Figure 1.
Cyclic stretch-induced stress fiber alignment depends on stretch frequency.
A–C: Representative images of confluent BAECs subjected to 4 hours of 10% cyclic uniaxial stretch at frequencies of 0.01 (A), 0.1 (B) and 1 Hz (C) in the direction indicated. After the experiments, cells were fixed and then stained with Alexa 488-phalloidin to identify stress fibers. D: Order parameters were computed for each image to quantify the extent of stress fiber alignment and the results were summarized (mean ± S.D.; n = 25). Order parameters were also computed for simulated stretch-induced stress fiber reorganization under the same stretch conditions for comparison to the experimental results. Scale bar length is 20 µm.
Figure 2.
Stress fiber stretch amplitude depends on stretch frequency.
The relative fiber stretch amplitude α* was estimated by solving the model for the initial cycle of 10% cyclic uniaxial or equibiaxial stretch for frequencies ranging from 0.01 to 10 Hz. The simulations predict that the stress fibers behave elastically above a saturation frequency of ∼1Hz and that the value of α* decreases exponentially as frequency decreases below the saturation frequency.
Figure 3.
Cell retraction caused by 1Hz, but not 0.01Hz, cyclic equibiaxial stretch.
Representative images are shown of U2OS cells expressing GFP-actin imaged immediately before (A, C) and 5 min after applying 10% equibiaxial stretch at 1Hz (B) or 0.01 Hz (D). The outlines of the cells prior to stretching (red) are shown for reference. Bar = 20 µm. E: The ratio of cell areas (after/before stretch) for the indicated durations of stretch at 1Hz and 0.01Hz are summarized (mean±S.D.; n = 6). * indicates significant difference from unity (P<0.01).
Figure 4.
JNK, ERK and p38 phosphorylation depend on the frequency of cyclic stretch.
Representative Western blots of phospho-specific and total JNK (A, D), ERK (B, E) and p38 (C, F) were obtained from confluent BAECs kept as static controls or subjected to 10% cyclic uniaxial (A–C) or equibiaxial (D–F) stretch for 30 min at the indicated frequencies. Optical density measurements were quantified to determine relative amounts of phosphorylated MAPK normalized by the respective total MAPK. The values (means±S.D.; n = 6 for uniaxial and n = 7 for equibiaxial) indicate the fold change in phosphorylation relative to the static control for each individual experiment. * and ** indicate significant difference from static controls (* P<0.05, ** P<0.01). † indicates significant difference between groups stretched at different frequencies (P<0.05).
Figure 5.
Transient changes in strain rate induce transient changes in the levels of JNK, ERK and p38 phosphorylation.
Confluent BAECs were subjected to a stretch release maneuver consisting of 10% equibiaxial stretch λ, followed by the release of the stretch 12 hr later (A), which was predicted to generate a transient increase and subsequent transient decrease in fiber stretch α (B). Representative Western blots of phospho-specific and total JNK (C, F), ERK (D, G) and p38 (E, H) from cell lysates collected at the indicated times after the initial stretch (C–E) and subsequent stretch release (F–H). Optical density measurements were quantified to determine relative amounts of phosphorylated MAPK normalized by the respective total MAPK. The values (means±S.D.; n = 4) indicate the fold change in phosphorylation relative to static controls for each individual experiment. * indicates significant difference from static control (P<0.05).
Figure 6.
JNK and ERK phosphorylation is decreased by inhibitors of Rho kinase and actin polymerization.
Representative Western blots are shown of phospho-specific and total JNK (A), ERK (B) and p38 (C) from cell lysates collected from confluent BAECs treated for 30 min with vehicle (0.1% DMSO; lane 1), 50 nM cytochalasin D, 10 nM jasplakinolide or 10 µM Y27632. Optical density measurements were quantified to determine relative amounts of phosphorylated MAPK normalized by the respective total MAPK. The values (means±S.D.; n = 4) indicate the fold change in phosphorylation relative to the DMSO-treated controls for each individual experiment. * and ** indicate significant difference from the control (* P<0.05, ** P<0.01).
Figure 7.
FAK is not necessary for stretch-induced stress fiber alignment.
Representative micrographs are shown from experiments in which non-confluent FAK-null (A, C) and FAK-expressing (B, D) MEFs were subjected to 3 hours of 10% cyclic uniaxial stretch at 0.01 (A, B) or 1 Hz (C, D) in the direction indicated, fixed and stained to measure stress fiber reorganization. E: Experiments were performed at frequencies of 0.01, 0.1 and 1 Hz and the order parameters for individual cells from three different experiments were quantified (mean±SD; n = 75). Bar = 20 µm. * indicates a significant difference between frequency-matched groups, (P<0.05).
Figure 8.
FAK is not necessary for stretch-induced increases in JNK, ERK and p38 phosphorylation.
Representative immunoblots are shown from experiments in which confluent FAK-expressing and FAK-null MEFs were kept as static controls or subjected to 10% cyclic uniaxial stretch at 1 Hz for 0.5 and 6 hr. Immunoblot pairings for JNK (A), ERK (B) and p38 (C) for the FAK-expressing and FAK-null MEFs were taken from two locations on the same blot. Optical density measurements were quantified to determine relative amounts of phosphorylated MAPK normalized by the respective total MAPK. The values (means±S.D.; n = 4) indicate the fold change in phosphorylation relative to the FAK-null static control for each individual experiment. * and ** indicates significant difference between groups (* P<0.05, ** P<0.01).