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Fig. 8. PACSIN2 over-expression perturbs the recruitment of integrin β1 but not αv to focal adhesion in XTC cells. XTC cells transfected with Wt-PACSIN2 or the ∂CC mutant were processed for immunofluorescence with a monoclonal antibody against integrin β1 (A) or αv (B) and biotinylated mAb 3D8 against PACSIN2. (A) In non-transfected cells (NT), β1 integrin localizes to focal adhesion (FA). The cells transfected with Wt-PACSIN2 (Wt) display an absence of integrin β1 localization to FA. The expression of the mutant ∂CC did not affect the localization of β1 integrin to FA (∂CC). The percentage of cells displaying integrin β1 positive focal adhesions is plotted on the histogram. The graph represents the mean of three independent experiments. The number of cells scored was as followed: NT = 230, Wt PACSIN2 = 226, ∂CC = 153. (B) Compared to the non-transfected cells (NT), the localization of integrin αv to FA is unchanged upon expression of Wt-PACSIN2 (Wt) or the ∂CC (∂CC). The histogram represents the percentage of cells displaying integrin αv positive focal adhesions and corresponds to the mean of two independent experiments. The number of cells scored was as followed: NT; n = 148, Wt-PACSIN2; n = 89, ∂CC; n = 85. The error bar represents standard deviation from the mean. ⁎P < 0.05. |
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Fig. 9. PACSIN2-MA does not prevent the recruitment of integrin β1 to focal adhesion. XTC cells were transfected with Wt-PACSIN2 (Wt) or PACSIN2-MA (Wt-MA) and processed as described in Fig. 8A. The percentage of cells displaying integrin β1 positive focal adhesions is plotted on the histogram. Non-transfected cells (NT) and cells expressing the mitochondria-anchored PACSIN2 displayed integrin β1 in focal adhesions. PACSIN2 over-expression inhibits integrin β1 localization to focal adhesion as described in Fig. 8. The colored inset represents a magnification of the region of the Wt-MA expressing cell indicated by the arrowhead (PACSIN2-MA in blue, β1 integrin in green and F-actin detected with phalloidin in red). The graph represents the mean of two independent experiments. The error bar represents standard deviation from the mean. The number of cells scored was as followed: NT = 80, Wt PACSIN2 = 79, PACSIN2-MA = 80. ⁎P < 0.05. |
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Fig. 10. PACSIN2 alters the distribution of filamin in XTC cells. (A) Co-immunoprecipitation of filamin with PACSIN2 and integrin β1. The proteins extracted from XTC cells were immunoprecipitated using antibodies to filamin (Fln) or integrin β1. Negative controls were performed by immunoprecipitation with non-immune mouse serum (IP Ctl). The immunoprecipitates as well as total extracts were analyzed by western blot for the presence of PACSIN2 and filamin respectively. In XTC cells, filamin co-precipitate with both integrin β1 and PACSIN2. (B, C) XTC cells were transfected with Wt-PACSIN2 (Wt), the ∂CC mutant (∂CC) or the PACSIN2-MA (Wt-MA) and processed by immunofluorescence with antibodies against filamin (red), integrin β1 (green) and PACSIN2 (blue). A merged image between the Fln and integrin β1 staining is presented (merge). Optical sections were performed using a structured light illumination system (ApoTome) at the level of the cell-substrate interaction (B) and 1 above (C). (B) In non-transfected cells, filamin is mostly localized to stress fibers (arrow) and β1 integrin is localized to FA in which the stress fibers are anchored to (arrowhead). The over-expression of Wt-PACSIN2 inhibits both β1 integrin localization to FA and filamin localization to stress fibers while the ∂CC and the PACSIN2-MA mutant do not. (C) At 1 above the substratum level, both β1 integrin and filamin are localized at the cell membrane of non-transfected cells (arrowhead). The over-expression of neither Wt-PACSIN2 nor the ∂CC mutant seems to perturb this localization. However, PACSIN2-MA appears to decrease filamin at the cell membrane. The graphs represent the mean of 4 (NT, Wt, Wt-MA) and 2 (∂CC) independent experiments. The error bar represents standard deviation from the mean. The number of cells scored was as followed: NT = 28, Wt PACSIN2 = 66, ∂CC = 77, PACSIN2-MA = 70. ⁎P < 0.05. Arrowhead: focal adhesions (B) and membrane (C). Arrow: stress fiber. |
