The intermediate filament protein nestin serves as a molecular hub for smooth muscle cytoskeletal signaling

Figures

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  Fig. 1. Nestin regulates actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. A Protein expression of human airway smooth muscle (HASM) cells expressing control (Ctrl) or nestin shRNA was evaluated by immunoblot analysis. Nestin shRNA reduces nestin expression in HASM cells. Data are means ± SE (n = 4 batches of culture from 3 donors). B Ctrl and nestin knockdown (KD) HASM cells were treated with 10–4 M acetylcholine (ACh) for 5 min, or left untreated (UT). F-actin/G-actin ratios in cells were evaluated using the fractionation assay. Data are means ± SE (n = 5 batches of culture from 3 donors). C Myosin light chain (MLC) phosphorylation at Ser-19 in Ctrl cells and nestin KD cells was assessed by immunoblot analysis. Basal and ACh-induced MLC phosphorylation was similar in Ctrl cells and nestin KD cells (NS, not significant). Data are means ± SE (n = 4–5 batches of culture from 3 donors). D Immunoblots showing the effects of nestin siRNA on protein expression in human PLCS. Data are means ± SE (n = 3 slices from 3 donors). E Nestin KD reduces airway constriction of human PLCS. Data are means ± SE (n = 4–6 slices from 3 donors).…

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  Fig. 2. Nestin modulates the recruitment of cortactin and Pfn-1 to the membrane. Ctrl and nestin knockdown (KD) HASM cells were treated with 10–4 M ACh for 5 min, or left untreated (UT). Spatial distribution of cortactin and Pfn-1 was evaluated by immunfluorescence microscopy. A Representative micrographs illustrating the effects of ACh on the spatial localization of cortactin and Pfn-1 in HASM cells. The arrows indicate a single line scan to quantify the fluorescent signals in cells. The line scan graphs show relative fluorescent intensity. B Image analysis for protein localization was described in the section of Materials and Methods. Protein distribution in cells is expressed as ratio of the intensity at the cell periphery to cell interior. Data are means ± SE (n = 6 batches of culture from 3 donors). *p < 0.05; ** p < 0.01

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  Fig. 3. Contractile stimulation enhances nestin phosphorylation at Thr-315 and nestin/Plk1 coupling. HASM cells were treated with 10–4 M ACh for different time points, or left untreated (UT). A Nestin phosphorylation at Thr-315 was determined by immunoblot analysis. Data are means ± SE (n = 5 batches of culture from 3 donors). B ACh stimulation enhances the ratios of Plk1/immunoprecipitated nestin without affecting protein expression in input fraction. IgG serves as a control. Data are means ± SE (n = 4 batches of culture from 3 donors). *p < 0.05; ** p < 0.01

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  Fig. 4. Nestin regulates Plk1 phosphorylation at Thr-210 and vimentin phosphorylation at Ser-56. Ctrl and nestin KD HASM cells were treated with 10–4 M ACh for 5 min, or left untreated (UT). Protein phosphorylation in HASM cells was determined by immunoblot analysis. Nestin KD diminishes ACh-induced Plk1 phosphorylation at Thr-210 (A) and vimentin phosphorylation at Ser-56 (B). Data are means ± SE (n = 4 batches of culture from 3 donors). ** p < 0.01

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  Fig. 5. Role of nestin phosphorylation at Thr-315 in actin polymerization, MLC phosphorylation and smooth muscle contraction. A Extracts of HASM cells treated with wild type (WT) or T315A nestin were separated by SDS PAGE and immunobotted with antibodies against nestin and GAPDH. Data are means ± SE (n = 4–5 batches of culture from 3 donors). B HASM cells expressing WT or T315A nestin were treated with 10–4 M ACh for 5 min, or left untreated (UT). F-actin/G-actin ratios in cells were evaluated using the fractionation assay. Data are means ± SE (n = 4–5 batches of culture from 3 donors). C MLC phosphorylation at Ser-19 in cells treated with WT or T315A nestin was assessed by immunoblot analysis. Basal and ACh-induced MLC phosphorylation was similar in cells expressing WT and T315A nestin (NS, not significant). Data are means ± SE (n = 4–5 batches of culture from 3 donors). D Expression of T315A nestin reduces airway constriction of human PLCS. Data are means ± SE (n = 4–6 slices from 3 donors). *p < 0.05; ** p < 0.01. One-way ANOVA was used for statistical analysis of A Two-way ANOVA was used for statistical analysis of B and C. Student’s t-test was used for statistical analysis of D

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  Fig. 6. Nestin phosphorylation at Thr-315 orchestrates redistribution of cortactin and Pfn-1 to the membrane. Cells expressing WT or T315A nestin were treated with 10–4 M ACh for 5 min, or left untreated (UT). Cellular localization of cortactin and Pfn-1 was evaluated by immunfluorescence microscopy. A Representative micrographs illustrating the effects of T315A nestin on the spatial localization of cortactin and Pfn-1 in HASM cells. The arrows indicate a single line scan to quantify the fluorescent signals in cells. The line scan graphs show relative fluorescent intensity. B Image analysis for protein localization was described in the section of Materials and Methods. Protein distribution in cells is expressed as ratio of the intensity at the cell periphery to cell interior. Data are means ± SE (n = 6 batches of culture from 3 donors). *p < 0.05; ** p < 0.01

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  Fig. 7. Plk1 regulates nestin phosphorylation at Thr-315 in smooth muscle. A Protein expression of Ctrl and Plk1 KD HASM cells were evaluated by immunoblot analysis. B Ctrl and Plk1 KD HASM cells were treated with 10–4 M ACh for 5 min, or left untreated. Nestin phosphorylation was determined by immunoblot analysis. Plk1 KD inhibits ACh-induced nestin phosphorylation at Thr-315. Data are means ± SE (n = 4–5 batches of culture from 3 donors). * p < 0.05

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  Fig. 8. Role of nestin in cytoskeletal signaling. Contractile stimulation promotes the association of nestin with Plk1, which facilitates nestin phosphorylation at Thr-315, and renders Plk1 in active state. This nestin/Plk1 interaction forms a positive activation loop. Activated Plk1 catalyzes vimentin phosphorylation at Ser-56, which subsequently promotes the recruitment of cortactin and Pfn-1 to the membrane, actin polymerization, and smooth muscle contraction