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Fig. 5. The two actin-binding sites in myosin IB heavy chain account for the inhibition of phagocytosis without changes in the F-actin content. (A) Overexpression of myosin IB does not change the F-actin/G-actin ratio compared with the treatment of cells with jasplakinolide. Whole-cell lysates from equivalent cell numbers of the indicated amoeba strain were separated into a Triton-X-100-insoluble fraction, containing actin filaments (F-actin), and a Triton-X-100-soluble fraction, containing globular actin (G-actin). The fractions obtained were analysed by immunoblotting with a monoclonal antibody against actin. The effect of Jas in the wild-type strain on the amount of actin in each fraction was also examined. The MyoIB+ and 
SH3 strains showed a significant 10% increase (P<0.01) in the proportion of F-actin on total actin in the cell compared with the other strains examined in this study (P>0.5). By contrast, after incubation with Jas, the proportion of F-actin in the treated amoebae was drastically increased from 22% to 82%. (B) MyoIB+ and SH3 strains show a defect in phagocytosis. Amoebae were incubated with human erythrocytes for 10 minutes at 37°C, lysed and analysed for their haem concentration at 400 nm. The graph depicts the mean optical densities±s.d. of three independent experiments. Only cells overproducing myosin IB or myosin IB deleted for the SH3 domain present a threefold decrease in their phagocytic activity compared with the control strain or the strains overproducing myosin IB deleted for one of the two actin-binding sites.
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