Cytosolic calcium levels and translocation of rhoptry protein CLAG3.1 to merozoite surface in response to interaction with glyA.

(A) Expression of CLAG3.1 on merozoite surface following interaction with glyA. Expression of CLAG3.1 was detected on surface of P. falciparum merozoites isolated in buffer mimicking intracellular conditions (IC – 5 mM NaCl, 140 mM KCl, 1 mM EGTA, red), or after transfer from IC buffer to buffer mimicking extracellular conditions (EC - 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, blue) or after transfer from IC buffer to EC buffer containing glyA (EC + GlyA, green) using specific sera by flow cytometry. Merozoites isolated in IC buffer and stained with pre-immune sera (PIS, black) were used as controls. (B) Changes in cytosolic calcium levels in P. falciparum 3D7 merozoites following transfer from intracellular to extracellular ionic conditions and following engagement with glyA. P. falciparum 3D7 merozoites were isolated in IC buffer. Merozoites were labeled with Fluo-4AM and cytosolic calcium levels [Ca⁺²] were measured by flow cytometry before and after transfer from IC to EC buffer (IC-EC) and from EC buffer to EC buffer containing glyA (EC + GlyA). (C) Translocation of CLAG3.1 to surface of P. falciparum 3D7Δ175 merozoites following receptor engagement. Expression of CLAG3.1 was detected on surface of P. falciparum 3D7Δ175 merozoites isolated in IC buffer (IC, red), after transfer from IC buffer to EC buffer (EC, blue), after transfer from IC buffer to EC buffer containing glyA (EC + GlyA, green) or after transfer from IC buffer to EC buffer containing red blood cell (RBC) ghosts (EC + RBC ghosts, pink) using specific sera by flow cytometry. P. falciparum 3D7Δ175 merozoites in IC buffer stained with pre-immune sera (PIS, black) were used as control. (D) Changes in cytosolic calcium levels in P. falciparum 3D7Δ175 merozoites after transfer from intracellular to extracellular conditions and following binding to erythrocyte ghosts. P. falciparum 3D7Δ175 merozoites were isolated in IC buffer. Merozoites were labeled with Fluo-4AM and cytosolic calcium levels were measured by flow cytometry. Merozoites were transferred first from IC buffer to EC buffer (IC-EC) and from EC buffer to EC buffer containing red blood cell ghosts (EC + RBC ghosts). Mean fluorescence intensities (MFI), which reflect relative levels of free cytosolic calcium measured by flow cytometry are shown as a function of time in seconds (B, D). Arrows indicate time-points at which merozoites were transferred from IC buffer to EC buffer (IC-EC) (B, D) or from EC buffer to EC buffer containing gly A (EC + Gly A) (B) or from EC buffer to EC buffer containing RBC ghosts (EC + RBC ghosts) (D).