Demonstration of actin association with enolase in P. falciparum : (A) Effect of cytochalasin D (actin depolymerizing drug) on sub-cellular distribution of enolase in (a) gametocyte (sexual stage) and (b) rings (asexual stage). Cells were treated with 50 μM cytochalasin D or DMSO (control) and IFA was performed with anti-r-Pfen antibodies (green) and rabbit anti-T. gondii actin antibody (red). Disruption of actin cytoskeleton led to accumulation of enolase at the two ends of the gametocyte cell (a), whereas in asexual stage translocation of enolase to nucleus was disrupted (b). (B) Direct interaction of GST tagged-r-Pfen with rabbit muscle G-actin and tubulin. GST-r-Pfen was adsorbed on glutathione-sepharose beads and was incubated with G-actin or tubulin for 2 hours at room temperature. Beads were collected by centrifugation and washed with appropriate buffers. The samples were analyzed on 12% SDS-PAGE. Third lane (from left) showed a pull down of G-actin with r-Pfen.