Characterization of n-cofilin null macrophages.

<p>(A) Confocal sections through an adherent n-cof^null macrophage show the unusual cytoskeletal structure and the impaired leading edge formation. F-actin (in red) is shown by phalloidin staining. Note the short extensions emerging from the cell body and the lack of a lamellipodium; (B) Scanning EM of saponin extracted macrophages illustrate a loose network of short filaments in the lamellipodium of control cells, while n-cof^null BMM show densly packed cortical F-actin bundles; (C) Biochemical fractionation showed increased Triton X-100 stable F-actin levels in n-cof^{flx/Δ,Mx1-cre} macrophage cultures (n_culture = 6, p<0.05). ADF^−/− macrophages showed normal F-actin levels; (D) FACS overlay of phalloidin stained macrophages from a day 6 control (grey histogram) and n-cof^{flx/Δ,Mx1-cre} (black line histogram) culture. About 10-fold increase in phalloidin signal intensity was observed in n-cof^null cells, while the escaper cells are represented in the low intensity F-actin peak, similar to the control cells. (E) Cell adhesion on laminin is not altered in n-cofilin mutant macrophage cultures (n-cof^{flx/Δ,Mx1-cre}) at 4 to 24 hours after plating. The average of three independent experiments performed in quadruplicates is shown; (F) Cell spreading was significantly impaired in n-cofilin mutant macrophages (n-cof^null). The spreading area was compared in control (n-cof^{wt/wt,Mx1-cre}), ADF^-/-, ADF^+/− and n-cofilin mutant BMM (n_cells>100, student's test, n-cof^null:control p<0,0001; ADF^−/−:ADF^+/− p = 0,15); (G) N-cof^null BMM fail to polarize. The shape of more than 90% of n-cof^null BMM was apolar, while 75% of control and ADF^+/− cells were either bipolar or multipolar (n_cells>130). ADF^−/− BMM showed a noticeable tendency to increase cell polarization.