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. 2021 Jun 24;220(9):e202012114. doi: 10.1083/jcb.202012114

Figure S5.

Figure S5.

α5β1 integrins are important for CYRI DBKO cell invasion.(A and B) Inverted invasion assay of control pLKO and CYRI DBKO A-673 cells with CMF plug treated with IgG (negative control), P1D6 (α5-blocking antibody), or P5D2 (β1-blocking antibody; A). The depth of invasion beyond 10 µm was quantified as an invasion index shown in B. Data from three independent experiments. Each experiment is color-coded. Mean ± SD. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Scale bar = 100 µm. (C and D) Immunofluorescence images of control pLKO or CYRI DBKO A-673 cells treated with 5 µg/ml of control IgG antibody or α5β1-blocking antibody IIA1 and stained for F-actin (cyan). Cell spread area was measured and plotted in D. Data from at least 10 random fields of view per experiment in a total of three independent experiments. Each experiment is color-coded. Mean ± SD. Statistical analysis using unpaired t test or one-way ANOVA with Tukey’s multiple comparison test where appropriate. Scale bar = 50 µm. (E) Treating DBKO A-673 cells with α5β1-blocking antibody IIA1 shows the reduction in their migration ability on 2D fibronectin substrate. Data from at least 30 cells per experiment in a total of three independent experiments. Each experiment is color-coded. Statistical analysis using unpaired t test or one-way ANOVA with Tukey’s multiple comparison test where appropriate. Mean ± SD. ****, P < 0.0001. (F) The current working model: (i) RAC1, the Scar/WAVE complex, the Arp2/3 complex, and actin drive the formation of macropinocytic cups. (ii) RAC1 activity increases lead to the recruitment of CYRI-A from a diffuse pool. (iii) CYRI-A is recruited to the nascent macropinocytic cups or just after the cups have closed by active RAC1, where it dampens down RAC1 activity, terminates actin polymerization, and allows for the completion of the macropinosomes, taking surface integrins into the cytoplasm. (iv) CYRI-A slowly disappears while RAB5A starts to appear. Cancer cells lacking both CYRIs retain more integrins on their surface, which can help them adhere to the surrounding matrix to assist their migration and invasion.