofluorescence images of WT myofibers treated with agrin for 10 days immunostained for DHPR (red), triadin (green), and DAPI (blue). On the right are 2× magnifications of rectangles. Bar, 15 μm.

5. Quantification of transversal triads in WT myofibers treated or untreated with agrin and differentiated for 5 or 10 days. Error bars, s.e.m., n = 3. P-values from t-test (untreated versus treated condition).

6. Representative electron microscopy image of WT primary myofibers, transfected with GAPDH siRNA and treated with agrin for 10 days. Bar 1 µm.

7. Magnification of the rectangle in (F). Arrows indicate triads located between myofibrils at the level of A–I border. Bar 500 nm. Inset: high magnification of the right lower triad. Arrowheads indicate the RyR feet. Bar 100 nm. SR: sarcoplasmic reticulum.

Figure 2. Amph2 is required for triad formation and peripheral localization of nuclei in in vitro myofibers

1. Representative immunofluorescence images of WT primary myofibers treated with agrin for 10 days and immunostained for amph2 (red), RyR (green), and DAPI (blue). Bar, 15 μm.

2. Representative immunofluorescence images of WT primary myofibers treated with agrin for 10 days and immunostained for amph2 (red), Cav3 (green), and DAPI (blue). Bar, 15 μm.

3. Representative immunofluorescence images of WT primary myofibers transfected with GAPDH siRNA, treated with agrin for 10 days, and immunostained for DHPR (red), triadin (green), and DAPI (blue). On the right 2× magnifications of rectangles. Bar, 15 μm.

4. Representative immunofluorescence images of WT primary myofibers transfected with Amph2 siRNA, treated with agrin for 10 days, and immunostained for DHPR (red), triadin (green), and DAPI (blue). On the right 2× magnifications of rectangles. Bars, 15 μm.

5. Representative immunofluorescence images of WT primary myofibers transfected with GAPDH siRNA, treated with agrin for 10 days, and immunostained for α-actinin (red), F-Actin (green). Bar, 15 μm.

6. Representative immunofluorescence images of WT primary myofibers transfected with Amph2 siRNA, treated with agrin for 10 days, and immunostained for α-actinin (red), F-Actin (green). Bar, 15 μm.

7. Quantification of peripheral nuclei in myofibers transfected with GAPDH, Amph2 siRNA, or Amph2 ex11 siRNA, and treated with agrin for 10 days. Error bars, s.e.m., n = 3. P-values from t-test (GAPDH siRNA versus Amph2 siRNA condition).

8. Quantification of transversal triads in myofibers transfected with GAPDH, Amph2 siRNA, or Amph2 ex11 siRNA and treated with agrin for 10 days. Error bars, s.e.m., n = 3. P-values from t-test (GAPDH siRNA versus Amph2 siRNA condition).

9. Quantification of myofiber thickness in GAPDH siRNA, Amph2 siRNA, or Amph2 ex11 siRNA and treated with agrin for 10 days. Error bars, s.e.m., n = 3. P-values from t-test (GAPDH siRNA versus Amph2 siRNA condition).

Figure 3. Amph2 interacts with N-WASP and the interaction is disrupted by CNM-associated amph2 mutations

A Western blot with N-WASP and amph2 antibodies of endogenous amph2 immunoprecipitation in primary myofibers, treated with agrin for 10 days. B Western blot with N-WASP and amph2 antibodies of endogenous amph2 immunoprecipitation in mouse muscle or C2C12 myotubes. C Western blot with N-WASP and amph2 antibodies of endogenous N-WASP immunoprecipitation in primary myofibers. D Diagrams of the amph2 constructs used in (F). All constructs are N-terminally tagged with GFP. Numbers are amino acid position. E Diagrams of the amph2 full-length CNM mutants used in (G). Point mutation position is highlighted by a star. All constructs are N-terminal tagged with GFP. F, G Western blot with N-WASP and GFP antibodies of GFP immunoprecipitation in C2C12 cells expressing the indicated constructs. H GST pull down of N-WASP using the indicated GST-tagged amph2 proteins from muscle homogenates (top) or in vitro transcribed/translated GFP-N-WASP (middle). Coomassie blue staining of loaded gel is shown at the bottom. Source data are available online for this figure.

Figure 4. N-WASP is required for peripheral localization of nuclei and triad organization during myofiber formation

1. Representative immunofluorescence images of WT primary myofibers transfected with GAPDH siRNA, treated with agrin for 10 days, and immunostained for DHPR (red), triadin (green), and DAPI (blue). Bar, 15 μm.

2. Representative immunofluorescence images of WT primary myofibers transfected with N-WASP siRNA, treated with agrin for 10 days, and immunostained for DHPR (red), triadin (green), and DAPI (blue). Bars, 15 μm.

3. Representative immunofluorescence images of WT primary myofibers transfected with GAPDH siRNA, treated with agrin for 10 days, and immunostained for α-actinin (red) and F-Actin (green). Bar, 15 μm.

4. Representative immunofluorescence images of WT primary myofibers transfected with N-WASP siRNA, treated with agrin for 10 days, and immunostained for α-actinin (red) and F-Actin (green). Bar, 15 μm.

5. Quantification of peripheral nuclei in myofibers transfected with GAPDH or N-WASP siRNA and treated with agrin for 10 days. Error bars, s.e.m., n = 3. P-values from t-test.

6. Quantification of transversal triads in myofibers transfected with GAPDH or N-WASP siRNA and treated with agrin for 10 days. Error bars, s.e.m., n = 3. P-values from t-test.

7. Quantification of myofiber thickness in myofibers transfected with GAPDH or N-WASP siRNA and treated with agrin for 10 days. Error bars, s.e.m., n = 3. P-values from t-test.

Figure 5. N-WASP involvement downstream of amph2

1. Representative immunofluorescence images of myofibers transfected with GAPDH siRNA and GFP (top), GFP-N-WASP full-length (N-WASP FL, middle) or GFP-VCA (VCA, bottom), treated with agrin for 10 days, and stained for DHPR (red) and DAPI (blue). Bar, 15 μm.

2. Representative immunofluorescence images of myofibers transfected with Amph2 siRNA and GFP (top), GFP-N-WASP (middle), or GFP-VCA (bottom), treated with agrin for 10 days and stained for DHPR (red) and DAPI (blue). Bar, 15 μm.

3. Representative immunofluorescence images of myofibers transfected with GFP-VCA and GAPDH siRNA (top) or Amph2 siRNA (bottom), and stained for amph2 (red) and DAPI (blue). Bar, 15 μm.

4. Western blot with amph2 and β-tubulin antibodies of myofibers transfected with Amph2 siRNA or Amph2 siRNA and GFP-VCA.

5. Quantification of peripheral nuclei in myofibers transfected with GAPDH (light blue) or Amph2 (blue) siRNA and with GFP, GFP-N-WASP-FL, or GFP-VCA, and treated with agrin for 10 days. Error bars, s.e.m., n = 3. P-values from t-test (GFP versus GFP-N-WASP-FL or GFP-VCA).

6. Quantification of transversal triads in myofibers transfected with GAPDH (pink) or Amph2 (red) siRNA and with GFP, GFP-N-WASP-FL, or GFP-VCA, and treated with agrin for 10 days. Error bars, s.e.m., n = 3. P-values from t-test (GFP versus GFP-N-WASP-FL or GFP-VCA).

7. Quantification of myofiber thickness in myofibers transfected with GAPDH (light green) or Amph2 (green) siRNA and with GFP and GFP-VCA, and treated with agrin for 10 days. Error bars, s.e.m., P-values from t-test (GFP versus GFP-VCA conditions).

Source data are available online for this figure.

Figure 6. Role of microtubules and actin in nuclear positioning and triad organization during myofiber maturation

A–C Representative immunofluorescence images of WT primary myofibers transfected with GAPDH siRNA (A), Map7 siRNA (B), or Kif5b siRNA (C), treated with agrin for 10 days and subsequently immunostained for DHPR (red), triadin (green), and DAPI (blue). Bar, 15 μm. D Quantification of peripheral nuclei in myofibers transfected with GAPDH, Map7, or Kif5b siRNA. Error bars, s.e.m., n = 3. P-values from t-test. E Quantification of transversal triads in myofibers transfected with GAPDH, Map7, or Kif5b siRNA. Error bars, s.e.m., n = 3. P-values from t-test. F Quantification of myofiber thickness in myofibers transfected with GAPDH, Map7, or Kif5b siRNA. Error bars, s.e.m., n = 3. P-values from t-test. G–I Representative immunofluorescence images of WT primary myofibers untreated (G) or treated with 75 nM of nocodazole (H) or 10 μM of latrunculin B (I) at day 5 after agrin treatment, and immunostained at day 10 for DHPR (red), triadin (green), and DAPI (blue). Bar, 15 μm. J Quantification of peripheral nuclei in myofibers untreated or treated with nocodazole and latrunculin B as described in (G–I). Error bars, s.e.m., n = 3. P-values from t-test. K Quantification of transversal triads in myofibers untreated or treated with nocodazole and latrunculin B as described in (G–I). Error bars, s.e.m., n = 3. P-values from t-test. L Quantification of myofiber thickness in myofibers untreated or treated with nocodazole and latrunculin B as described in (G–I). Error bars, s.e.m., n = 3. P-values from t-test. M, N Representative immunofluorescence images of WT primary myofibers transfected with RFP-KASHΔL (M) and RFP-SR-KASH (N), 10 days after agrin addition immunostained for triadin (green) and DAPI (blue). Bar, 15 μm. O Quantification of peripheral nuclei in myofibers transfected with RFP-KASHΔL or RFP-SR-KASH as described in (M, N). Error bars, s.e.m., n = 3. P-values from t-test. P Quantification of transversal triads in myofibers transfected with RFP-KASHΔL or RFP-SR-KASH as described in (M, N). Error bars, s.e.m., n = 3. P-values from t-test. Q Quantification of myofibers thickness in myofibers transfected with RFP-KASHΔL or RFP-SR-KASH as described in (M, N). Error bars, s.e.m., n = 3. P-values from t-test.

Figure 7. N-WASP and amph2 localization in adult muscle and its role on the maintenance of triad organization

1. Left panel: representative immunofluorescence images of isolated WT mouse muscle fibers immunostained for amph2 (red) or N-WASP (green). Right panel: line-scan of indicated region in the left panels showing average intensity of N-WASP (green) and amph2 (red), respectively. Bar, 1 μm.

2. Representative immunofluorescence images of longitudinal sections of WT mouse muscle electroporated with GAPDH siRNA and immunostained after 10 days for GAPDH (red) and amph2 (green). A star indicates a myofiber depleted for GAPDH. Bar, 50 μm.

3. Representative immunofluorescence images of longitudinal sections of WT mouse muscle electroporated with GAPDH siRNA and immunostained after 10 days for GAPDH (red) and N-WASP (green). A star indicates a myofiber depleted for GAPDH. Bar, 50 μm.

4. Representative immunofluorescence images of longitudinal sections of WT mouse muscle electroporated with Amph2 siRNA and immunostained after 10 days for amph2 (red), N-WASP (green). A star indicates a myofiber depleted for amph2. Bar, 50 μm.

5. Representative immunofluorescence images of longitudinal sections of WT mouse muscle electroporated with N-WASP siRNA and immunostained after 10 days for N-WASP (red), and amph2 (green). A star indicates a myofiber depleted for N-WASP. Bar, 50 μm.

6. Representative immunofluorescence images of longitudinal sections of WT mouse muscle electroporated with Amph2 siRNA and immunostained after 10 days for amph2 (red), DHPR (green). A star indicates a myofiber depleted for amph2. Bar, 50 μm.

7. Representative immunofluorescence images of longitudinal sections of WT mouse muscle electroporated with N-WASP siRNA and immunostained after 10 days for, N-WASP (red) and DHPR (green). A star indicates a myofiber depleted for N-WASP. Bar, 50 μm.

8. Representative immunofluorescence images of longitudinal sections of WT mouse muscle electroporated with Amph2 siRNA and immunostained after 10 days for amph2 (red) and RyR (green). A star indicates a myofiber depleted for amph2. Bar, 50 μm.

9. Representative immunofluorescence images of longitudinal sections of WT mouse muscle electroporated with N-WASP siRNA and immunostained after 10 days for N-WASP (red), and RyR (green). A star indicates a myofiber depleted for N-WASP. Bar, 50 μm.

10. Left panels: representative immunofluorescence images of isolated mouse muscle fibers, immunostained for triadin (red) and DHPR (green) in GAPDH siRNA, Amph2 siRNA, and N-WASP siRNA electroporated fibers, respectively. Right panel: line-scan of indicated regions in the left panel showing average intensity of N-WASP staining (green) compared to amph2 (red), respectively. Bar, 1 μm.

Figure 8. N-WASP and amph2 localization in muscle from healthy and ARCNM patients

1. Representative immunofluorescence images of transversal sections of human muscle from healthy donor (top) or ARCNM patient carrying AMPH2 R154Q mutation, immunostained for DHPR (red), N-WASP (green), and DAPI (blue). Bars, 15 μm.

2. Representative immunofluorescence images of transversal sections of human muscle from healthy donor (top) or ARCNM patient carrying AMPH2 R154Q mutation, immunostained for amph2 (red), N-WASP (green), and DAPI (blue). Bars, 15 μm.

3. Representative immunofluorescence images of transversal sections of human muscle from healthy donor (top) or DM1 patient, immunostained for amph2 (red), N-WASP (green), and DAPI (blue). Bars, 15 μm.

4. Schematic representation of the pathways that regulate triad organization and peripheral nuclear position downstream of amph2 and N-Wasp.

5. Model for peripheral nuclear positioning during myofiber formation. After fusion of myoblast, nuclei (red) of myotubes cluster in the center of the myotube before nuclear spreading. After nuclei spread along the myotube, they become peripheral located at the myofiber periphery.