Figure 6.
BCAR1 Regulates Environment Sensing at Filopodia Tips
(A) Efficiency of siRNA-mediated (oligos nos. 5 and 6) silencing of BCAR1 in U2OS cells.
(B and C) BCAR1-silenced (oligos nos. 5 and 6) U2OS (B) and MDA-MB-231 (C) cells transiently expressing MYO10-GFP were plated on fibronectin for 2 hr, fixed, and the number of MYO10-positive filopodia per cell was quantified (n > 65 cells, three biological repeats; ∗∗∗p value < 5.4 × 10−6).
(D) BCAR1-silenced (oligo no. 6) U2OS cells transiently expressing MYO10-mScarlet together with GFP, GFP-BCAR1, or GFP-BCAR1ΔCCHD were plated on fibronectin for 2 hr, fixed, and the number of MYO10-positive filopodia per cell was quantified (n > 69 cells, three biological repeats; ∗∗∗p value < 1.1 × 10−5).
(E) BCAR1-silenced (oligo no. 6) U2OS cells transiently expressing MYO10-GFP were plated on fibronectin and imaged live using an Airyscan confocal microscope (1 picture every 5 s; scale bar, 20 μm). Representative images at different time points are shown. For each condition, MYO10-positive particles were automatically tracked, and MYO10 spot lifetime (calculated as a percentage of the total number of filopodia generated per cell) was plotted and displayed as Tukey boxplots (see STAR Methods for details; three biological repeats, more than 40 cells per condition, ∗∗∗p value < 8.78 × 10−5).
(F–H) U2OS cells expressing MYO10-mScarlet (F) or MYO10-GFP (G and H) were plated on fibronectin-coated polyacrylamide gels of defined stiffness (0.5 kPa, soft; 50 kPa, stiff) for 2 hr.
(F) Cells were stained for F-actin and phospho-BCAR1 (Y410) and imaged using a spinning disk confocal microscope. MIPs are displayed; yellow squares highlight ROI, which are magnified; yellow arrows highlight filopodia tips; scale bars: (main) 20 μm; (inset) 5 μm.
(G) Cells were stained for F-actin, imaged using an Airyscan confocal microscope, and the number of MYO10-positive filopodia per cell was quantified (n > 81 cells, three biological repeats; ∗∗∗p value < 5.5 × 10−20). MIPs are displayed in Figure S5C.
(H) Live-cell imaging on an Airyscan confocal microscope (scale bars: 20 μm). Representative images at different time points are shown. For each condition, MYO10 spot lifetime was analyzed as in (E) (three biological repeats, more than 34 cells per condition; ∗∗∗p value < 9.4 × 10−4).