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

<p>(<b>A</b>) <i>Expression of CLAG3.1 on merozoite surface following interaction with glyA</i>. Expression of CLAG3.1 was detected on surface of <i>P. falciparum</i> 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 CaCl<sub>2</sub>, 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>B</b>) <i>Changes in cytosolic calcium levels in</i> P. falciparum <i>3D7 merozoites following transfer from intracellular to extracellular ionic conditions and following engagement with glyA</i>. <i>P. falciparum</i> 3D7 merozoites were isolated in IC buffer. Merozoites were labeled with Fluo-4AM and cytosolic calcium levels [Ca<sup>+2</sup>] 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). (<b>C</b>) <i>Translocation of CLAG3.1 to surface of</i> P. falciparum <i>3D7Δ175 merozoites following receptor engagement</i>. Expression of CLAG3.1 was detected on surface of <i>P. falciparum</i> 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. <i>P. falciparum</i> 3D7Δ175 merozoites in IC buffer stained with pre-immune sera (PIS, black) were used as control. (<b>D</b>) <i>Changes in cytosolic calcium levels in</i> P. falciparum <i>3D7Δ175 merozoites after transfer from intracellular to extracellular conditions and following binding to erythrocyte ghosts</i>. <i>P. falciparum</i> 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>B</b>, <b>D</b>). Arrows indicate time-points at which merozoites were transferred from IC buffer to EC buffer (IC-EC) (<b>B</b>, <b>D</b>) or from EC buffer to EC buffer containing gly A (EC + Gly A) (<b>B</b>) or from EC buffer to EC buffer containing RBC ghosts (EC + RBC ghosts) (<b>D</b>).</p>