Nestin Modulates Airway Smooth Muscle Cell Migration by Affecting Spatial Rearrangement of Vimentin Network and Focal Adhesion Assembly

Abstract

Airway smooth muscle cell migration plays a role in the progression of airway remodeling, a hallmark of allergic asthma. However, the mechanisms that regulate cell migration are not yet entirely understood. Nestin is a class VI intermediate filament protein that is involved in the proliferation/regeneration of neurons, cancer cells, and skeletal muscle. Its role in cell migration is not fully understood. Here, nestin knockdown (KD) inhibited the migration of human airway smooth muscle cells. Using confocal microscopy and the Imaris software, we found that nestin KD attenuated focal adhesion sizes during cell spreading. Moreover, polo-like kinase 1 (Plk1) and vimentin phosphorylation at Ser-56 have been previously shown to affect focal adhesion assembly. Here, nestin KD reduced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation), vimentin phosphorylation at Ser-56, the contacts of vimentin filaments to paxillin, and the morphology of focal adhesions. Moreover, the expression of vimentin phosphorylation-mimic mutant S56D (aspartic acid substitution at Ser-56) rescued the migration, vimentin reorganization, and focal adhesion size of nestin KD cells. Together, our results suggest that nestin promotes smooth muscle cell migration. Mechanistically, nestin regulates Plk1 phosphorylation, which mediates vimenitn phosphorylation, the connection of vimentin filaments with paxillin, and focal adhesion assembly.

Keywords: intermediate filament protein; migration; nestin; smooth muscle.

Figure 1

Knockdown (KD) of nestin reduces human ASM cell migration. (A) Protein expression of human ASM cells stably expressing control (Ctrl) shRNA or nestin shRNA was evaluated by immunoblotting. Data are mean values of experiments from 4 batches of cell culture. Error bars indicate SE. (B) Cell migration was tracked using a time-lapse microscope. Images were taken every 10 min for 12 h. Migration plots (32 cells from each group) were generated using the NIH ImageJ software. Red tracks indicate downward migration whereas black tracks indicate upward migration. (C–E) Nestin (Nes) KD reduced accumulated distance, Euclidean distance, and speed of cell migration. (F) Nestin KD inhibits the migration of human ASM cells as evidenced by the wound healing assay, which was described in detail in Material and Methods. Data are mean values of six experiments. Error bars indicate SE. * p < 0.05. t-test was used for statistical analysis.

Figure 2

Nestin KD reduces size of focal adhesions. (A) Ctrl and nestin KD human ASM cells were plated on collagen-coated coverslips for 45 min and immunostained for paxillin and pY397-FAK. Cell images were captured using a confocal microscope and the Imaris software was used to render and analyze images. Scale bar, 15 µm. The white arrows point spots colocalized with paxillin and pY397-FAK. The cyan arrows point to pY397-FAK labeling spots. (B–I) The area, BoundingBoxOO Length-A/B/C of paxillin and pY397-FAK staining were calculated using the Imaris software. BoundingBox OO length-A measures the length of the shortest principal axis. BoundingBox OO length-B measures the length of the second longest principal axis. BoundingBox OO length-C measures the length of the longest principal axis inside of the object. Data are means ± SE (n = 5, * p < 0.05). t-test was used for statistical analysis. NS, not significant.

Figure 3

Nestin KD affects Plk1 phosphorylation at Thr-210, vimentin phosphorylation at Ser-56, and vimentin network. (A) Extracts of Ctrl and nestin KD cells were immunoblotted with antibodies against phospho-Plk1 (Thr-210) and total Plk1. Plk1 phosphorylation is normalized to the level obtained from Ctrl cells. Data are mean ± SE (n = 4). (B) Extracts of Ctrl and nestin KD cells were immunoblotted with antibodies against phospho-vimentin (Ser-56) and total vimentin. Vimentin phosphorylation is normalized to the level obtained from Ctrl cells. Data are mean ± SE (n = 4). (C) Ctrl and nestin KD ASM cells were plated on collagen-coated coverslips for 45 min and immunostained for vimentin and paxillin. Cell images were taken using a Zeiss LSM880 microscope with Airyscan. The images of vimentin filaments and paxillin staining in cell protrusions were used for Imaris quantitative analysis. Scale bar: 15 μm. Imaris software was utilized to 3D-render vimentin filaments and paxillin surfaces. 3D-rendered vimentin is green, paxillin closed to vimentin is cyan, and paxillin alone is red. (D) The Imaris software was utilized to quantify the length of filament segments. (E,F) The percent of paxillin clusters closed to or away from vimentin filaments was assessed using the Imaris software. Data are mean ± SE (n = 5). t-test was used for statistical analysis. * p < 0.05; ** p < 0.01.

Figure 4

Vimentin S56D affects migration and vimentin network of nestin KD cells. To assess the role of vimentin phosphorylation at Ser-56, nestin KD cells were transfected with S56A or S56D vimentin. Cell migration was evaluated using a time-lapse microscope. (A–C) S56D, but not S56A, increases accumulated distance, Euclidean distance, and speed of nestin KD cell migration (n = 32–34 cells/each group). (D–G) Cell images were captured and analyzed using the methods described in Figure 3 legend. Expression of S56D in nestin KD cells increases vimentin filament length and the connection of the filaments to paxillin. Data are mean ± SE (n = 5). * p < 0.05; ** p < 0.01. t-test was used for statistical analysis. Scale bar, 15 µm.

Figure 5

Vimentin S56D regulates focal adhesions. (A) Nestin KD human ASM cells were transfected with S56A or S56D vimentin, and cell images were collected and analyzed using the methods described in Figure 2 legend. Scale bar: 15 μm. The white arrows point spots colocalized with paxillin and pY397-FAK. The cyan arrows point to pY397-FAK labeling spots. (B–I) The area, BoundingBoxOO Length-A/B/C of paxillin and pY397-FAK staining were calculated using the Imaris software. S56D increases the areas and BoundingBox OO length-C of paxillin and pY397-FAK. Data are means ± SE (n = 5, * p < 0.05). t-test was used for statistical analysis. NS, not significant.

Figure 6

Nestin KD does not affect the recruitment of c-Abl and cortactin, myosin phosphorylation, and F-actin. (A) Ctrl and nestin KD human ASM cells were plated on collagen-coated coverslips for 45 min and immunostained for c-Abl and cortactin, Scale bar: 10 µm. The arrows point to the cell edge. The relative intensity of c-Abl and cortactin was calculated using the following formula: intensity of each cell/average intensity of control cells. Data are mean values of at least 20 cells from three experiments. Error bars indicate SE. NS, not significant. (B) Myosin light chain phosphorylation at Ser-19 of Ctrl and nestin KD human ASM cells was evaluated by immunoblot analysis. The phosphorylation levels of nestin KD cells are normalized to Ctrl cells. Data are mean values of 4 batches of cell cultures. Error bars indicate SE. (C–E) Ctrl and nestin KD human ASM cells were plated on collagen-coated coverslips for 45 min and stained with phalloidin for F-actin. Scale bar: 10 µm. The arrows point to the cell edge. The arrow heads point to stress fibers. Data are mean values of 4 batches of cell cultures. Error bars indicate SE. NS, not significant.

Figure 7

Proposed mechanism: During migration, nestin regulates the activation of Plk1, which mediates the phosphorylation of vimentin at Ser-56. The phosphorylation of vimentin promotes the connection of vimentin filaments with paxillin, focal adhesion assembly, and cell migration.