Fig 2: Integrin avß5 is necessary in the formation of FCL. (A) U2Os-AP2-GFP/halo, HeLa-AP2-GFP/halo, and MCF7-AP2-halo were plated on FN-coated or non-coated dishes, allowed to settle overnight, stained for integrin avß5 and p-Pax, imaged using TIRF, and analyzed for FA coverage. N (images): U2Os glass/FN = 37, HeLa glass = 32, Hela FN = 29, MCF7 glass = 28, and MCF7 FN = 32. (B) U2Os-AP2-GFP cells silenced for integrin ß5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control were imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (C) Analysis of FCL proportions from time-lapse videos in A. N (videos): control = 8, shITGB5 #1 = 11, shITGB5 #2 = 6, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F(2, 22) = 44.46, P < 0.0001. (D) U2Os-AP2-GFP cells silenced for integrin ß5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control, were stained for integrin avß5 and p-Pax. Representative TIRF images. (E) Analysis of integrin ß5 silencing efficiency, n = 10, from one representative experiment. Similar results were observed from three independent experiments. One-way ANOVA with Tukey’s multiple comparison F(2, 27) = 63.29, P < 0.0001. (F) U2Os-AP2-GFP cells plated on non-coated dishes for 20 h were treated with the integrin avß5 inhibitor Cilengitide (10 µM) for 15 or 45 min and stained for integrin avß5 and p-Pax. Representative TIRF images. (G) Analysis of AP2 signal colocalizing with p-Pax or integrin avß5 over time from samples in E. N (images): control = 14, Cil 15 min = 13, Cil 45 min = 10, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA and Tukey’s multiple comparison F(2, 99) = 63.38, P < 0.0001. Data are the mean ± SD, ns. non-significant P-value; *** P-value < 0.001. Scale bars, 10 µm.

Fig 3: FCLs and RAs presence correlates with FN production in multiple cells lines. (A–E) The following knock-in cell lines were used in this figure: U2Os-AP2-GFP, HeLa-AP2-GFP, hMEC-AP2-GFP, U2Os-AP2-halo, HeLa-AP2-halo, MCF7-AP2-halo, HDF-AP2-halo, and Caco2-AP2-halo. (A) Cell lines indicated were plated to non-coated (nc) dishes, allowed to settle overnight, and stained for integrin avß5 and FN. Representative TIRF images. (B) Analysis of FN fluorescent intensity from samples in A. N (images): U2Os = 21, HeLa = 20, MCF7 = 23, HDF = 33, Caco2 = 16, hMEC = 23, from two independent experiments. F(5, 130) = 56.16, P < 0.0001. (C) Cells were plated on FN-coated or nc dishes, allowed to settle overnight, and stained for integrin avß5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C. Cell lines with an AP2 halo tag, n (images): U2Os (nc/FN) = 20, HeLa (nc) = 33, HeLa (FN) = 29, MCF7 (nc) = 29, MCF7 (FN) = 32, HDF (nc/FN) 32, Caco2 (nc/FN) = 32. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F(9, 273) = 64.96, P < 0.0001. (E) Analysis of RA coverage from samples in C. Cell lines with an AP2 GFP tag, n (images): U2Os (nc) = 31, U2Os (FN) = 18, HeLa (nc) = 35, HeLa (FN) = 38, and hMEC (nc/FN) = 38. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F(5, 189) = 89.97, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm.

Fig 4: Optimazition experiments for visualizing FCL and RA co-assembly. (A) U2Os-AP2-ITGB5-mScarlet cells plated on non-coated dishes were treated with Cilengitide (10 µM) or DMSO for 25 min. Samples given Cilengitide were washed twice with fresh complete medium (except no-wash control), fixed at the respective recovery time points, and stained for p-Pax. Representative TIRF images. (B) Analysis of RA coverage for samples in A. N (images): control = 31, no wash = 32, 15-min recovery = 30, 30-min recovery = 30, 1-h recovery = 31, 2-h recovery = 30, 4-h recovery = 30, from one representative experiment. Similar results were observed in three independent experiments. (C and D) Additional examples of individual ITGB5 and AP2 events from experiments shown in Fig. 5. In C, an event where an RA grows from a stabilized FCL/ITGB5 cluster. A kymograph for the line on time zero is shown on the right. In D, three events are shown. One FCL-stabilized ITGB5 cluster (yellow arrowheads) and two non-stabilized ITGB5 clusters (blue arrowheads). Open blue arrows represent frames post-disappearance of ITGB5 clusters. Scale bars, 10 µm.

Fig 5: FN inhibits RA formation in a local manner. (A) U2Os-AP2-GFP cells plated on FN, VTN, Col I, Col IV, LN111 (all 10 µg/ml) -coated or non-coated dishes, stained for p-Pax and integrin avß. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N = 21 from two independent experiments. F(5, 120) = 48.05, P < 0.0001. (C) U2Os-AP2-GFP cells were plated on FN/glass patterned imaging dishes overnight, stained for integrin avß5 and p-Pax, and imaged using TIRF microscopy; representative TIRF images. (D) Quantification of integrin avß5 fluorescent intensity on FN- and glass-side of the pattern; n = 10 from one representative experiment. Similar results were observed in five similar experiments. Two-tailed Student’s t test, P < 0.01. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.