Figure 2. Vcn variants deficient in actin binding affect spreading and cell adhesion in MEFs.

(A) RTCA using the xCELLigence system shows that Vin−/− cells expressing Vcn^WT have higher cell impedance, hence more spread, than cells expressing Vcn^V1001A, Vcn^I997A or Vin−/−.MEFs. A representative trace of cell impedance (graphed as cell index (CI)) taken every 15 seconds for 13 hours; lower impedance indicates less contact with the sensor. Each data point represents an average CI of at least triplicate wells for each condition. (B) A graph showing the relative CI of cells spread on FN two hours following plating, which corresponds to the same time as the pictures shown in (C). Data is the average ± SEM combined from four independent experiments. *p≤ 0.05, in comparison to Vcn^WT. (C) Vin−/− MEFs transfected with GFP-tagged Vcn^WT, Vcn^I997A, or Vcn^V1001A and plated on FN for two hours. Vcn^I997A and Vcn^V1001A exhibit the same localization as Vcn^WT. (D,E,F) Box and whisker plots of FA area (D), FA number (E) and cell area (F). Areas were calculated using Matlab (Methods) (n=25). Cells expressing Vcn^I997A and Vcn^V1001A had fewer and larger FAs, *p-value≤0.05. Cells expressing Vcn^I997A were significantly smaller, but those expressing Vcn^V1001A were not. Scale bar is 25 µm.