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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.


Fascin Interacts with the C-Terminal Region of Nesprin-2(A) Co-immunoprecipitation (coIP) of GFP, GFP-nes1(66–74) and GFP-nes2(49–56) with FLAG-tagged fascin expressed in HEK293T cells. Densitometry values normalized to GFP IP protein content from three independent experiments ± SEM are depicted below. ∗p < 0.05 compared with GFP only.(B) HeLa cells co-expressing GFP-nes1(66–74), GFP-nes2(49–56), or GFP-SUN2 with mRFP-fascin. Center panels show pseudocolored FLIM images. Graph shows mean FRET efficiency of GFP-tagged proteins and mRFP-tagged fascin. n = 18 cells from three experiments ± SEM. ∗∗p < 0.01 compared with GFP-SUN2.(C) Western blot of endogenous fascin pulled down from NIH 3T3 fibroblast lysates with GST alone and GST-tagged nesprin-1 and nesprin-2 SR triplets. Densitometry values normalized to GST ± SEM from three experiments are shown below. ∗∗p < 0.01 compared with GST.(D) Western blot of recombinant His-tagged fascin pulled down with GST-tagged nesprin-1 and nesprin-2 SR triplets. Mean densitometry values normalized to GST ± SEM from three experiments are shown below. ∗∗p < 0.01 compared with GST.(E) CoIP of endogenous fascin with GFP, GFP-mini-N2G-ΔSR3-54, and GFP-mini-N2G-ΔSR3-50 expressed in NIH 3T3. Densitometry values normalized to GFP ± SEM from three experiments are shown below. ∗p < 0.05 compared with GFP.(F) Representative single confocal slice showing co-localization of endogenous fascin and nesprin-2 in NIH 3T3 acquired by structured illumination microscopy. Arrowheads show enrichment of fascin at the NE; arrows show fascin localized at fibers over the nucleus. Scale bars, 2.5 μm.(G) Endogenous fascin and nesprin-2 in NIH 3T3 expressing a non-targeting scrambled SCR shRNA (SCR), nesprin-2-targeting shRNA (Nes2 KD), or fascin-targeting shRNA (Fascin KD). Graphs show fluorescence line scan analysis averaged from at least 15 cells in two experiments. Yellow arrows represent sites of line scan analysis. Scale bars, 10 μm.See also Figure S1.
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fig1: Fascin Interacts with the C-Terminal Region of Nesprin-2(A) Co-immunoprecipitation (coIP) of GFP, GFP-nes1(66–74) and GFP-nes2(49–56) with FLAG-tagged fascin expressed in HEK293T cells. Densitometry values normalized to GFP IP protein content from three independent experiments ± SEM are depicted below. ∗p < 0.05 compared with GFP only.(B) HeLa cells co-expressing GFP-nes1(66–74), GFP-nes2(49–56), or GFP-SUN2 with mRFP-fascin. Center panels show pseudocolored FLIM images. Graph shows mean FRET efficiency of GFP-tagged proteins and mRFP-tagged fascin. n = 18 cells from three experiments ± SEM. ∗∗p < 0.01 compared with GFP-SUN2.(C) Western blot of endogenous fascin pulled down from NIH 3T3 fibroblast lysates with GST alone and GST-tagged nesprin-1 and nesprin-2 SR triplets. Densitometry values normalized to GST ± SEM from three experiments are shown below. ∗∗p < 0.01 compared with GST.(D) Western blot of recombinant His-tagged fascin pulled down with GST-tagged nesprin-1 and nesprin-2 SR triplets. Mean densitometry values normalized to GST ± SEM from three experiments are shown below. ∗∗p < 0.01 compared with GST.(E) CoIP of endogenous fascin with GFP, GFP-mini-N2G-ΔSR3-54, and GFP-mini-N2G-ΔSR3-50 expressed in NIH 3T3. Densitometry values normalized to GFP ± SEM from three experiments are shown below. ∗p < 0.05 compared with GFP.(F) Representative single confocal slice showing co-localization of endogenous fascin and nesprin-2 in NIH 3T3 acquired by structured illumination microscopy. Arrowheads show enrichment of fascin at the NE; arrows show fascin localized at fibers over the nucleus. Scale bars, 2.5 μm.(G) Endogenous fascin and nesprin-2 in NIH 3T3 expressing a non-targeting scrambled SCR shRNA (SCR), nesprin-2-targeting shRNA (Nes2 KD), or fascin-targeting shRNA (Fascin KD). Graphs show fluorescence line scan analysis averaged from at least 15 cells in two experiments. Yellow arrows represent sites of line scan analysis. Scale bars, 10 μm.See also Figure S1.

Mentions: To define potential unexplored regulators of fascin function, we performed mass spectrometry analysis of specific isolated proteins in complex with GFP-fascin immunoprecipitated from MDA MB 231 human breast carcinoma cells. Within the reproducible hits that were significantly represented in the resulting peptide analysis, we identified nesprin-1, a member of the LINC complex (Figure S1A). We have previously shown that fascin localizes to the nucleus and the nuclear periphery both during Drosophila development and in mammalian cells (Groen et al., 2015). The function associated with this specific localization remains unknown; therefore, we chose to further characterize the potential interaction with the nesprin family of proteins. Initial experiments did not reveal a detectable interaction with endogenous full-length nesprin-1, but as this analysis was performed at low detergent concentration, we cannot exclude that this was due to retained co-association between other cytoskeletal-associated NE proteins in complex with fascin. Based on our previously published data on fascin subcellular localization, we then focused on the C-terminal domains of the proteins nesprin-1 and -2, as these regions are present in all nesprin-1 and -2 isoforms anchored to the NE and encompass the most conserved regions across the whole sequence (Autore et al., 2013) (Figure S1B). Co-immunoprecipitation (coIP) experiments with either GFP-nes1(66–74) or GFP-nes2(49–56) demonstrated that fascin was able to specifically form a complex with the nesprin-2 C-terminal region (Figure 1A). We confirmed this interaction in intact cells by quantifying fluorescence resonance energy transfer (FRET) using fluorescence lifetime imaging microscopy (FLIM) between GFP-tagged nesprins and monomeric RFP (mRFP)-tagged fascin. This analysis demonstrated a specific and direct interaction in whole cells between GFP-nes2(49–56), but not GFP-nes1(66–74) or the inner NE protein SUN2 (Figure 1B). To define the region of nesprin-2 that associates with fascin, we used recombinant glutathione S-transferase (GST)-tagged regions of tandem SRs from nesprin-1 and nesprin-2 as bait to pull down endogenous fascin from lysates of NIH 3T3 fibroblasts. Western blotting revealed that fascin was only found in a complex with SR51–53 from nesprin-2 (Figure 1C). We verified that fascin directly interacted with SR51–53 of nesprin-2 through in vitro binding assays (Figure 1D) and far-western blot analysis of GST-nesprin SR proteins probed with His-tagged fascin (Figure S1C). Moreover, GFP-mini-N2G-ΔSR3-50 containing the SR51–53 region (schematic representation in Figure S1B) showed specific coIP with endogenous fascin in NIH 3T3cells (Figure 1E). Collectively these data demonstrate that fascin forms a complex specifically with nesprin-2 in vitro and in cells, and this binding occurs directly and through SR51–53 in nesprin-2.


Fascin Regulates Nuclear Movement and Deformation in Migrating Cells
Fascin Interacts with the C-Terminal Region of Nesprin-2(A) Co-immunoprecipitation (coIP) of GFP, GFP-nes1(66–74) and GFP-nes2(49–56) with FLAG-tagged fascin expressed in HEK293T cells. Densitometry values normalized to GFP IP protein content from three independent experiments ± SEM are depicted below. ∗p < 0.05 compared with GFP only.(B) HeLa cells co-expressing GFP-nes1(66–74), GFP-nes2(49–56), or GFP-SUN2 with mRFP-fascin. Center panels show pseudocolored FLIM images. Graph shows mean FRET efficiency of GFP-tagged proteins and mRFP-tagged fascin. n = 18 cells from three experiments ± SEM. ∗∗p < 0.01 compared with GFP-SUN2.(C) Western blot of endogenous fascin pulled down from NIH 3T3 fibroblast lysates with GST alone and GST-tagged nesprin-1 and nesprin-2 SR triplets. Densitometry values normalized to GST ± SEM from three experiments are shown below. ∗∗p < 0.01 compared with GST.(D) Western blot of recombinant His-tagged fascin pulled down with GST-tagged nesprin-1 and nesprin-2 SR triplets. Mean densitometry values normalized to GST ± SEM from three experiments are shown below. ∗∗p < 0.01 compared with GST.(E) CoIP of endogenous fascin with GFP, GFP-mini-N2G-ΔSR3-54, and GFP-mini-N2G-ΔSR3-50 expressed in NIH 3T3. Densitometry values normalized to GFP ± SEM from three experiments are shown below. ∗p < 0.05 compared with GFP.(F) Representative single confocal slice showing co-localization of endogenous fascin and nesprin-2 in NIH 3T3 acquired by structured illumination microscopy. Arrowheads show enrichment of fascin at the NE; arrows show fascin localized at fibers over the nucleus. Scale bars, 2.5 μm.(G) Endogenous fascin and nesprin-2 in NIH 3T3 expressing a non-targeting scrambled SCR shRNA (SCR), nesprin-2-targeting shRNA (Nes2 KD), or fascin-targeting shRNA (Fascin KD). Graphs show fluorescence line scan analysis averaged from at least 15 cells in two experiments. Yellow arrows represent sites of line scan analysis. Scale bars, 10 μm.See also Figure S1.
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fig1: Fascin Interacts with the C-Terminal Region of Nesprin-2(A) Co-immunoprecipitation (coIP) of GFP, GFP-nes1(66–74) and GFP-nes2(49–56) with FLAG-tagged fascin expressed in HEK293T cells. Densitometry values normalized to GFP IP protein content from three independent experiments ± SEM are depicted below. ∗p < 0.05 compared with GFP only.(B) HeLa cells co-expressing GFP-nes1(66–74), GFP-nes2(49–56), or GFP-SUN2 with mRFP-fascin. Center panels show pseudocolored FLIM images. Graph shows mean FRET efficiency of GFP-tagged proteins and mRFP-tagged fascin. n = 18 cells from three experiments ± SEM. ∗∗p < 0.01 compared with GFP-SUN2.(C) Western blot of endogenous fascin pulled down from NIH 3T3 fibroblast lysates with GST alone and GST-tagged nesprin-1 and nesprin-2 SR triplets. Densitometry values normalized to GST ± SEM from three experiments are shown below. ∗∗p < 0.01 compared with GST.(D) Western blot of recombinant His-tagged fascin pulled down with GST-tagged nesprin-1 and nesprin-2 SR triplets. Mean densitometry values normalized to GST ± SEM from three experiments are shown below. ∗∗p < 0.01 compared with GST.(E) CoIP of endogenous fascin with GFP, GFP-mini-N2G-ΔSR3-54, and GFP-mini-N2G-ΔSR3-50 expressed in NIH 3T3. Densitometry values normalized to GFP ± SEM from three experiments are shown below. ∗p < 0.05 compared with GFP.(F) Representative single confocal slice showing co-localization of endogenous fascin and nesprin-2 in NIH 3T3 acquired by structured illumination microscopy. Arrowheads show enrichment of fascin at the NE; arrows show fascin localized at fibers over the nucleus. Scale bars, 2.5 μm.(G) Endogenous fascin and nesprin-2 in NIH 3T3 expressing a non-targeting scrambled SCR shRNA (SCR), nesprin-2-targeting shRNA (Nes2 KD), or fascin-targeting shRNA (Fascin KD). Graphs show fluorescence line scan analysis averaged from at least 15 cells in two experiments. Yellow arrows represent sites of line scan analysis. Scale bars, 10 μm.See also Figure S1.
Mentions: To define potential unexplored regulators of fascin function, we performed mass spectrometry analysis of specific isolated proteins in complex with GFP-fascin immunoprecipitated from MDA MB 231 human breast carcinoma cells. Within the reproducible hits that were significantly represented in the resulting peptide analysis, we identified nesprin-1, a member of the LINC complex (Figure S1A). We have previously shown that fascin localizes to the nucleus and the nuclear periphery both during Drosophila development and in mammalian cells (Groen et al., 2015). The function associated with this specific localization remains unknown; therefore, we chose to further characterize the potential interaction with the nesprin family of proteins. Initial experiments did not reveal a detectable interaction with endogenous full-length nesprin-1, but as this analysis was performed at low detergent concentration, we cannot exclude that this was due to retained co-association between other cytoskeletal-associated NE proteins in complex with fascin. Based on our previously published data on fascin subcellular localization, we then focused on the C-terminal domains of the proteins nesprin-1 and -2, as these regions are present in all nesprin-1 and -2 isoforms anchored to the NE and encompass the most conserved regions across the whole sequence (Autore et al., 2013) (Figure S1B). Co-immunoprecipitation (coIP) experiments with either GFP-nes1(66–74) or GFP-nes2(49–56) demonstrated that fascin was able to specifically form a complex with the nesprin-2 C-terminal region (Figure 1A). We confirmed this interaction in intact cells by quantifying fluorescence resonance energy transfer (FRET) using fluorescence lifetime imaging microscopy (FLIM) between GFP-tagged nesprins and monomeric RFP (mRFP)-tagged fascin. This analysis demonstrated a specific and direct interaction in whole cells between GFP-nes2(49–56), but not GFP-nes1(66–74) or the inner NE protein SUN2 (Figure 1B). To define the region of nesprin-2 that associates with fascin, we used recombinant glutathione S-transferase (GST)-tagged regions of tandem SRs from nesprin-1 and nesprin-2 as bait to pull down endogenous fascin from lysates of NIH 3T3 fibroblasts. Western blotting revealed that fascin was only found in a complex with SR51–53 from nesprin-2 (Figure 1C). We verified that fascin directly interacted with SR51–53 of nesprin-2 through in vitro binding assays (Figure 1D) and far-western blot analysis of GST-nesprin SR proteins probed with His-tagged fascin (Figure S1C). Moreover, GFP-mini-N2G-ΔSR3-50 containing the SR51–53 region (schematic representation in Figure S1B) showed specific coIP with endogenous fascin in NIH 3T3cells (Figure 1E). Collectively these data demonstrate that fascin forms a complex specifically with nesprin-2 in vitro and in cells, and this binding occurs directly and through SR51–53 in nesprin-2.

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.