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Fascin Regulates Nuclear Movement and Deformation in Migrating Cells

View Article: PubMed Central - PubMed

ABSTRACT

Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.

No MeSH data available.


Related in: MedlinePlus

Fascin Couples F-Actin and the Nuclear Envelope(A) GFP-mini-N2G-Δ3-54, myc-tagged fascin, and F-actin in TAN lines. Starved NIH 3T3 monolayer was wounded, constructs microinjected, and cells stimulated with 10 μM LPA for 1 hr before fixation. Scale bar, 10 μm. Graph shows fluorescence line scan analysis of white arrows shown in the left panel. Yellow arrow indicates points of colocalization. Black arrows on graph show points of GFP-mini-N2G-Δ3-50, myc-fascin, and F-actin co-localization at TAN lines.(B) NIH 3T3 fibroblasts during nucleo-centrosomal axis formation 2 hr after scratch wound and LPA stimulation. Upper panels show merged channels for DNA, tubulin, and F-actin. Lower panels show GFP and GFP-fascin (WT and S39A mutant) channels in the same cells. Representative of four experiments. Scale bar, 20 μm.(C) Quantification of relative nuclear and centrosomal position respective to the cell centroid in SCR and fascin KD fibroblasts expressing GFP, and GFP-fascin (WT and S39A) after LPA stimulation. Mean of four independent experiments, at least 90 cells per group. ∗∗p < 0.01, ∗∗∗p < 0.001 compared with SCR GFP.(D) Quantification of actin fibers retrograde displacement speed during LPA-induced fibroblast polarization. Each value represents the speed of a single fiber, two fibers from >10 cells per condition from three experiments. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 compared with SCR.(E) Kymographs of actin fiber retrograde displacement during LPA-induced fibroblast polarization in fascin KD cells expressing GFP, GFP-fascin WT, or S39A mutant. Wound located at top of kymographs. Arrows indicate example actin cables and dashed lines represent nuclear outline. Representative of three experiments. Scale bars, 10 μm.(F) NIH 3T3 expressing mCherry, mCherry-β-trefoil1, and mCherry-β-trefoil3 after scratch wound and LPA stimulation. Upper panels show DNA (DAPI), tubulin, and F-actin (phalloidin). Lower panels show mCherry and mCherry-β-trefoil expression in the same cells. Representative of four experiments. Scale bar, 20 μm.(G) Quantification of relative nuclear and centrosomal position with respect to the cell centroid in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains 2 hr after LPA stimulation. Average of three experiments, >90 cells per condition. ∗∗p < 0.01 compared with SCR mCherry.(H) Quantification of actin fiber retrograde displacement speed during LPA-induced polarization in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains. Each value represents the speed of a single fiber, >30 cells per condition in three experiments. ns, not significant compared with SCR mCherry.(I) Kymographs of dorsal actin fiber displacement during LPA-induced fibroblast polarization in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains. Arrows indicate example actin fibers. Dashed lines indicate nuclear outline. Representative of three experiments. Scale bars, 10 μm.See also Figure S3; Movies S1 and S2.
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fig3: Fascin Couples F-Actin and the Nuclear Envelope(A) GFP-mini-N2G-Δ3-54, myc-tagged fascin, and F-actin in TAN lines. Starved NIH 3T3 monolayer was wounded, constructs microinjected, and cells stimulated with 10 μM LPA for 1 hr before fixation. Scale bar, 10 μm. Graph shows fluorescence line scan analysis of white arrows shown in the left panel. Yellow arrow indicates points of colocalization. Black arrows on graph show points of GFP-mini-N2G-Δ3-50, myc-fascin, and F-actin co-localization at TAN lines.(B) NIH 3T3 fibroblasts during nucleo-centrosomal axis formation 2 hr after scratch wound and LPA stimulation. Upper panels show merged channels for DNA, tubulin, and F-actin. Lower panels show GFP and GFP-fascin (WT and S39A mutant) channels in the same cells. Representative of four experiments. Scale bar, 20 μm.(C) Quantification of relative nuclear and centrosomal position respective to the cell centroid in SCR and fascin KD fibroblasts expressing GFP, and GFP-fascin (WT and S39A) after LPA stimulation. Mean of four independent experiments, at least 90 cells per group. ∗∗p < 0.01, ∗∗∗p < 0.001 compared with SCR GFP.(D) Quantification of actin fibers retrograde displacement speed during LPA-induced fibroblast polarization. Each value represents the speed of a single fiber, two fibers from >10 cells per condition from three experiments. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 compared with SCR.(E) Kymographs of actin fiber retrograde displacement during LPA-induced fibroblast polarization in fascin KD cells expressing GFP, GFP-fascin WT, or S39A mutant. Wound located at top of kymographs. Arrows indicate example actin cables and dashed lines represent nuclear outline. Representative of three experiments. Scale bars, 10 μm.(F) NIH 3T3 expressing mCherry, mCherry-β-trefoil1, and mCherry-β-trefoil3 after scratch wound and LPA stimulation. Upper panels show DNA (DAPI), tubulin, and F-actin (phalloidin). Lower panels show mCherry and mCherry-β-trefoil expression in the same cells. Representative of four experiments. Scale bar, 20 μm.(G) Quantification of relative nuclear and centrosomal position with respect to the cell centroid in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains 2 hr after LPA stimulation. Average of three experiments, >90 cells per condition. ∗∗p < 0.01 compared with SCR mCherry.(H) Quantification of actin fiber retrograde displacement speed during LPA-induced polarization in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains. Each value represents the speed of a single fiber, >30 cells per condition in three experiments. ns, not significant compared with SCR mCherry.(I) Kymographs of dorsal actin fiber displacement during LPA-induced fibroblast polarization in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains. Arrows indicate example actin fibers. Dashed lines indicate nuclear outline. Representative of three experiments. Scale bars, 10 μm.See also Figure S3; Movies S1 and S2.

Mentions: We next sought to determine the functional consequences of fascin localization to the NE and its association with nesprin-2. Nesprin-2 has been shown to be important for active positioning of the nucleus during cell migration. Starved fibroblasts require stimulation with serum factors such as LPA to establish their nucleo-centrosomal axis during cell polarization after scratch wounding (Gomes et al., 2005). This process relies in the rearward displacement of actin cables that associate with nesprin-2 at the transmembrane actin-associated (TAN) lines. These structures are responsible for coupling the nuclei to the moving actin cables, inducing the nuclear positioning at the cell rear while the centrosome remains stationary at the cell centroid (Luxton et al., 2010, Kutscheidt et al., 2014, Lombardi et al., 2011). We therefore hypothesized that the fascin-nesprin complex may play a role in nuclear positioning during migration. To test this, we first determined whether fascin localized to TAN lines in NIH 3T3cells. Confocal microscopy and fluorescence intensity line scan analysis demonstrated that overexpressed myc-tagged fascin showed significant overlap with both F-actin and GFP-mini-N2G-ΔSR3-54 in cables positioned over the nucleus (Figure 3A), endogenous fascin colocalized with GFP-mini-N2GΔSR3-50 (Figure S3A), and GFP-fascin and F-actin cable displacement both correlated with nuclear movement during cell polarization (Figure S3B). Moreover, stable KD of fascin in NIH 3T3cells (Figure S3C) resulted in a significant reduction in nuclear rearward movement during polarization, and this defect was restored by re-expression of human WT GFP-fascin but not S39A mutant (Figures 3B and 3C) correlating with fascin binding to nesprin-2. The reduction in rearward nuclear displacement was also coupled with a reduction in the speed of actin fiber moving over the nucleus (Figures 3D and 3E; Movie S1). This demonstrates that fascin localization at the NE is required for not only efficient F-actin-nuclear coupling but also normal actin rearward movement during cell polarization.


Fascin Regulates Nuclear Movement and Deformation in Migrating Cells
Fascin Couples F-Actin and the Nuclear Envelope(A) GFP-mini-N2G-Δ3-54, myc-tagged fascin, and F-actin in TAN lines. Starved NIH 3T3 monolayer was wounded, constructs microinjected, and cells stimulated with 10 μM LPA for 1 hr before fixation. Scale bar, 10 μm. Graph shows fluorescence line scan analysis of white arrows shown in the left panel. Yellow arrow indicates points of colocalization. Black arrows on graph show points of GFP-mini-N2G-Δ3-50, myc-fascin, and F-actin co-localization at TAN lines.(B) NIH 3T3 fibroblasts during nucleo-centrosomal axis formation 2 hr after scratch wound and LPA stimulation. Upper panels show merged channels for DNA, tubulin, and F-actin. Lower panels show GFP and GFP-fascin (WT and S39A mutant) channels in the same cells. Representative of four experiments. Scale bar, 20 μm.(C) Quantification of relative nuclear and centrosomal position respective to the cell centroid in SCR and fascin KD fibroblasts expressing GFP, and GFP-fascin (WT and S39A) after LPA stimulation. Mean of four independent experiments, at least 90 cells per group. ∗∗p < 0.01, ∗∗∗p < 0.001 compared with SCR GFP.(D) Quantification of actin fibers retrograde displacement speed during LPA-induced fibroblast polarization. Each value represents the speed of a single fiber, two fibers from >10 cells per condition from three experiments. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 compared with SCR.(E) Kymographs of actin fiber retrograde displacement during LPA-induced fibroblast polarization in fascin KD cells expressing GFP, GFP-fascin WT, or S39A mutant. Wound located at top of kymographs. Arrows indicate example actin cables and dashed lines represent nuclear outline. Representative of three experiments. Scale bars, 10 μm.(F) NIH 3T3 expressing mCherry, mCherry-β-trefoil1, and mCherry-β-trefoil3 after scratch wound and LPA stimulation. Upper panels show DNA (DAPI), tubulin, and F-actin (phalloidin). Lower panels show mCherry and mCherry-β-trefoil expression in the same cells. Representative of four experiments. Scale bar, 20 μm.(G) Quantification of relative nuclear and centrosomal position with respect to the cell centroid in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains 2 hr after LPA stimulation. Average of three experiments, >90 cells per condition. ∗∗p < 0.01 compared with SCR mCherry.(H) Quantification of actin fiber retrograde displacement speed during LPA-induced polarization in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains. Each value represents the speed of a single fiber, >30 cells per condition in three experiments. ns, not significant compared with SCR mCherry.(I) Kymographs of dorsal actin fiber displacement during LPA-induced fibroblast polarization in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains. Arrows indicate example actin fibers. Dashed lines indicate nuclear outline. Representative of three experiments. Scale bars, 10 μm.See also Figure S3; Movies S1 and S2.
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fig3: Fascin Couples F-Actin and the Nuclear Envelope(A) GFP-mini-N2G-Δ3-54, myc-tagged fascin, and F-actin in TAN lines. Starved NIH 3T3 monolayer was wounded, constructs microinjected, and cells stimulated with 10 μM LPA for 1 hr before fixation. Scale bar, 10 μm. Graph shows fluorescence line scan analysis of white arrows shown in the left panel. Yellow arrow indicates points of colocalization. Black arrows on graph show points of GFP-mini-N2G-Δ3-50, myc-fascin, and F-actin co-localization at TAN lines.(B) NIH 3T3 fibroblasts during nucleo-centrosomal axis formation 2 hr after scratch wound and LPA stimulation. Upper panels show merged channels for DNA, tubulin, and F-actin. Lower panels show GFP and GFP-fascin (WT and S39A mutant) channels in the same cells. Representative of four experiments. Scale bar, 20 μm.(C) Quantification of relative nuclear and centrosomal position respective to the cell centroid in SCR and fascin KD fibroblasts expressing GFP, and GFP-fascin (WT and S39A) after LPA stimulation. Mean of four independent experiments, at least 90 cells per group. ∗∗p < 0.01, ∗∗∗p < 0.001 compared with SCR GFP.(D) Quantification of actin fibers retrograde displacement speed during LPA-induced fibroblast polarization. Each value represents the speed of a single fiber, two fibers from >10 cells per condition from three experiments. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 compared with SCR.(E) Kymographs of actin fiber retrograde displacement during LPA-induced fibroblast polarization in fascin KD cells expressing GFP, GFP-fascin WT, or S39A mutant. Wound located at top of kymographs. Arrows indicate example actin cables and dashed lines represent nuclear outline. Representative of three experiments. Scale bars, 10 μm.(F) NIH 3T3 expressing mCherry, mCherry-β-trefoil1, and mCherry-β-trefoil3 after scratch wound and LPA stimulation. Upper panels show DNA (DAPI), tubulin, and F-actin (phalloidin). Lower panels show mCherry and mCherry-β-trefoil expression in the same cells. Representative of four experiments. Scale bar, 20 μm.(G) Quantification of relative nuclear and centrosomal position with respect to the cell centroid in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains 2 hr after LPA stimulation. Average of three experiments, >90 cells per condition. ∗∗p < 0.01 compared with SCR mCherry.(H) Quantification of actin fiber retrograde displacement speed during LPA-induced polarization in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains. Each value represents the speed of a single fiber, >30 cells per condition in three experiments. ns, not significant compared with SCR mCherry.(I) Kymographs of dorsal actin fiber displacement during LPA-induced fibroblast polarization in fibroblasts expressing mCherry and mCherry-tagged β-trefoil domains. Arrows indicate example actin fibers. Dashed lines indicate nuclear outline. Representative of three experiments. Scale bars, 10 μm.See also Figure S3; Movies S1 and S2.
Mentions: We next sought to determine the functional consequences of fascin localization to the NE and its association with nesprin-2. Nesprin-2 has been shown to be important for active positioning of the nucleus during cell migration. Starved fibroblasts require stimulation with serum factors such as LPA to establish their nucleo-centrosomal axis during cell polarization after scratch wounding (Gomes et al., 2005). This process relies in the rearward displacement of actin cables that associate with nesprin-2 at the transmembrane actin-associated (TAN) lines. These structures are responsible for coupling the nuclei to the moving actin cables, inducing the nuclear positioning at the cell rear while the centrosome remains stationary at the cell centroid (Luxton et al., 2010, Kutscheidt et al., 2014, Lombardi et al., 2011). We therefore hypothesized that the fascin-nesprin complex may play a role in nuclear positioning during migration. To test this, we first determined whether fascin localized to TAN lines in NIH 3T3cells. Confocal microscopy and fluorescence intensity line scan analysis demonstrated that overexpressed myc-tagged fascin showed significant overlap with both F-actin and GFP-mini-N2G-ΔSR3-54 in cables positioned over the nucleus (Figure 3A), endogenous fascin colocalized with GFP-mini-N2GΔSR3-50 (Figure S3A), and GFP-fascin and F-actin cable displacement both correlated with nuclear movement during cell polarization (Figure S3B). Moreover, stable KD of fascin in NIH 3T3cells (Figure S3C) resulted in a significant reduction in nuclear rearward movement during polarization, and this defect was restored by re-expression of human WT GFP-fascin but not S39A mutant (Figures 3B and 3C) correlating with fascin binding to nesprin-2. The reduction in rearward nuclear displacement was also coupled with a reduction in the speed of actin fiber moving over the nucleus (Figures 3D and 3E; Movie S1). This demonstrates that fascin localization at the NE is required for not only efficient F-actin-nuclear coupling but also normal actin rearward movement during cell polarization.

View Article: PubMed Central - PubMed

ABSTRACT

Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in&nbsp;vitro and in&nbsp;vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.

No MeSH data available.


Related in: MedlinePlus