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Further investigation: Effects of Asef2 on cell migration when cells are…

- - - - Rho, Cdc42 activity - no change
      - Rac activity - increased apx. 1.8-fold
    - - GFP –Asef2 cells - significantly enhanced level of active Rac
    - - GEF-deficient mutant cells (Pulldown assay)
        
        GFP–Asef2 cells - higher level of active Rac
        
        Lower migration speed
      - Hypothesis: Slower migration promoted by Asef2 results from impaired adhesion turnover
        
        Immunostaining for paxillin (adhesion marker)
        
        GFP–Asef2 cells - larger adhesion than in controls
        
        Quantification of adhesion turnover
        
        Half-life of adhesion assembly and disassembly - increased
        
        Hypothesis: Asef2 affects adhesion turnover by increasing the amount of active integrins at the cell membrane
        
        Flow citometry
        HUTS4 antibody detecting active beta-1 integrin
        
        GFP–Asef2 cells - increased cell-surface active b1 integrin, no change in total integrin quantity
        
        Rac inhibitor NSC23766; Knockdown of Asef2 - decrease levels of active b1 integrin
        
        TIRF microscopy
        
        GFP–Asef2 cells - higher levels of active b1 integrin in adhesions
        
        Hypothesis: Traction force and cell contractility are enhanced by Asef2
        
        Traction force microscopy; vector maps of the traction stresses
        
        GFP–Asef2 cells - higher traction stress
        
        Gel contraction assay
        
        GFP–Asef2 cells - greater gel contraction
        
        Hypothesis: Asef2 regulates migration by increasing MyoII activity
        
        Immunostaining
        (AB against active MyoII)
        
        GFP–Asef2 cells - greater amount of active MyoII
        
        Inhibition of MyoII ATPase activity (Blebbistatin)
        
        GFP–Asef2 cells with blebbistatin - increase in migration, counteract Asef2 activity