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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. |
