Fig. 5. Effect of EhPAK on F-actin distribution. (A) Micrographs represent confocal analysis of the different labelling obtained in optical planes through the middle of the cells. Moving amoebae (control and C-PAK⁺) were fixed on cover slips, filamentous actin was decorated with phallacidine (green) and PAK was stained with specific anti-EhPAK polyclonal antibodies (red). As observed by DIC, the control strain was elongated, polarised and presented a unique pseudopod, and the strain overexpressing C-PAK is a rounded cell presenting multiple membrane extension. F-actin was diffusely distributed in the cytoplasm of the C-PAK⁺ strain. For the control strain, F-actin was enriched at one pole of polarised parasites. EhPAK concentrated in the membranous protrusion for both C-PAK⁺ and the control amoeba. Bars, 10 µm. (B) Cell fractionation in the presence of Triton X100 was performed on C-PAK⁺ and control strains. The fractions were electrophoresed and analysed by western blot with anti-PAK or anti-actin antibodies. The revealed protein was analysed with a phosphoimager, bands were quantified in arbitrary units with the IQ Mac V12 molecular imaging system. An equivalent amount of PAK was found in both TritonX100-soluble (s) and TritonX100-insoluble (i) fractions. The C-PAK peptide, carried by the C-PAK⁺ strain, was present in the TritonX100-insoluble fraction. The total amount of PAK remained unchanged in each strain. In the control cells, the total actin partitioned corresponded to 70% TritonX100-soluble and 30% TritonX100-insoluble fractions, whereas in the C-PAK⁺ strain actin was at 10% in the TritonX100-insoluble and 90% in the TritonX100-soluble fractions.

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