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First published online 22 February 2005
doi: 10.1242/jcs.01695

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Sphingosine 1-phosphate induces cytoskeletal reorganization in C2C12 myoblasts: physiological relevance for stress fibres in the modulation of ion current through stretch-activated channels

¹ Department of Anatomy, Histology and Forensic Medicine, Interuniversitary Institute of Miology (IIM), 85 50134 Florence, Italy
² Department of Biochemical Sciences, Interuniversitary Institute of Miology (IIM), 85 50134 Florence, Italy
³ Department of National Institute of Applied Optics and Interuniversitary Institute of Miology (IIM), 85 50134 Florence, Italy
⁴ Department of Physiological Sciences, Interuniversitary Institute of Miology (IIM), 85 50134 Florence, Italy

View larger version (103K): [in a new window]   Fig. 1. Effects of S1P on F-actin polymerization. C2C12 cells transfected with GFP-tagged -and ß-actin were stimulated with 1 µM S1P under live video two-photon excitation fluorescence microscope. Images were taken at 1 minute (A) and 30 minutes (B) after treatment, respectively. Arrows indicate regions of prominent actin microfilament organization. The images are representative of independent experiments (n=12) with similar results.

View larger version (108K): [in a new window]   Fig. 2. Effects of S1P on stress fibre and focal adhesion formation. Untreated C2C12 myoblasts (A) and treated with 1 µM S1P (B) were fixed and double stained with TRITC-phalloidin (red) and anti-vinculin (green). S1P induces formation of massive stress fibres coincident with the redistribution of vinculin-rich focal adhesion sites at the end of stress fibres. The images are representative of at least three independent experiments with similar results.

View larger version (128K): [in a new window]   Fig. 3. Role of S1PR in the cytoskeletal response of S1P. Intracellular S1P stimulation of native C2C12 myoblasts: cells were microinjected with a mixture of 500 µM S1P and 5 mg/ml PI (A). Extracellular stimulation of native C2C12 myoblasts with 1 µM S1P: cells were microinjected with PI solely as a tracking agent (B). Extracellular stimulation of native C2C12 myoblasts after pretreatment with PTx (C). In all the experimental conditions, the cells were fixed after 30 minutes of stimulation and F-actin organization was visualized by Alexa488-phalloidin. Note that, in contrast to the extracellular stimulation (B), the intracellular delivery of S1P (A) is unable to modify the assembly of stress fibres. However, the inhibition of Gi-coupled receptor pathway by PTx reduces the formation of F-actin filaments by S1P. The images are representative of at least three independent experiments with similar results.

View larger version (178K): [in a new window]   Fig. 4. Effects of Rho kinase inhibitor on S1P-induced stress fibres. C2C12 myoblasts were stimulated with 1 µM S1P for 30 minutes in the absence (A) or presence of 50 µM Y-27632 (B), fixed and observed under a confocal laser microscope. Note that, after the pretreatment with the Rho kinase inhibitor, cells have only a few peripheral stress fibres and scattered focal adhesions. Time-lapse analysis of myoblasts expressing GFP-actins by two-photon excitation fluorescence microscopy (C and D). Images were taken 1 minute (C) and 30 minutes after (D) the application of 1 µM S1P. The pretreatment with the Rho kinase inhibitor causes shortening and disruption of actin filaments. The images are representative of at least three independent experiments with similar results.

View larger version (59K): [in a new window]   Fig. 5. Overexpression of a catalytically inactive PLD mutant affects stress fibre formation. (A) C2C12 cells were transfected with HA-tagged catalytically inactive DN-PLD or empty vector. Following serum starvation, the cells were stimulated with S1P in the presence of ethanol and processed as described in the Materials and Methods. PLD activity is reported as percentage of PtdEtOH on PtdCho. Data are mean±s.e.m. of three independent experiments (*P<0.05). Inset: western blot shows the expression of recombinant protein. Blot representative of three experiments with similar results. Time-lapse analysis of myoblasts expressing GFP-actin transfected with DN-PLD by two-photon excitation fluorescence microscopy (B,C). Images were taken 1 minute (B) and 30 minutes after (C) the application of 1 µM S1P. A clear reduction of stress fibres is evident in cells with reduced PLD activity.

View larger version (167K): [in a new window]   Fig. 6. Effects of reduced PLD and Rho kinase activities on S1P-induced stress fibres and focal adhesions. C2C12 myoblasts transfected with DN-PLD were stimulated with 1 µM S1P for 30 minutes (A) or pretreated with 50 µM Y-27632 prior to stimulation (B). The cells were then fixed and double stained with TRITC-phalloidin (red) and anti-vinculin (green). Of interest, the combined treatment is able to abrogate completely stress fibre and focal adhesion formation in response to S1P. (C,D) Time-lapse analysis of C2C12 myoblasts expressing GFP-actins transfected with DN-PLD by two-photon excitation fluorescence microscopy. Images were taken 1 minute (C) and 30 minutes (D) after the application of 1 µM S1P. A complete filament dispersion is visible after 30 minutes from stimulation. The images are representative of at least three independent experiments with similar results.

View larger version (35K): [in a new window]   Fig. 7. Effects of S1P and of chemical treatments on membrane current and conductance. Voltage-clamp step pulses between –80 and 0 mV in 10 mV steps from a holding potential of –60 mV were applied to the cells for 100 milliseconds. Representative current traces in control (A) and after S1P (B), GdCl3 (C), S1P + GdCl3 (D), DHCB (E) and S1P + DHCB (F) treatments. G, normalized Im versus Vm plots in Control condition () and after S1P () or GdCl3 () or S1P + GdCl3 () or DHCB() or S1P + DHCB() treatment. The best linear regression lines are superimposed on control (continuous line) and the other experimental conditions (dashed lines). The calculated Cm, Gm and Gm/Cm values for all experimental conditions are reported in Table 1. Data are the mean±s.e.m.

View larger version (34K): [in a new window]   Fig. 8. Effects of S1P and of the inhibition of Rho kinase, PLD and Gi-coupled receptor on membrane current and conductance. (A-E) Voltage-clamp step pulses as in Fig. 7. Representative current traces (A-E) and normalized Im versus Vm plots (F) after S1P stimulation in: empty vector transfected myoblast (A, in F); DN-PLD-transfected myoblasts (B, in F); Y-27362-treated myoblasts (C, in F); DN-PLD-transfected myoblasts in the presence of Y-27362 (D, in F); PTx-treated myoblasts (A, in F). The best linear regression lines are superimposed on the data (continuous lines). For comparison, the data and linear regression (dashed line) of control from Fig. 7 are reported. The calculated Cm, Gm and Gm/Cm values for all experimental conditions are reported in Table 2. Data are the mean±s.e.m.

View larger version (19K): [in a new window]   Fig. 9. Ca2+ influx after the application of stretching forces. Native C2C12 cells were pre-loaded with Fluo-3 (1 µM) and mechanically stimulated using the cantilever (white line) of an AFM. Arrow points to the cell to be stretched. The images were recorded by a cooled CCD camera prior and after the cantilever had been lifted off the cell at the indicated time points. The myoblast responds to the mechanical stimulus by a rise in intracellular Ca2+ that propagates from the stretched cell to the adjacent one. The images are representative of at least three independent experiments with similar results.