Regulation of lamellipodial persistence, adhesion turnover, and motility in macrophages by focal adhesion kinase

Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion

Breast cancer antiestrogen resistance 3 (BCAR3) promotes cell motility by regulating actin cytoskeletal and adhesion remodeling in invasive breast cancer cells.

Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process

BCAR3 promotes Rac1 activity.

<p>(<b>A</b>) BT549 cells transfected with siCtl (lane 1) or siB3-1 (lane 2) siRNA oligonucleotides were incubated for 72 hours, held in suspension for 90 minutes, then plated on 10 µg/ml fibronectin for 1 hour. GTP-bound Rac1 was isolated from whole cell lysates by incubation with PAK-1-binding domain agarose. Bound proteins (top panel) and total Rac1 (middle panel) were detected by immunoblotting with a Rac1 antibody, and BCAR3 knockdown was confirmed with a BCAR3-specific antibody (bottom panel). Quantification of the relative GTP-Rac1 level is shown. Data represent the mean ± SEM of 3 independent experiments (*, p<0.05). (<b>B</b>) BT549 cells were transfected with siCtl or siB3-1 oligonucleotides, incubated for 24 hours, followed by transfection with plasmids encoding Myc-RacL61 for an additional 48 hours. Cells were plated onto 10 µg/ml fibronectin-coated coverslips for 1–3 hours and processed for immunofluorescence as described in the methods. Actin is stained with Texas red-conjugated phalloidin (red) and Myc (RacL61) with fluorescein isothiocyanate (FITC) (green). Arrows indicate Myc-RacL61 expressing cells. Arrowheads indicate actin-rich stress fibers. The images shown are representative of 6 separate experiments. Scale bar = 15 µm. (<b>C</b>) The percentage of cells exhibiting actin-rich ruffles was determined for non-transfected and RacL61-expressing cells. Data represent the mean ± SEM of at least 36 cells per condition over to 2 separate experiments (*, p<0.05).</p

BCAR3 promotes membrane protrusiveness in breast cancer cells.

<p>(<b>A</b>) BT549 cells were transfected with control (siCtl; lane 1) or BCAR3-specific (siB3-1; lane 2) siRNA oligonucleotides and incubated for 72 hours prior to lysis. Representative immunoblots of total cell lysates are shown. (<b>B</b>) BT549 cells transfected as in (A) were plated on 10 µg/ml fibronectin for 4 hours and imaged by time-lapse phase microscopy for 12.5 minutes. Cell outlines of the first and last frames (pseudo-colored gray and black, respectively) of representative cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065678#pone.0065678.s002" target="_blank">Videos S1 and S2</a> are shown.(<b>C</b>) The average protrusive area was determined by measuring the area shown in black. Data represent the mean ± SEM of at least 12 cells over at least 4 videos (*, p<0.005). (<b>D</b>) The average time to maximal membrane extension was determined by kymography. Data represent the mean ± SEM of at least 12 kymographs over 3 separate videos (*, p<0.005). (<b>E</b>) MCF-7 cells expressing BCAR3 under the control of a tetracycline-inducible (Tet-off) promoter were treated in the presence (lane 1) or absence (lane 2) of 1 µg/ml doxycycline (Dox) for 72 hours. Total cell protein and Crk immune complexes were immunoblotted with the designated antibodies. (<b>F</b>) MCF-7 cells were treated with or without Dox as described in (E), then plated on 10 µg/ml fibronectin overnight and subjected to time-lapse DIC microscopy for 1 hour. Tracings generated as in (B) for representative cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065678#pone.0065678.s004" target="_blank">Videos S3 and S4</a> are shown. (<b>G</b>) The average protrusive area per cell was determined as in (C). Data represent the mean ± SEM of 31 cells per condition over 3 separate videos (*, p<0.02). (<b>H</b>) Cell motility was measured by tracing the movement of the nucleus over time (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065678#pone.0065678.s001" target="_blank">Fig. S1</a>). The average rate of migration was calculated by dividing the total distance traveled by time for each cell. Data represent the mean ± SEM of at least 72 cells per condition over 3 separate movies (*, p<0.0001).</p

BCAR3 regulates actin cytoskeletal and adhesion remodeling in response to growth factor.

<p>(<b>A</b>) MDA-MB-231 cells were transfected with siCtl, siB3-1, or siB3-2 oligonucleotides and incubated for 24 hours prior to plating onto 10 µg/ml fibronectin-coated glass coverslips. Cells were serum-starved for 16–18 hours, stimulated with 100 ng/ml EGF for 0 or 30 minutes, and then fixed and processed for immunofluorescence as described in the methods. Actin and vinculin-containing adhesions were visualized with phalloidin (red) and vinculin (green) antibodies, respectively. Merged images are shown in the right panels; insets show higher magnifications of actin and adhesion structures. Scale bars = 15 µm. (<b>B</b>) The percentage of siCtl (black bars), siB3-1 (gray bars), and siB3-2 (white bars) treated cells containing actin-rich stress fibers was determined. Data represent the mean ± SEM of at least 730 cells per condition from 3 separate experiments (*, p<0.04). (<b>C</b>) MDA-MB-231 cells were transfected as described in (A). Representative immunoblots are shown confirming knockdown of BCAR3 using 2 separate siRNA oligonucleotides.</p