NudCL2 regulates cell migration by stabilizing both myosin-9 and LIS1 with Hsp90

Abstract

Cell migration plays pivotal roles in many biological processes; however, its underlying mechanism remains unclear. Here, we find that NudC-like protein 2 (NudCL2), a cochaperone of heat shock protein 90 (Hsp90), modulates cell migration by stabilizing both myosin-9 and lissencephaly protein 1 (LIS1). Either knockdown or knockout of NudCL2 significantly increases single-cell migration, but has no significant effect on collective cell migration. Immunoprecipitation-mass spectrometry and western blotting analyses reveal that NudCL2 binds to myosin-9 in mammalian cells. Depletion of NudCL2 not only decreases myosin-9 protein levels, but also results in actin disorganization. Ectopic expression of myosin-9 efficiently reverses defects in actin disorganization and single-cell migration in cells depleted of NudCL2. Interestingly, knockdown of myosin-9 increases both single and collective cell migration. Depletion of LIS1, a NudCL2 client protein, suppresses both single and collective cell migration, which exhibits the opposite effect compared with myosin-9 depletion. Co-depletion of myosin-9 and LIS1 promotes single-cell migration, resembling the phenotype caused by NudCL2 depletion. Furthermore, inhibition of Hsp90 ATPase activity also reduces the Hsp90-interacting protein myosin-9 stability and increases single-cell migration. Forced expression of Hsp90 efficiently reverses myosin-9 protein instability and the defects induced by NudCL2 depletion, but not vice versa. Taken together, these data suggest that NudCL2 plays an important role in the precise regulation of cell migration by stabilizing both myosin-9 and LIS1 via Hsp90 pathway.

Fig. 1. NudCL2 is required for single-cell migration in vitro.

a A549 cells transfected with siRNAs targeting different NudCL2 mRNA regions (siNudCL2-1 and siNudCL2-2) were subjected to western blotting analysis with anti-NudCL2 antibody. β-actin was used as a loading control. b, c Transwell migration assays revealed the cell motility of control and NudCL2-depleted cells. Scale bar, 200 μm. Cells that migrated to the undersides of the filters were counted. d–f The migration tracks of individual cells transfected with the indicated siRNAs were traced by Imaris 9.1.2 software. Representative single-cell migration paths are shown. Euclidean distance and migration velocity were calculated. g, h The wound healing assays showed collective cell migration at different time points. Dashed lines indicate the wound edges. Scale bar, 200 μm. The distance of the wound was measured by ImageJ software. i–k Cells transfected with the indicated siRNAs and Flag-NudCL2* (siRNA-resistant NudCL2) or Flag were subjected to the following analyses. Western blotting analysis showed the expression of NudCL2 and Flag-NudCL2. β-actin was used as a loading control. Transwell migration assays revealed cell motility. Scale bar, 200 μm. Cells that migrated to the undersides of the filters were counted. l–n Cells transfected with the indicated siRNAs and vectors for 72 h were subjected to a migration experiment. The migration paths of the individual cells were analyzed with Imaris 9.1.2 software. Representative single-cell migration tracks are shown. Euclidean distance and migration velocity were measured. Quantitative data from at least three independent experiments are shown as the mean ± SD. n, sample size. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (P > 0.05). Student’s t-test.

Fig. 2. NudCL2 binds to and stabilizes myosin-9.

a Schematic representation of the IP-Mass approach. b HeLa cells transfected with either Flag-NudCL2 or Flag were subjected to immunoprecipitation analysis with anti-Flag antibody-coupled beads. c The top 10 proteins ranked by relative abundance from IP-Mass analysis are shown. d A549 cells transfected with control or NudCL2 siRNA were subjected to western blotting analysis with the indicated antibodies. GAPDH was used as a loading control. e Quantitative RT-PCR analysis of MYH9 mRNA in control and NudCL2-depleted cells. GAPDH was used as an internal control. f A549 cells transfected with the indicated siRNAs and vectors were subjected to western blotting analysis with anti-NudCL2 and anti-Myosin-9 antibodies. β-actin was used as a loading control. g Cells were treated with 10 µM MG132 or DMSO for 2 h. Cell lysates were used for western blotting analysis with anti-Myosin-9 and anti-NudCL2 antibodies. β-actin was used as a loading control. h Purified GST or GST-NudCL2 protein was incubated with A549 cell lysates and subjected to immunoblotting with anti-Myosin-9 antibody. Five percent of the total input is shown. GST and GST-NudCL2 input was stained with Coomassie brilliant blue. i, j A549 cells were harvested and lysed. Immunoprecipitation analyses were carried out using the indicated antibodies. Three percent of the total input is shown. Quantitative data are expressed as the mean ± SD (at least three independent experiments). ns, no significance (P > 0.05). Student’s t-test.

Fig. 3. Depletion of NudCL2 impairs actin dynamics.

A549 cells transfected with siRNAs and vectors were subjected to the following analyses: a Western blotting analysis of the expression of NudCL2. β-actin was used as a loading control. b Cells were fixed and stained with phalloidin (red). DNA was visualized with DAPI (blue). Scale bar, 20 μm. c The percentage of cells with lamellipodia in (b) was calculated. More than 100 cells were counted in each experiment. d A sequence of phase-contrast time-lapse images of the cells were obtained with a LSM880 confocal microscope using a ×63 objective. Kymographs were produced and analyzed using MetaMorph software. The minimum intensity projection of a 250-frame movie (3 s per frame) is presented on the left. Pixel intensities along a one-pixel-wide line (white) were used to generate the kymograph presented on the right. Cells are outlined with dashed lines. Scale bar, 20 μm. e, f The velocity and persistence of lamellipodia protrusions in (d) are shown. g Cells were fixed and subjected to immunofluorescence staining with anti-paxillin (green) antibody. Scale bar, 20 μm. h Focal adhesions between cells in (g) were counted. About 20 cells were counted in each experiment. i Cells transfected with the indicated siRNA and vectors were subjected to western blotting analysis using the antibodies as shown. β-actin was used as a loading control. j–l Cells were fixed and stained with phalloidin (red) and anti-paxillin (green) antibody. DNA was visualized with DAPI (blue). Scale bar, 20 μm. The percentage of cells with lamellipodia and number of cellular focal adhesions were plotted respectively. More than 100 cells were counted in each experiment. m–o A sequence of phase-contrast time-lapse images of cells were obtained with a LSM880 confocal microscope using a ×63 objective. Kymographs were produced and analyzed using MetaMorph software. The minimum intensity projection of a 250-frame movie (3 s per frame) is presented on the left. Pixel intensities along a one-pixel-wide line (white) were used to generate the kymograph presented on the right. Cells are outlined with dashed lines. Scale bar, 20 μm. The velocity and persistence of lamellipodia protrusions are calculated. Quantitative data derived from at least three independent experiments are shown as the mean ± SD. n, sample size. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t-test.

Fig. 4. Exogenous expression of myosin-9 reverses the defects caused by NudCL2 depletion.

A549 cells transfected with siRNAs and vectors were subjected to the following analyses: a Western blotting analysis of the expression of NudCL2, myosin-9, and GFP-myosin-9. β-actin was used as a loading control. b ImageJ software was used to quantify protein levels in (a). The relative amounts of myosin-9 and GFP-myosin-9 compared with the control were calculated and shown. c, d Cells were fixed and stained with phalloidin, anti-paxillin antibody and DAPI. Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. e Transwell migration assays revealed cell motility. Cells that migrated to the undersides of the filters were counted. f, g The migration tracks of individual cells were traced by Imaris 9.1.2 software. Euclidean distance and migration velocity were measured. h A549 cells transfected with the indicated siRNAs and vectors were subjected to the following analyses: western blotting analysis of the expression of myosin-9 and Myc-NudCL2. β-actin was used as a loading control. i, j Cells were fixed and stained with phalloidin, anti-paxillin antibody, and DAPI. Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. k Transwell migration assays revealed cell motility. Cells that migrated to the undersides of the filters were counted. l, m The migration tracks of individual cells were traced by Imaris 9.1.2 software. Euclidean distance and migration velocity were measured. Quantitative data derived from at least three independent experiments are shown as the mean ± SD. More than 100 cells were counted in each experiment. n, sample size. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (P > 0.05). Student’s t-test.

Fig. 5. Depletion of LIS1 inhibits cell migration.

A549 cells transfected with siRNAs were subjected to the following analyses: a Western blotting analysis of the expression of NudCL2, myosin-9, and LIS1. β-actin was used as a loading control. b ImageJ software was used to quantify protein levels in (a). The relative amounts of myosin-9 and LIS1 compared with the control were calculated and shown. c Western blotting analysis of the expression of LIS1. d, e Transwell migration assays revealed the cell motility of control and LIS1-depleted cells. Scale bar, 200 μm. Cells that migrated to the undersides of the filters were counted. f–h The migration tracks of individual cells were traced by Imaris 9.1.2 software. Representative single-cell migration paths are shown. Euclidean distance and migration velocity were measured. i, j Wound healing assays showed collective cell migration at different time points. Dashed lines indicate wound edges. Scale bar, 200 μm. The distance of the wound was measured by ImageJ software. Quantitative data derived from at least three independent experiments are shown as the mean ± SD. n, sample size. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t-test.

Fig. 6. Co-depletion of myosin-9 and LIS1 promotes single-cell migration.

A549 cells transfected with siRNAs were subjected to the following analyses: a Western blotting analysis of the expression of LIS1 and myosin-9. β-actin was used as a loading control. b, c Transwell migration assays revealed cell motility. Scale bar, 200 μm. Cells that migrated to the undersides of the filters were counted. d–f The migration tracks of individual cells were traced by Imaris 9.1.2 software. Representative single-cell migration paths are shown. Euclidean distance and migration velocity were measured. g, h Wound healing assays showed collective cell migration at different time points. Dashed lines indicate wound edges. Scale bar, 200 μm. The distance of the wound was measured by ImageJ software. Quantitative data derived from at least three independent experiments are shown as the mean ± SD. n, sample size. *P < 0.05; ***P < 0.001; ns, not significant (P > 0.05). Student’s t-test.

Fig. 7. Hsp90 is involved in NudCL2-meidated myosin-9 stability and cell migration.

A549 cells transfected with or without the indicated siRNAs and vectors were treated with 3.78 µM geldanamycin (GA) or DMSO for 48 h and subjected to the following analyses: a A549 cells were harvested and lysed. Immunoprecipitation analyses were carried out using the indicated antibodies. Three percent of the total input is shown. b Western blotting analysis of the expression of myosin-9, LIS1, and Hsp90. β-actin was used as a loading control. c Relative protein levels compared with the control at the same time point of GA treatment in (b) were measured using ImageJ software and shown. d–f Cells were fixed and stained with phalloidin (red) and anti-paxillin (green) antibody. DNA was visualized with DAPI (blue). Scale bar, 20 μm. Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. g, h Transwell migration assays revealed cell motility. Scale bar, 200 μm. Cells that migrated to the undersides of the filters were counted. i Western blotting analysis of the expression of myosin-9, LIS1, Myc-Hsp90, and NudCL2. β-actin was used as a loading control. j ImageJ software was used to quantify protein levels in (i). The relative amounts of myosin-9 and LIS1 compared with the control were calculated and shown. k, l Cells were stained as described in (d). Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. m Western blotting analysis of the expression of myosin-9, LIS1, Hsp90, and Myc-NudCL2. β-actin was used as a loading control. n ImageJ software was used to quantify protein levels in (m). The relative amounts of myosin-9 and LIS1 compared with the control were calculated and shown. o, p Cells were stained as described in (d). Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. q A549 cells transfected with the indicated siRNAs were treated with GA and subjected to western blotting analysis of the expression of myosin-9, LIS1, Hsp90, and NudCL2. β-actin was used as a loading control. r ImageJ software was used to quantify protein levels in (q). The relative amounts of myosin-9 and LIS1 compared with the control were calculated and shown. s, t Cells were stained as described in (d). Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. Quantitative data derived from at least three independent experiments are shown as the mean ± SD. More than 100 cells were counted in each experiment. n, sample size. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (P > 0.05). Student’s t-test. u Working model for the role of NudCL2 in cell migration. NudCL2 stabilizes myosin-9 and LIS1 proteins by Hsp90 and plays an important role in the precise regulation of cell migration. Depletion of NudCL2 leads to myosin-9 and LIS1 degradation and increases single-cell migration but not collective cell migration.