Fig 2: Filopodia Tip Adhesions(A) U2OS cells expressing Lifeact-mTurquoise2, PXN-mEmerald, and MYO10-mScarlet were plated on fibronectin and imaged live using an Airyscan confocal microscope (1 picture every 30 s; scale bar, 5 μm; Video S2). Images highlight time points of interest in a magnified area; yellow arrows highlight filopodia tips.(B) U2OS cells expressing MYO10-mScarlet were plated on fibronectin for 2 hr; stained for F-actin, phospho-BCAR1 (Y410), and endogenous PXN; and imaged using SIM. A representative MIP is displayed. The blue and yellow squares highlight ROIs, which are magnified; scale bars: (main) 20 μm; (inset) 2 μm.(C–E) Live-cell imaging of U2OS cells transiently expressing MYO10-mScarlet together with either Lifeact-mTurquoise2 and BCAR1-eGFP (C; Video S3), Lifeact-mTurquoise2 and TLN1-eGFP (D; Video S4), or ITGA5-GFP (E; Video S5). Cells were plated on fibronectin and imaged live using an Airyscan confocal microscope. Yellow arrows highlight filopodia tips; scale bars: 5 μm.(F) U2OS cells expressing MYO10-mScarlet together with either GFP-BCAR1 full-length (FL), BCAR1 deletion constructs, or GFP-PTK2-FAT (PTK2FAT) were plated on fibronectin for 2 hr, stained for F-actin, and imaged using SIM. BCAR1ΔSH3, BCAR1 SH3 domain deletion; BCAR1ΔCCHD, BCAR1 CCHD domain deletion; BCAR1ΔCCHDΔSH3, BCAR1 SH3 domain and CCHD deletion; BCAR1CCHD, BCAR1 CCHD domain alone. Images highlight magnified areas of interest; scale bar: 2 μm; full fields of view are available in Figure S4. Distribution of each construct within filopodia was analyzed and displayed as described in Figures 1 and 2.See also Figures S2, S3, and S4.

Fig 3: ECM Sensing in Distinct Filopodia Types(A) RAT2 cells expressing BCAR1-GFP were plated on fibronectin for 2 hr, and their endogenous filopodia were stained for F-actin and imaged using SIM. A representative MIP is displayed.(B–D) Cells were plated on fibronectin (B, RAT2 cells, 2 hr; C, MCF10DCIS.com, 2 hr; D, U2OS, 20 min) fixed, stained for F-actin and phospho-BCAR1 (Y410), and endogenous filopodia imaged using SIM. Representative MIPs are displayed.(E–G) U2OS cells expressing GFP-FSCN1 (E), GFP-BAIP2 (F), or GFP-FMNL3 (G; all known filopodia-inducing proteins) were plated on fibronectin for 2 hr, stained for F-actin and phospho-BCAR1 (Y410), and imaged using SIM. Representative MIPs are displayed.(H) U2OS cells expressing GFP-FMNL3 and mCherry-TLN2 were plated on fibronectin for 2 hr, stained for F-actin, and imaged using SIM. A representative MIP is displayed.(I) Live-cell imaging of U2OS cells transiently expressing Lifeact-mTurquoise2 and MYO10-GFP or Lifeact-mTurquoise2 and FMNL3-GFP (Video S6). Cells plated on fibronectin were imaged live using an Airyscan confocal microscope. Images display a time point of interest as well as color-coded time projections. Scale bars: 20 µm.(J and K) U2OS cells expressing GFP-FMNL3 (J) or GFP-MYO10 (K) were plated on fibronectin for 2 hr, fixed and stained for F-actin together with inactive (clone 4b4) or active (clone 12G10) ß1 integrin, and imaged using SIM. MIPs are displayed.For all panels, the yellow squares highlight ROIs, which are magnified; yellow arrows highlight filopodia tips; scale bars: (main) 20 µm; (inset) 2 µm. See also Figure S6.

Fig 4: CRL5 regulates BCAR3 protein turnover.(a) Cas and BCAR3 structures. (b) Ratio of BCAR3 pY117 and pY266 phosphopeptide abundance in Cul5-deficient (shCul5) relative to control (Ctrl) MCF10A cells. n=three biological replicates. (c) BCAR3 protein and RNA levels in Ctrl and shCul5 cells. Representative immunoblot of cells cultured in assay or growth media (see Methods). Quantification of BCAR3 immunoblots and RNA analysis by qPCR, both normalized to GAPDH. Mean ± SD ; n=3 biological replicates. ***p < 0.001 (t-test). (d) BCAR3 degradation. Control and Cul5-deficient cells were treated with cycloheximide (CHX) for various times. Representative immunoblot and quantification. Mean ± SEM ; n=3 biological replicates. (e) Proteasome-dependent BCAR3 degradation. MCF10A cells were treated for 8 hr with CHX and either MLN4924 (cullin neddylation inhibitor), MG132 (proteasome inhibitor), or bafilomycin A1 (lysosome inhibitor). Mean ± SD; the number of biological replicates is noted on the graph. *p < 0.05; ****p < 0.0001 (One-way ANOVA).

Fig 5: Cul5 requires Y117 and Cas association to regulate BCAR3 protein level.(a) Y117F mutation increases BCAR3 protein level. SNAP-V5-BCAR3WT and Y117F MCF10A cells were induced with various dox concentrations (ng/mL). Lysates were immunoblotted with BCAR3 antibody to detect endogenous and induced SNAP-V5-BCAR3. (b) Cul5 regulates BCAR3 protein level through Y117. Representative immunoblot and quantification of BCAR3 protein in siRNA-treated, dox-induced SNAP-V5-BCAR3WT and Y117F cells. Mean ± SEM; n=3 biological replicates. ***p<0.001 by t-test. (c) Cul5 does not regulate SNAP-V5-BCAR3 F4. Mean ± SEM; n=3. ns, not significant. (d) Cul5 regulates SNAP-V5-BCAR3R177K but not EE. Mean ± SEM; n=3. **p<0.005 by t-test. Vertical lines indicate different immunoblots, each run with its own wild-type control.