Figures and data
Spatiotemporal coordination of macrophages and SCMs during skeletal muscle regeneration.
(A) Diagram of the TA muscle injury scheme. The TA muscles of the wild-type mice were injured by intramuscular injection of BaCI2. The injured TA muscles were collected at dpi 2.5, 3.5, and 4.5 for cross and longitudinal sectioning and immunostaining. (B) Immunostaining with anti-Laminin, anti-NACM, and anti-MAC-2 of the cross and longitudinal sections of TA muscles at the indicated time points. Note the decrease in the macrophage number within the ghost fiber at dpi 3.5 (compared to dpi 2.5), and the fusion of SCMs between dpi 3.5 and 4.5. Scale bars: 20 μm. (C) Quantification of the percentage of macrophages and differentiated SCMs within ghost fibers at the indicated time points. n = 3 mice were analyzed for each time point and > 98 ghost fibers in each mouse were examined. Mean ± s.d. values are shown. (D) Quantification of the number of differentiated SCMs in a single cross section of a ghost fiber at indicated time points. n = 3 mice were analyzed for each time point and > 98 ghost fibers in each mouse were examined. Mean ± s.e.m values are shown.
Branched actin polymerization is required for skeletal muscle regeneration.
(A) Schematic diagram of tamoxifen and BaCI2 treatment and subsequent CSA analysis at dpi 14. (B) Dystrophin and DAPI staining of the cross sections of TA muscles at dpi 14 from the control (Ctrl) and mutant mice. Note that the myofiber CSA is moderately decreased in N-WASPcKO and CYFIP1cKO mice, and severely reduced in dcKO, ArpC2cKO and MymXcKO mice. Scale bar: 100 μm. (C) The fold change of myofiber CSA in mutant mice vs. control mice. n = 3 mice were analyzed for each time point and > 200 fibers in each mouse were examined. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. ****: p < 0.0001. (D) Frequency distribution of myofiber CSA of TA muscles in the control and mutant mice at dpi 14. n = 3 mice of each genotype were examined and > 200 ghost fibers in each mouse were examined.
Branched actin polymerization is required for SCM fusion.
(A) Schematic diagram of tamoxifen and BaCI2 treatment and subsequent SCM number analysis at dpi 4.5. (B) Immunostaining with anti-laminin, anti-NCAM, and anti-MAC-2 of the cross sections of TA muscles at dpi 4.5 from the control and mutant mice. Note that each ghost fiber in the control mice contained 1-2 centrally nucleated myofiber at dpi 4.5, indicating the near completion of SCM fusion. The ghost fibers in N-WASPcKO and CYFIP1cKO mice contained more SCMs, indicating impaired SCM fusion. Note that even more SCMs were seen in dcKO, ArpC2cKO and MymXcKO mice. Scale bar: 20 μm. (C) Quantification of the SCM number in a single cross section of a ghost fiber from TA muscles of the control and mutant mice at dpi 4.5. n = 3 mice were analyzed for each time point and > 80 ghost fibers in each mouse were examined. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. ***: p < 0.001 and ****: p < 0.0001. (D) Frequency distribution of SCM number in a single cross section of a ghost fiber from TA muscles of the mutant mice and their littermate control. n = 3 mice of each genotype were analyzed and > 80 ghost fibers in each mouse were examined. (E) ArpC2 is required for SCM fusion in cultured cells. The satellite cells isolated from ArpC2cKO mice were maintained in GM without or with 2 μM 4OH-tamoxifen (4OHT) for 10 days. Subsequently, the cells were plated at 70% confluence in GM. After 24 hours, the cells were cultured in DM for 48 hours, followed by immunostaining with anti-MHC and DAPI. Note the robust fusion of the control (–4OHT) SCMs and the severe fusion defects in ArpC2 KO (+4OHT) SCMs. Scale bar: 100 μm. (F, G) Quantification of the differentiation index (% of nuclei in MHC+ cells vs. total nuclei) and fusion index (% of nuclei in MHC+ myotubes with ≥ 3 nuclei vs. total nuclei) of the two types of cells shown in (E). n = 3 independent experiments were performed. Mean ± s.d. values are shown in the bar graphs, and significance was determined by two-tailed student’s t-test. ****: p < 0.0001; n.s: not significant. (H) ArpC2 is required in both fusion partners. Fluorescence images from cell-mixing experiments using differentially labelled SCMs are shown. The satellite cells isolated from ArpC2cKO mice were infected with retroviruses encoding GFP or mScarleti (mScar). Next, the GFP+ cells were maintained in GM for 10 days (Ctrl GFP+ cells), and the mScar+ cells were maintained in GM without (Ctrl mScar+ cells) or with 2 μM 4OH-tamoxifen (ArpC2cKO mScar+ cells) for 10 days. Subsequently, the Ctrl GFP+ cells were mixed with Ctrl mScar+ cells or with ArpC2cKO mScar+ cells with a ratio of 1:1 and plated at 70% confluence in GM. After 24 hours, the cells were cultured in DM for 48 hours followed by direct fluorescent imaging. Arrowheads indicate syncytia derived from both GFP and mScar cells. Scale bar: 100 μm. (I) Percentage of GFP+mScar+ syncytia in total cells shown in (H). Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. **: p < 0.01.
Branched actin polymerization is required for invasive protrusion formation during SCM fusion.
(A) Still images of a fusion event between two LifeAct-mScar and Arp2-mNG co-expressing SCMs (see Supplemental Video 4). The boxed area is enlarged in (A’). Note the presence of two invasive protrusions (16 minute, arrowheads) enriched with LifeAct-mScar and Arp2-mNG at the fusogenic synapse. n = 8 fusion events were observed with similar results. Scale bar: 5 μm. (B) TEM of TA muscle cells in wild-type control, ArpC2cKO, and MymXcKO mice at dpi 3.5. The invading SCMs are pseudo-colored in light magenta. Note the finger-like protrusions projected by SCMs invading their neighboring cells in control and MymXcKO, but not in the ArpC2cKO, mice. Scale bars: 500 nm. (C) Quantification of the percentage of SCMs with invasive protrusions in a single cross section of a ghost fiber in the mice with genotypes shown in (B) at dpi 3.5. At least 83 SCMs from n = 20 ghost fibers in each genotype were quantified. Mean ± s.d. values are shown in the dot plots, and significance was determined by two-tailed student’s t-test. ***p < 0.001; n.s.: not significant. (D) A model depicting the function of Arp2/3-mediated branched actin polymerization in promoting invasive protrusion formation to promote SCM fusion during skeletal muscle regeneration. BM: basement membrane.
Macrophages extravasate the ghost fibers by traversing the BM.
(A and B) Macrophages traversing the BM of ghost fibers shown by confocal microscopy. TA muscle cross sections of wild-type mice at dpi 3 were immunostained with anti-laminin, anti-NCAM, and anti-MAC-2, followed by confocal imaging. The confocal z-stacks were reconstructed to 3D images. Two examples are shown here. For each traversing macrophage (delineated by cyan dotted lines), images at two rotational angles are shown (r1 and r2). Note the small opening (arrowhead) on the BM through which a macrophage was passing (see supplementary video 1 & 2). (C) Macrophages traversing the BM of ghost fibers shown by TEM. The TA muscles as described in (A) and (B) were subjected to TEM processing. The BM is outlined by black dotted lines. The traversing macrophage is delineated by red dotted lines in the left panel. MAC: macrophage; SCM: SC-derived muscle cell. Scale bars: 2 μm.
The knockout efficiency of targeted proteins in the mouse models.
Mice with indicated genotypes were treated with tamoxifen as described in Figure 1A. The skeletal muscles were isolated and digested one day after the 5th tamoxifen injection. The satellite cells were enriched in vitro for two days, yielding a culture composed of > 90% satellite cells, followed by western blot for the targeted proteins. For each genotype, skeletal muscle from n = 6 mice were pooled for satellite cell isolation and western blot analysis.
The TA muscle weight and size are not affected by satellite cell-specific deletion of branched actin polymerization regulators before injury.
(A) Schematic diagram of tamoxifen treatment and TA muscle harvest. (B) Quantification of the TA muscle weight. Mice with the indicated genotypes were treated as described in (A). The whole body and TA muscle weights were measured. N ≥ 3 mice of each genotype were examined. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. ns: not significant. (C) The TA myofiber size appeared normal in control and mutants with indicated genotypes. Cross sections of TA muscles were stained with anti-Laminin and DAPI. n = 3 mice of each genotype were examined with similar results. Scale bar: 100 μm.
Impaired muscle regeneration in ArpC2cKO and MymXcKO mice persists to dpi 28.
(A) Schematic diagram of tamoxifen and BaCI2 treatment and subsequent CSA analysis at dpi 28. (B) Dystrophin and DAPI staining in TA muscle cross sections at dpi 28 in the control and mutant mice. Note that the myofiber CSA is severely reduced in ArpC2cKO and MymXcKO mice compared to that of the control mice. Scale bar: 100 μm.
Branched actin polymerization is not required for satellite cell proliferation, differentiation, or fusogenic protein expression.
(A) Schematic diagram of tamoxifen and BaCI2 treatment and subsequent cell proliferation and differentiation analyses at dpi 2.5 and 4, respectively. (B) Immunostaining with anti-Laminin, anti-Pax7 and anti-Ki67 of cross sections of TA muscles from control and mutant mice at dpi 2.5. The boxed the areas are shown on the right. Scale bar: 30 μm. (C) Immunostaining with anti-Laminin and anti-MyoG of cross sections of TA muscles from control and mutant mice at dpi 4. The boxed areas are shown at the top right corner. Scale bar: 30 μm. (D) Quantification of the percentage of proliferating satellite cells (% of Ki67+ cells in the Pax7+ cells) in TA muscles from control and mutant mice of the indicated genotypes. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. ns: not significant. (E) Quantification of the percentage of MyoG+ nuclei in the total cells in TA muscles from control and mutant mice of the indicated genotypes. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. ns: not significant. (F) Western blot analysis for MymX and MymK in TA muscles from control and mutant mice of the indicated genotypes at dpi 4. n = 3 mice of each genotype were examined. One sample of each control and mutant genotype were loaded. (G, H) Quantification of MymX and MymK protein expression in the control and littermate mutant mice as shown in (F). The band intensity of each protein was normalized against β-tubulin. The y axis indicates the expression of MymX or MymK in different mutants relative to the control mice. Mean ± s.d. values are shown in the bar graphs, and significance was determined by two-tailed student’s t-test. *p < 0.05; ns: not significant.
Branched actin polymerization is required for SCM fusion.
(A) Immunostaining with anti-Laminin and anti-NCAM of longitudinal sections of TA muscles from control and ArpC2cKO mice at dpi 4.5. Scale bar: 10 μm. (B) Pharmacological inhibition of the Arp2/3 complex during differentiation blocked SCM fusion. The wild-type SCMs were plated at 60% confluence in GM. After a day. The cells were then incubated in DM supplemented with DMSO as a control (0.1%) or the Arp2/3 complex inhibitor CK666 (100 μM) for 48 hours, followed by anti-MHC and DAPI staining. Note the robust fusion of control SCMs vs. the severe fusion defects in SCMs treated with CK666. Scale bar: 100 μm. (C and D) Quantification of the differentiation and fusion indexes of the cells shown in (B). n = 3 independent experiments were performed. Mean ± s.d. values are shown in the bar graphs, and significance was determined by two-tailed student’s t-test. ****: p < 0.0001; n.s: not significant.
Fusogenic protein MymX is not required for invasive protrusion formation
Quantification of the length (A) and width (B) of the invasive protrusions in control and MymXcKO mice at dpi 3.5 imaged by TEM. The width was measured at the midpoints of the invasive protrusions. Mean ± s.d. values are shown in dot plots, and statistical analysis was performed for each parameter in n ≥ 29 invasive protrusions in each genotype. Significance was determined by two-tailed student’s t-test. n.s.: not significant.
