Figure 1.
Immunofluorescence micrographs of labeled three-day human myotube culture developing the cross-striated myofibrils.
Monoclonal anti-titin antibodies recognising titin epitopes in the Z-disk (A), A/I junction (B), A-band (C) and M-band (D). Three-day differentiated human culture cells were identified in an advanced developed state by a striated staining pattern for slow and fast IIa MyHCs, located in the A-band (E; arrow). Nuclei are labeled with DAPI (blue). Cells were imaged using a Zeiss Axio Observer microscope. Scale bar = 10 µm.
Figure 2.
Expression of wild-type β-TMEGFP and E41K-β-TMEGFP in human cells.
(A–B) Wild-type β-TMEGFP and (C–D) E41K-β-TMEGFP mutants were transfected in human (A and C) myoblasts and (B and D) myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) WT-β-TM was expressed in human myoblasts or (B) myotubes and incorporated well into endogenous filamentous actin as visualised with phalloidin (long arrows). The E41K-β-TM mutant induced diffuse intranuclear and cytoplasmic clouds in transfected myoblasts (C–Ć ´’’) and perinuclear aggregates in myotubes (D–D´ ´’’; short arrows). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.
Figure 3.
The expression of K49del-β-TMEGFP in human cells.
(A–D) The K49del-β-TMEGFP mutant was transfected in human myoblasts and (E) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The K49del-β-TM co-localised with cytoplasmic and nuclear aggregates of endogenous actin, detectable with phalloidin labeling (A–C”; short arrows). It produced clouds around the nucleus (A and D) but was also incorporated into stress fibres and filamentous lamellipodia (A and A”, B and B”, and D and D”; long arrows). In addition, it induced thickened filamentous structures of endogenous actin (A’–A”; arrow heads). The K49del-β-TM produced small, long rod-shaped intranuclear structures. A subset of aggregates labeled with phalloidin but was not detectably composed of EGFP-tagged mutant K49del-β-TM (D’–D”; short arrows). Frequently, cytoplasmic thickened filamentous structures with no co-localisation of F-actin were found in myoblasts transfected with K49del-β-TM (D and D”; arrow heads). The K49del-β-TMEGFP mutant produced rod-shaped filamentous actin, cytoplasmic aggregates and cloud patterns in human myotubes (E). The rod-shaped structures were labeled with phalloidin, indicating co-localisation with F-actin (E–E”; short arrows). Cytoplasmic thickened filamentous structures were also observed (E and E”; arrow heads). The K49del-β-TMEGFP mutant was also incorporated into filamentous actin (E; long arrow). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.
Figure 4.
The expression of G53ins-β-TMEGFP in human cells.
The G53ins-β-TMEGFP mutant was transfected in human (A) myoblasts and (B) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The G53ins-β-TMEGFP mutant produced delocalisation and endogenous actin aggregates in human myoblasts, labeled with phalloidin (A–A”). The cells transfected with the G53ins-β-TM mutant differentiated into myotubes in an advanced, developed state, identified by their elongated shape and multiple nuclei (B–B”). Transfected myotubes showed good integration of the mutant TM into sarcomeric structures (B; long arrow). The G53ins mutant produced diffuse cytoplasmic labeling at the far end of the myotubes (B; short arrow). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.
Figure 5.
The expression of E122K-β-TMEGFP in human cells.
The E122K-β-TMEGFP mutant was transfected in human (A–C) myoblasts and (D) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The E122K-β-TMEGFP mutant formed aggregates of endogenous actin in human myoblasts (A–A”; inset (B–B”)). Moreover, the E122K-β-TMEGFP incorporated in clouds with an unorganised filamentous structure (C–C”). E122K-β-TMEGFP formed intranuclear rods in human myoblasts that were only detectable with phalloidin labeling (C–C”; short arrows). It also integrated well with stress fibres (A–A” and C–C”; long arrows). The transfection of the E122K-β-TMEGFP construct generally resulted in the less well-defined phalloidin labeling of actin filaments at the far end of the transfected myotubes with the appearance of small rod-like structures located at the periphery (D–D”; inset, short arrows). The rod-shaped structures did not label with phalloidin, indicating that they were not accessible to phalloidin or were not composed of filamentous actin. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.
Figure 6.
The expression of E202K-β-TMEGFP and empty EGFP constructs in human cells.
The E202K-β-TMEGFP mutant was transfected in human (A–B) myoblasts and (C) myotubes. (D) Empty EGFP construct was transfected in myoblasts. Cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The transfection of human myoblasts with mutant N202K-β-TM induced the diffuse cytoplasmic labeling of stress fibres (A–A’; long arrows) and small phalloidin-labeled aggregates in the cytoplasm (A’ and A”; short arrows). Mutant N202K-β-TM formed clouds around the nucleus (B and B”; short arrows), in addition to a well-defined organised filamentous structure of stress fibres (B–B”; long arrows). In many N202K-β-TMEGFP-transfected myotubes, more marked changes in actin structures were observed (C–C”). A large accumulation of mutant N202K-β-TM with the co-localisation of polymerised actin appeared, suggesting the disruption of endogenous actin filaments (C–C”; short arrows). Human myoblasts transfected with empty EGFP vector formed well-organised filamentous structures (D–D”). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.
Figure 7.
The expression of WT and mutant β-TMs-EGFP in human myotubes.
WT and mutant β-TMs were transfected in human myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) WT-β-TM was expressed in human myotubes and incorporated well into endogenous sarcomeric thin filaments as visualised with phalloidin (arrows). (B) The E41K-β-TM mutant induced diffuse perinuclear aggregates in myotubes (arrows). (C) The K49del-β-TMEGFP mutant produced cytoplasmic rod-shaped filamentous actin in human myotubes. (D) The cells transfected with G53ins-β-TM mutant differentiated into myotubes, showed the integration of the mutant TM into sarcomeric structures and developed the cross-striated myofibrils in an advanced and developed state (arrow). (D’) The G53ins mutant produced diffuse cytoplasmic labeling at the far end of the transfected myotubes (arrow). (E) The transfection of the E122K-β-TMEGFP construct generally resulted in the appearance of cytoplasmic rod-like structures located at the periphery of the transfected myotubes (arrow). (F) The cells transfected with N202K-β-TMEGFP differentiated into myotubes, showed an accumulation of mutant N202K-β-TM, co-localisation with polymerised actin (arrows). Cells were imaged using a Zeiss Axio Observer microscope. Scale bar = 10 µm.
Figure 8.
Untagged β-TM constructs form abnormal aggregates in human myoblasts.
Human myoblasts transfected with untagged WT-, E41K-, K49del- and G53ins-β-TM constructs and labeled with phalloidin (green) and β-TM (red) and DAPI (blue) to highlight cell nuclei. Stress fibres appeared well aligned in human myoblasts transfected with WT-β-TM (A). Abnormal aggregates were observed in human myoblasts transfected with untagged E41K-, K49del- and G53ins-β-TM constructs (B–D). Intranuclear rod-shaped aggregates labeled by phalloidin are detected in human myoblasts transfected with the untagged K49del-β-TM construct, demonstrating that intranuclear aggregation is an inherent property of K49del mutation and does not result from EGFP-tagging (C; arrows).
Figure 9.
Incorporation of β-TM mutants into cytoskeleton and sarcomeric filaments in cultured human myoblasts and differentiated myotubes.
(A) Western blot images from a single experiment showing levels of β-TMEGFP within the insoluble (Insol) and soluble (Sol) protein pools in myoblasts (Myo) and in six-day differentiated cultures (D6). Bands intensity was quantified through densitometric analysis. The mean data from triplicate experiments is shown in (B).
Figure 10.
The expression of wild-type-β-TMEGFP and E41K-β-TMEGFP in C2C12.
(A–B) Wild-type β-TMEGFP and (C–D) E41K-β-TMEGFP mutant were transfected in (A and C) the myoblasts and (B and D) myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. WT-β-TM expressed in C2C12 (A) myoblasts or (B) myotubes incorporated well into C2C12 stress fibres (A) and endogenous filamentous actin (B; long arrow) as visualised with phalloidin. The E41K-β-TM mutant appeared to be diffused in transfected myoblasts and intense EGFP aggregates co-localised with endogenous actin in the peripheral area of the cells (C–C”; short open arrows). The E41K-β-TM mutant formed perinuclear aggregates in myotubes that did not show phalloidin labeling (D–D’’; long open arrows). C2C12 cells appeared as fused myoblasts rather than differentiated myotubes in six-day differentiated cultures. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.
Figure 11.
The expression of K49del and G53ins β-TM-EGFP in C2C12.
The K49del β-TM-EGFP mutant was transfected in (A) myoblasts and (B) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) The K49del-β-TM mutant was mislocalised and showed the diffuse labeling of phalloidin in transfected myoblasts. It induced nuclear and cytoplasmic aggregates (A–A’; long open arrows). The K49del-β-TM mutant produced intense EGFP aggregates, co-localised with endogenous actin in the peripheral area of both myoblasts (A–A”; short arrows) and differentiated cells (B–B”; short open arrows), in addition to perinuclear aggregates in differentiated C2C12 (B and B”; short closed arrows). (B) The K49del-β-TM mutant showed some integration with actin filaments of differentiated C2C12 (B–B”; long arrows). The G53ins mutant appeared mislocalised, showed the diffuse labeling of phalloidin in transfected myoblasts (C–C”) and formed cytoplasmic aggregates (C–C”; long open arrows). Intense aggregates in the peripheral area of both myoblasts (C–C”; short arrows) and differentiated cells (D–D”; short arrows) were observed. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.
Figure 12.
The expression of E122K and N202K-β-TMEGFP and empty EGFP in C2C12.
The cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The E122K-β-TMEGFP mutant was transfected in (A) myoblasts and (B) myotubes. (A) The E122K-β-TM mutant was mislocalised and produced intense EGFP aggregates, co-localised with endogenous actin in the peripheral area of both myoblasts (A–A”; short arrows) and differentiated cells (B–B”; short arrows). The E122K mutant induced the perinuclear aggregates in differentiated C2C12 (B and B”; long arrows). C2C12 myoblasts transfected with N202K-β-TMEGFP appeared cytopathic, with a thickened, ruffled cell surface (C–C”; long open arrows). Aggregates in the peripheral (D–D”; short closed arrows) and perinuclear (D–D”; long open arrows) areas of differentiated cells were detected. The transfection of C2C12 myoblasts with empty EGFP vector resulted in the formation of well-organised stress fibres (E–E”). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.
Figure 13.
Immunofluorescent labeling of p62 in human myoblasts and C2C12 transfected with E41K-β-TMEGFP and E122K-β-TMEGFP constructs.
The cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A–A’”) The co-localisation of p62 (red, A’; arrow) in aggregates induced by the transfection of E41K-β-TMEGFP in human myoblasts (A and A’”; arrows). (B–B’”) The co-localisation of p62 (red, B’; arrow) in aggregates induced by the transfection of E122K-β-TMEGFP in human myoblasts (B and B’”; arrows). (C–C’”) C2C12 myoblasts transfected with E41K-β-TMEGFP showed positive immunoreactivity with p62 (red, C’; arrow). (D–D’”) The co-localisation of p62 (red, D’; arrow) in aggregates induced by the transfection of E122K-β-TMEGFP in C2C12 myoblasts (D and D’”; arrows). The p62 labeling closely resembled EGFP-positive protein aggregates in the transfected cells with mutant TM (i.e. yellow in the merged images A’”, B’”, C’” and D’”; arrows). Confocal microscopy was performed using an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.