Limits...
Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

Gazarini ML, Thomas AP, Pozzan T, Garcia CR - J. Cell Biol. (2003)

Bottom Line: This allowed selective loading of the Ca2+ probes within the PV.The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism.We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Biosciences, University of São Paulo, São Paulo 05508-900, Brazil.

ABSTRACT
Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

Show MeSH

Related in: MedlinePlus

Trapping of Fluo-3 (free acid) in the PV of infected RBCs. (A) Confocal fluorescence image of a human RBC invaded by P. falciparum in the presence of 10 μM Fluo-3 (acid) in the medium. (B) Phase-contrast image of the same cell. P refers to the parasite, and E to the invaded RBC. (C) Intensity histogram of the fluorescence image shown in A. The fluorescence intensity (expressed as arbitrary units) was measured along the axis shown in D. The numbers 1 and 2 refer to the position along the axis.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172890&req=5

fig1: Trapping of Fluo-3 (free acid) in the PV of infected RBCs. (A) Confocal fluorescence image of a human RBC invaded by P. falciparum in the presence of 10 μM Fluo-3 (acid) in the medium. (B) Phase-contrast image of the same cell. P refers to the parasite, and E to the invaded RBC. (C) Intensity histogram of the fluorescence image shown in A. The fluorescence intensity (expressed as arbitrary units) was measured along the axis shown in D. The numbers 1 and 2 refer to the position along the axis.

Mentions: Malaria parasites were allowed to invade RBCs in vitro in a medium containing the fluorescent calcium indicator Fluo-3 in its cell-impermeant, free acid form. This approach allows the Ca2+ indicator dye to be trapped in the PV during invasion. If the PVM is permeable to high mol wt solutes, the dye should be at about the same concentration in both the PV and the RBC cytoplasm. On the contrary, if the PVM is impermeable to Fluo-3, the dye should remain in the PV and thus allow selective measurement of [Ca2+] within that microenvironment. Fig. 1 A shows a confocal image of a human RBC invaded by Plasmodium falciparum in the presence of Fluo-3. The fluorescence of Fluo-3 forms a ring around the parasite, although no staining is observed in either the RBC cytosol or in the parasite itself. Fig. 1 B shows the phase-contrast image of the same Plasmodium-infected RBC, and Fig. 1 C shows the fluorescence intensity profile along the white line overlaying the parasite and the RBC (inset). The fluorescence intensity profile shows two large peaks on opposite sides of the parasite (corresponding to the PV), and a fluorescence level indistinguishable from that of background in the RBC cytoplasm. The mean values of fluorescence intensity obtained in two independent experiments (and 10 different cells) performed with either P. falciparum or Plasmodium chabaudi are shown in Table 1. Control experiments where RBCs were incubated for the same period of time with Fluo-3 (but without parasites) resulted in no trapping of the dye, nor was any Fluo-3 sequestered if the indicator was added after invasion (unpublished data). Similarly, isolated parasites incubated with Fluo-3 (acid) did not accumulate any dye (unpublished data). When RBCs infected by the parasites in the presence of Fluo-3 were allowed to stay at RT for 30 min–1 h, the staining pattern did not change significantly.


Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

Gazarini ML, Thomas AP, Pozzan T, Garcia CR - J. Cell Biol. (2003)

Trapping of Fluo-3 (free acid) in the PV of infected RBCs. (A) Confocal fluorescence image of a human RBC invaded by P. falciparum in the presence of 10 μM Fluo-3 (acid) in the medium. (B) Phase-contrast image of the same cell. P refers to the parasite, and E to the invaded RBC. (C) Intensity histogram of the fluorescence image shown in A. The fluorescence intensity (expressed as arbitrary units) was measured along the axis shown in D. The numbers 1 and 2 refer to the position along the axis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172890&req=5

fig1: Trapping of Fluo-3 (free acid) in the PV of infected RBCs. (A) Confocal fluorescence image of a human RBC invaded by P. falciparum in the presence of 10 μM Fluo-3 (acid) in the medium. (B) Phase-contrast image of the same cell. P refers to the parasite, and E to the invaded RBC. (C) Intensity histogram of the fluorescence image shown in A. The fluorescence intensity (expressed as arbitrary units) was measured along the axis shown in D. The numbers 1 and 2 refer to the position along the axis.
Mentions: Malaria parasites were allowed to invade RBCs in vitro in a medium containing the fluorescent calcium indicator Fluo-3 in its cell-impermeant, free acid form. This approach allows the Ca2+ indicator dye to be trapped in the PV during invasion. If the PVM is permeable to high mol wt solutes, the dye should be at about the same concentration in both the PV and the RBC cytoplasm. On the contrary, if the PVM is impermeable to Fluo-3, the dye should remain in the PV and thus allow selective measurement of [Ca2+] within that microenvironment. Fig. 1 A shows a confocal image of a human RBC invaded by Plasmodium falciparum in the presence of Fluo-3. The fluorescence of Fluo-3 forms a ring around the parasite, although no staining is observed in either the RBC cytosol or in the parasite itself. Fig. 1 B shows the phase-contrast image of the same Plasmodium-infected RBC, and Fig. 1 C shows the fluorescence intensity profile along the white line overlaying the parasite and the RBC (inset). The fluorescence intensity profile shows two large peaks on opposite sides of the parasite (corresponding to the PV), and a fluorescence level indistinguishable from that of background in the RBC cytoplasm. The mean values of fluorescence intensity obtained in two independent experiments (and 10 different cells) performed with either P. falciparum or Plasmodium chabaudi are shown in Table 1. Control experiments where RBCs were incubated for the same period of time with Fluo-3 (but without parasites) resulted in no trapping of the dye, nor was any Fluo-3 sequestered if the indicator was added after invasion (unpublished data). Similarly, isolated parasites incubated with Fluo-3 (acid) did not accumulate any dye (unpublished data). When RBCs infected by the parasites in the presence of Fluo-3 were allowed to stay at RT for 30 min–1 h, the staining pattern did not change significantly.

Bottom Line: This allowed selective loading of the Ca2+ probes within the PV.The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism.We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Biosciences, University of São Paulo, São Paulo 05508-900, Brazil.

ABSTRACT
Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

Show MeSH
Related in: MedlinePlus