Figure 4. Loss of Tsc2 results in Pak2 dependent wound healing defects.

(A) Filamentous actin was visualised by performing rhodamin-phalloidin stainings. Wild-type MEFs are characterised by a relatively modest amount of filamentous actin and highly organised stess fibers (osf; an example is indicated in the figure). Loss of Tsc2 coincides with robust cytoskeletal rearrangements, including the formation of numerous lamellipodium-like networks (lln), increased and disorganised cytoskeletal actin, the appearance of prominent cortical actin structures (ca) and the formation of many small microspikes (μs). Scale bars 20 μm. (B) Scratch wound assays performed in Tsc2^−/− and wild-type MEFs shows that Tsc2^−/− cells migrate slower than wild-type MEFs. Reducing Pak2 by siRNA results in partial restoration of migration in Tsc2^−/− MEFs, whereas control siRNA does not. (C) Quantification of the difference in lebesgue measure between the initial (0 hours) and final (24 hours) of the apparent scratch area confirms impaired migration in Tsc2^−/− MEFs (p < 0.001) and restoration thereof by Pak2 siRNA (p = 0.0137). (D) Quantitative PCR results demonstrating that levels of Pak2 mRNA expression are not affected in Tsc2^−/− MEFs, and are reduced upon siPAK2 treatment, but not control siRNA (*<0.0005).