PRR11 induces filopodia formation and promotes cell motility via recruiting ARP2/3 complex in non-small cell lung cancer cells

Abstract

Filopodia, a finger-like structure and actin-rich plasma-membrane protrusion at the leading edge of the cell, has important roles in cell motility. However, the mechanisms of filopodia generation are not well-understood via the actin-related protein 2/3 (ARP2/3) complex in Non-Small Cell Lung Cancer (NSCLC) cells. We previously have demonstrated that PRR11 associates with the ARP2/3 complex to regulate cytoskeleton-nucleoskeleton assembly and chromatin remodeling. In this study, we further demonstrate that PRR11 involves in filopodia formation, focal adhesion turnover and cell motility through ARP2/3 complex. Cell phenotype assays revealed that the silencing of PRR11 increased cellular size and inhibited cell motility in NSCLC cells. Mechanistically, PRR11 recruited and co-localized with Arp2 at the membrane protrusion to promote filopodia formation but not lamellipodia formation. Notably, PRR11 mutant deletion of the proline-rich region 2 (amino acid residues 185-200) abrogated the effect of filopodia formation. In addition, PRR11-depletion inhibited filopodial actin filaments assembly and increased the level of active integrin β1 in the cell surface, whereas reduced the phosphorylation level of focal adhesion kinase (FAK^Y397) to repress focal adhesion turnover and cell motility in NSCLC cells. Taken together, our findings indicate that PRR11 has critical roles in controlling filopodia formation, focal adhesion turnover and cell motility by recruiting ARP2/3 complex, thus dysregualted expression of PRR11 potentially facilitates tumor metastasis in NSCLC cells.

Keywords: ARP2/3 complex; Cell motility; FAK; Filopodia; Focal adhesion; Integrin; NSCLC cells; PRR11.

Figure 1

Silencing of PRR11 inhibits cell motility in NSCLC cells. (A) siRNA-mediated silencing of PRR11. A549 or H1299 cells were transiently transfected with a negative control siRNA (siNC) or with siRNA against PRR11. Forty-eight hours after transfection, whole-cell lysates were prepared and analyzed the expression of the indicated proteins. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) The effects of PRR11 depletion with the cell morphology. A549 and H1299 cells were transiently transfected as in (A). Forty-eight hours after transfection, cells were photographed. Scale bar, 50 μm. (C) Quantified cellular area in siRNA treatment cells. A549 cells were transiently transfected as in (A). Forty-eight hours after transfection, cells were placed into the 6 cm dish. Cellular area were quantified with ImageJ and shown as line graphs. n = 3. Greater than 50 cells were counted per condition in every repeat. ∗∗∗,P < 0.001. (D) Quantified of attached cells/field in PRR11 depletion cells. A549 and H1299 cells were transiently transfected as in (A). Forty-eight hours after transfection cells were placed into the 6 cm dish. Attached cells/field were quantified with ImageJ and shown as line graphs. n = 3. Greater than 50 cells were counted per condition in every repeat. ∗∗∗,P < 0.001. (E–G) Movements of individual A549 cells were traced by videomicroscopy. A549 cells were transiently transfected as in (A). Forty-eight hours after transfection, cells were placed into the confocal dish to photograph by Time-lapse confocal microscopy. Representative images are shown in (E). Cell migration trajectories were then recorded. n = 5 (F). The velocity of cell migration and the distance from (E) were analyzed with the ImageJ software and shown as the mean ± S.E. (n = 3) (G). Scale bar, 50 μm ∗∗∗,P < 0.001.

Figure 2

PRR11 recruits Arp2/3 complex moving toward the filopodia. (A) The subcellular localization of endogenous PRR11 and Arp2. A549 cells transiently transfected with siNC and siPRR11 were fixed after 48 h, and stained with PRR11 and Arp2 antibody. Cell nuclei were stained with DAPI (blue). Scale bars, 20 μm. And zoomed images of the boxed region are shown at the bottom-right corner. White arrowhead indicates that PRR11 and Arp2 co-localizes at the filopodia. Red arrowhead indicates that Arp2 localizes at the lamellipodia. (B) Co-localization analysis of PRR11 and Arp2 fluorescence signal from (A) was performed. The line graphs represent overlap between PRR11 and Arp2 signal at the filopodia (left panel) or PRR11 and Arp2 signal at the lamellipodia (right panel) (the white line from (A)).

Figure 3

PRR11 induces the filopodia formation depending on Arp2/3 complex. (A) siRNA-mediated silencing of PRR11 and ARPC1A. A549 cells transfected with siNC or siRNA directed against PRR11 and ARPC1A. Whole-cell lysates were prepared to analyze the expression of the indicated proteins 48 h after transfection. (B) PRR11 and ARPC1A knockdown suppressed actin filaments assembly of filopodia. A549 cells were transiently transfected as in (A). Forty-eight hours after transfection, the siNC-, siPRR11-and siARPC1A-transfected cells were fixed and stained with F-actin-binding phalloidin-TRITC (red). Representative images are shown. Cell nuclei were stained with DAPI (blue). Bars, 20 μm. (C) Quantification of the number of rearranged long filament actin following the siRNA treatment described as in (B). ∗∗∗,P < 0.001. (D, E) Quantified of the number and length of filopodia. The number and length of filopodia were quantified by automated scanning in the siNC-, siPRR11-and siARPC1A-transfected cells using confocal microscopy. We counted all filopodia from per cell, and the length of each tentacle was measured from the cell surface to the tip of the filopodia. Results are shown as the mean ± S.E. (n = 3). Greater than 50 cells were counted per condition in every repeat. n.s,P > 0.05; ∗∗∗,P < 0.001.

Figure 4

PRR11 induces the filopodia formation via proline rich region 2. (A) Western blot analysis for the indicated proteins. Cells were overexpressed with transient transfected pVN173, WT PRR11, Δ33-41 or Δ185-200, and then cells were fixed after 24 h. Cells were treated as (D) and then were lysed, and analyzed for expression of the indicated proteins by Western blotting. (B) The subcellular localization of WT PRR11 and Δ185-200. Cells were overexpressed with transient transfected pVN173, WT PRR11 or Δ185-200, and then cells were fixed after 24 h and stained for Flag and phalloidin. Representative images are shown. Bar, 10 μm. (C, D) Quantified of the number and length of filopodia in pVN173-, WT PRR11-, Δ33-41- or Δ185-200-expressing cells. The number and length of filopodia in pVN173-, WT PRR11-, Δ33-41-, or Δ185-200-transfected cells were evaluated by automated scanning using confocal microscopy. The length of filopodia was measured from the surface to the tip of the filopodia. Results are shown as the mean ± S.E, (n = 3). Greater than 50 cells were counted per condition in every repeat. n.s,P > 0.05; ∗∗∗,P < 0.001. (E) Quantified of the number of filopodia in pVN173- and WT PRR1-expressing cells combined with inhibitor treatment. Cells were transiently transfected pVN173- and WT PRR11. Four to 6 h after transfection, cells were treated with ARP2/3 complex inhibitor (CK666, final concentration of 84 μM) for 48 h. The number of filopodia in pVN173- and WT PRR11-transfected cells were evaluated by automated scanning using confocal microscopy. Results are shown as the mean ± S.E. (n = 3). Greater than 50 cells were counted per condition in every repeat. n.s,P > 0.05; ∗∗∗,P < 0.001.

Figure 5

PRR11 regulates focal adhesion turnover dynamic in NSCLC cells. (A) Immunolabeling for focal adhesions marker vinculin in the siNC-, siPRR11-and ARPC1A-transfected cells. Cells were transiently transfected with siNC or siRNA directed against PRR11 and ARPC1A. Forty-eight hours after transfection, cells were fixed and stained with vinculin antiboby. Representative images are shown. Cell nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Quantitated of the size and total fluorescence intensity of vinculin. A549 cells were transiently transfected as in (A). Box-and-whisker plot indicating the size and total fluorescence staining intensity of focal adhesions in the siNC-, siPRR11-and siARPC1A-transfected cells, respectively. n = 3. Greater than 20 cells were counted per condition in every repeat. a.u., arbitrary units. ∗∗∗,P < 0.001. (C–E) Focal adhesions turnover in the siRNA transfected cells. mCherry-Zyxin-expressing A549 cells were transiently transfected as in (A). Forty-eight hours after transfection, cells were placed into the confocal dish to photograph by confocal microscopy. (C) Representative time-lapse images (montages) of mCherry-Zyxin-expressing (the fluorescently labeled focal adhesions marker protein) in the siNC- and siPRR11-transfected cells. Scale bar, 10 μm. (D) Box-and-whisker plots revealing slow assembly and disassembly rates of focal adhesions in siPRR11 cells relative to their siNC counterparts. Note formation and dissolution of focal adhesions in the siNC transfected cells and very static focal adhesions in siPRR11-transfected cells. n = 3. Greater than 50 cells were counted per condition in every repeat. (E) Box-and-whisker plots revealing slow assembly and disassembly rates of focal adhesions in siARPC1A cells relative to their siNC counterparts in keeping with the siRNA-mediated knockdown PRR11. n = 3. Greater than 50 cells were counted per condition in every repeat. ∗∗∗,P < 0.001.

Figure 6

PRR11 regulates internalization of integrin β1 and phosphorylation of FAK^Y397 depending on ARP2/3 complex in NSCLC cells. (A) Western blot analysis for the total level of integrin β1. A549 cells were treated with siRNA transiently transfected (siNC, siPRR11 or siARPC1A) or ARP2/3 complex inhibitor treatment with a final concentration of 84 μM (CK666) for 48 h, respectively. And then cells were lysed and analyzed for the indicated proteins. (B) Quantitated of the surface expression of integrin β1 by flow cytometry. A549 cells were treated as in (A). Surface level of activated integrin β1 was immunofluorescence staining via mouse monoclonal antibodies against activated integrin β1 and was determined by flow cytometry. Results are shown as the mean ± S.E. (n = 3). a.u., arbitrary units. ∗∗∗,P < 0.001. (C) Western blot analysis for the indicated proteins. A549 cells were treated as in (A). And then cells were lysed and analyzed for the indicated proteins. (D) Immunofluorescence staining for active integrin β1 A549. Cells were treated as in (A), and then stained for active integrin β1 (green). Representative images are shown. Cell nuclei were stained with DAPI (blue). Bar, 10 μm. (E) Detected the level of pFAK^Y397 by immunofluorescence staining. A549 cells were treated as (A). Then cells were fixed and stained with pFAK^Y397 antibody. Representative images are shown. Cell nuclei were stained with DAPI (blue). Bar, 10 μm.

Figure 7

The expression of PRR11 and FAK^Y397 in NSCLC cells. (A) Expression of PRR11 and FAK^Y397 was examined by immunohistochemistry in NSCLC. Bar, 100 μm. (B) Proposed working model. PRR11 associates with and recruits Arp2/3 complex to facilitate F-actin assembly and rearrangement, thereby to control fiopodia formation, but not lamellipodia. PRR11-ARP2/3 complex also implicates in regulation nuclear lamina integrity, which might be mediated by the LINC complex coupling both nuclear lamina and actin cytoskeleton.