Quantitative Calcium Measurements in Subcellular Compartments of Plasmodium falciparum-infected Erythrocytes

Abstract

The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several antimalarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca 2؉ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca 2؉ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca 2؉ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca 2؉ imaging. Apparent free Ca 2؉ concentrations of ϳ90, ϳ350, and ϳ400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca 2؉ concentrations, we evaluated several Ca 2؉ -sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca 2؉ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca 2؉ , disfavoring a role as a major intracellular Ca 2؉ store.

A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3′,4′-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

“…Fluo-4 is a calcium probe that fluoresces green in the presence of Ca 2ϩ . The parasite DV is a calcium store, and Ca 2ϩ egress can be used as a proxy for DV permeabilization (9). A schematic representation of the assay work flow is shown in Fig.…”

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

“…Previous studies have shown that Ca 2ϩ indicator dyes accumulate in the parasite acidic food vacuole (49,50), complicating measurements of cytosolic Ca 2ϩ in the intraerythrocytic parasite. Consistent with this, in the absence of probenecid we observed no Ca 2ϩ response to photolysis of caged-IP 3 and little or no response to SERCA blockade with thapsigargin in P. falciparum-infected RBCs (Fig.…”

Biophotonic techniques for the study of malaria-infected red blood cells

“…This technique has been used in malaria research to quantify differences in acidity in chloroquine resistant and non-resistant parasite strains with pH sensitive dyes (e.g., pHluorin in [32]) or to assess Ca 2? accumulation in the parasite food vacuole with a Ca-sensitive dye (e.g., Fura Red in [49]). The selection of the dye requires careful attention and planning, as some ''standard'' ratiometric dyes for RBCs can be phototoxic for the parasite [49].…”

“…accumulation in the parasite food vacuole with a Ca-sensitive dye (e.g., Fura Red in [49]). The selection of the dye requires careful attention and planning, as some ''standard'' ratiometric dyes for RBCs can be phototoxic for the parasite [49].…”