Filamin-A-interacting protein 1 (FILIP1) is a dual compartment protein linking myofibrils and microtubules during myogenic differentiation and upon mechanical stress

Figures

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  Fig. 1. Localization of FILIP1 in adult mouse muscle. (a–b'') Immunolocalization of FLNc and FILIP1 in longitudinal cryosections of adult mouse soleus muscle indicates a localization of both proteins in the Z-disc region of the myofibrils (arrowheads). The boxed area in (a–a'') is shown enlarged in (b–b''). (c–c''') Localization of FLNc, FILIP1 and a Z-disc epitope of titin in a macrolesion and a microlesion (asterisk). FLNc and FILIP1, but not titin, are concentrated in lesions. FLNc shows a more filamentous staining than FILIP1 that shows a more diffuse localization and is only associated with individual filaments (arrows). Arrowheads indicate the position of Z-discs within the macrolesion. Due to the short exposure because of the enhanced signal in the lesions, Z-disc staining of FLNc and FILIP1 is not visible. Bars: 10 m

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  Fig. 2. FILIP1 expression and localization in differentiating cultured C2C12 myotubes. a, a' Representative western blots showing the levels of FLNc, FILIP1, EB3, tyrosylated tubulin (Tyr-tub) and acetylated tubulin (Ac-tub) in proliferating C2C12 myoblasts (0 days) and myotubes differentiated for the indicated time (1–8 days). GAPDH was used as loading control and for normalization. b–b'''' Quantification of the data presented in (a–a'), expressed as mean ± SD. n = 3. In all individual experiments maximum relative expression of each protein was set to 1.0. c–e''' Immunolocalization of FLNc, FILIP1 and EB3 in C2C12 myotubes differentiated for 2 (c–c'''), 4 (d–d''') and 6 (e–e''') days. c–c''' FILIP1 and EB3 partially colocalize (arrows), which becomes more evident later in differentiation (d–d''', arrows). This is still evident in mature myotubes 6 days after the start of differentiation (e–e''', arrows), when FILIP1 and FLNc are mainly present in Z-discs (e–e''' and f–f''', arrowheads). A magnification of the boxed area in (e–e''') is shown in (f–f'''). Bars: 10 μm

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  Fig. 3. FILIP1 is localized in EPS-induced sarcomeric lesions. a–a'' Immunolocalization of FLNc and FILIP1 in overnight mechanoadapted C2C12 myotubes (a–a''), followed by twitch stimulation for 2 h (b–b'') or 4 h (c–c''). Arrowheads indicate the presence of both FLNc and FILIP1 in EPS-induced lesions. Note that incidence and size of lesions as well as the presence of FILIP1 in the lesions increase with the intensity of the applied stimulus and duration of pacing. Bars: 10 μm. d Representative western blots showing the levels of FLNc and FILIP1 in mechanoadapted C2C12 myotubes treated with EPS for the indicated time periods. GAPDH was used as loading and normalization control. e–e' Quantification of the data shown in (d) expressed as mean ± S.E.M. Significance was assessed using One-way ANOVA and Tukey’s post hoc test or Kruskal–Wallis test and the post hoc Dunn’s multiple comparisons test. No significant differences were found

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  Fig. 4. FILIP1 mainly colocalizes with FLNc but not with EB3 after EPS-treatment. Immunolocalization of FLNc (a), FILIP1 (b) and EB3 (c) in EPS-treated myotubes reveals the presence of both, FLNc and FILIP1 in the remaining Z-discs (arrowheads) and in lesions (asterisks), while FILIP1 does not colocalize with EB3 (arrows). The merged picture is shown in panel d. Bar: 10 μm

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  Fig. 5. Tyrosylated microtubules flank EPS-induced sarcomeric lesions. a–a''''' Microtubules stained for beta-tubulin show the typical longitudinal orientation in mechanoadapted (mech. adapt.) myotubes (a–a'') while in twitch-paced myotubes many sarcomeric lesions are flanked by microtubules (a'''–a''''', arrowheads). b–b'' Immunolocalization of FLNc and EB3 in a C2C12 myotube treated with twitch EPS. The arrowheads indicate sarcomeric lesions marked by FLNc. EB3 is widely expressed throughout the myotube, and appears concentrated in the proximity of these lesions. c–c'' Immunolocalization of FLNc and tyrosylated tubulin (Tyr-tub) in C2C12 myotubes treated with basic EPS (c–c'') and twitch EPS (c'''–c'''''). Arrowheads indicate sarcomeric lesions, strongly positive for FLNc, which are flanked by tyrosylated microtubules. d Representative western blots showing the expression level of tyrosylated tubulin (Tyr-tub) and EB3 in mechanoadapted C2C12 myotubes that were twitch EPS treated for the indicated time periods. GAPDH was used as loading and normalization control. e–e' Quantification of the data shown in (d) expressed as mean ± S.E.M. n = 3. Significance was assessed using One-way ANOVA…

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  Fig. 6. Microtubule depolymerization does not affect the localization of FILIP1 at Z-discs and in sarcomeric lesions. a–a''''', b–b''''' Immunolocalization of FLNc and total tubulin (beta-tub) in C2C12 myotubes treated with (a–a''''') DMSO or (b–b''''') 4 μg/ml nocodazole (NZ). NZ treatment for 2 h induced complete depolymerization of the microtubule network, which was rescued within 2 h after washout of NZ, after the EPS treatment. c–c''''''' Representative images of C2C12 myotubes treated with EPS and DMSO or NZ and stained for FLNc, FILIP1 and total tubulin (beta-tub). The localization of FILIP1 at the Z-discs (indicated by arrowheads) and in sarcomeric lesions (indicated by arrows) is not affected by the treatment with NZ. d–d''''' Representative images of C2C12 myotubes pre-incubated for 2 h with DMSO (d) or NZ (d'), subsequently treated with twitch EPS for 1 h to induce sarcomeric lesions (d''–d'''), and rescued by a 2 h incubation with medium without DMSO or NZ (d''''–d'''''). Myotubes were stained for FLNc. The arrows indicate EPS-induced lesions. Note that treatment with NZ seems to reduce the number of lesions induced by EPS. e Histogram showing the number of sarcomeric lesions…