Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites
Figure 4
Cytosolic calcium levels and expression of EBA175 on merozoite surface in response to changes in ionic conditions.
(A) Cytosolic calcium levels in merozoites under different ionic conditions. P. falciparum merozoites were isolated in buffer mimicking intracellular conditions (IC – 5 mM NaCl, 140 mM KCl, 1 mM EGTA). Merozoites were labeled with Fluo-4AM and cytosolic calcium levels [Ca+2] were measured by flow cytometry before and after transfer from IC buffer to buffer mimicking extracellular conditions (EC – 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 (IC-EC, brown)) or IC Klow buffer (IC Klow - 5 mM NaCl, 5 mM KCl, 135 mM choline-Cl, 1 mM EGTA (IC-IC Klow, green)). Merozoites isolated in IC buffer were transferred to EC buffer after prior treatment with either BAPTA-AM (IC+BA-EC+BA, blue) or U73122 (IC+U73122-EC+U73122, red) or its inactive analog U73343 (IC+U73343-EC+U73343, purple). 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. Arrow indicates time-point at which merozoites were transferred from IC buffer. (B) Surface expression of EBA175 under different ionic conditions. Expression of EBA-175 was detected on surface of merozoites isolated in IC buffer (IC, red), following transfer from IC to EC buffer (EC, blue) or following transfer from IC to EC buffer after prior treatment with BAPTA-AM (EC + BA, green) using specific sera by flow cytometry. Merozoites isolated in IC buffer and stained with pre-immune serum (PIS, black) were used as controls. (C) Surface expression of EBA175 in response to change in [K+]. Expression of EBA-175 was detected on surface of merozoites isolated in IC buffer (IC, red), after transfer from IC to EC buffer (EC, blue), IC Klow buffer (IC Klow, pink) or EC w/o Ca+2 buffer (140 mM NaCl, 5 mM KCl, 1 mM EGTA (EC w/o Ca+2, green) using specific sera by flow cytometry. (D) Effect of PLC inhibitor U73122 on surface expression of EBA175. Expression of EBA175 was detected on surface of merozoites isolated in IC buffer (IC, red), following transfer from IC to EC buffer (EC, blue) or from IC to EC buffer following pre-treatment with either U73122 (EC+U73122, green) or its inactive analog U73343 (EC+U73343, pink) using specific sera by flow cytometry. (E) Invasion rates in presence of calcium signaling pathway inhibitors. Merozoites were allowed to invade erythrocytes in presence of calcium chelator BAPTA-AM (EC + BA), PLC inhibitor U73122 (EC+U73122) or its inactive analog U73343 (EC+U73343). Newly invaded rings were scored by Giemsa staining. Invasion rates in RPMI1640 medium in absence of any calcium modulating agents were used as controls to determine invasion inhibition rates in presence of BAPTA-AM, U73122 and U73343.