Endoplasmic reticulum

The largest store of Ca2+ in cells is usually found in the endoplasmic reticulum, with local concentration reaching millimolar levels. The endoplasmic reticulum also possesses two independent pathways for calcium influx and efflux. The influx is catalyzed by the very well-known sarco-endoplas-mic reticulum Ca2+-ATPase (SERCA), which actively translocates two Ca2+ for the hydrolysis of one ATP molecule. Evidence for the presence of a SERCA-type Ca2+-ATPase in T. gondii was first provided by experiments using fura-2-loaded tachyzoites in which thapsigargin, a very specific inhibitor of this pump when used at low concentrations (Thastrup et al., 1990), was shown to increase [Ca2+]i in tachyzoites (Moreno and Zhong, 1996). Molecular evidence for the presence of a

SERCA-type Ca2+-ATPase in T. gondii has been reported recently (Nagamune and Sibley, 2005; Nagume et al., 2005). The gene encoding this pump was able to complement yeast deficient in Ca2+ pumps, providing evidence of its function as a Ca2+ pump, and the encoded protein has an apparent molecular mass of 120 kDa. Inhibitors of this pump in other cells, such as thapsigargin (Thastrup et al., 1990) or artemisinin (Eckstein-Ludwig et al., 2003), were able to stimulate microneme secretion, a process that relies on elevated [Ca2+]i (see below, under 'Ca2+- signaling').

Ca2+ release from the endoplasmic reticulum of eukaryotic cells is mediated by ryanodine (RyR) and inositol 1,4,5-trisphosphate (InsP3R) channels. RyR are activated by a rise in [Ca2+]i (Ca2+-induced Ca2+ release, CICR). In addition, there are RyR-like channels activated by cyclic ADP-ribose (cADPR), sphingosine, and a distinct Ca2+-release pathway activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The T. gondii phosphoinositide-specific phospholipase C, the enzyme that generates the second messengers InsP3 and diacylglycerol, was recently cloned, sequenced, and expressed in E. coli, and its enzymatic characteristics were investigated (Fang et al., 2006). InsP3/ryanodine-sensitive stores had been postulated to be present in T. gondii, on the basis of pharmacological studies (Lovett et al., 2002). Treatment with ethanol increased InsP3 and [Ca2+]i, and this pathway was sensitive to inhibitors of InsP3R channels. T. gondii also responded to agonists of cADPR-gated channels, such as ryanodine and caffeine (Lovett et al., 2002). Evidence for the presence of cADPR cyclase and hydrolase activities, the two enzymes that control cADPR levels, has been found recently (Chini et al., 2005). T. gondii microsomes that were loaded with 45Ca2+ released Ca2+ when treated with cADPR, and the RyR antagonists 8-bromo-cADPR and ruthenium red blocked this response. Although T. gondii microsomes also responded to InsP3, the inhibition profiles of these calcium-release channels were mutually exclusive (Chini et al., 2005). Although there is no molecular evidence for the presence of InsP3R or RyR channels in T. gondii, this could be due to lack of homology with the channels of animal cells, as occurs in plants that otherwise respond to the same second messengers (Nagata et al., 2004).

10.4.2 Nucleus

The transport of Ca2+ across the nuclear membrane has been the subject of much controversy. Although even proteins permeate the nuclear membrane through the nuclear pores, some authors have shown that the movement of Ca2+ may be restricted and require a SERCA-type pump. The nuclear membrane of T. gondii is continuous with the endoplasmic reticulum (Hager et al., 1999), and a similar composition in channels and pumps would be expected.

10.4.3 Mitochondria

Mitochondria possess a high capacity to sequester Ca2+, although under physiologic conditions the total mitochondrial Ca2+ levels and free Ca2+ reflect and parallel cytosolic Ca2+. The inner mitochon-drial membrane possesses a uniport carrier for Ca2+, which allows the electrogenic entry of the cation driven by the electrochemical gradient generated by respiration or ATP hydrolysis. Calcium efflux, on the other hand, takes place by a different pathway, which appears to catalyze the electroneutral exchange of internal calcium by external sodium or protons. Biochemical evidence for mitochon-drial Ca2+ uptake is available in malaria parasites (Uyemura et al., 2000), and preliminary evidence suggests the presence of a uniport mechanism in T. gondii (Vercesi and Moreno, unpublished observations). Unlike the mammalian mitochondria, where intracellular Ca2+ regulates the activity of several dehydrogenases, no such Ca2+-regulated dehydrogenases have been reported in T. gondii. Figure 10.2 shows a scheme of the Ca2+ homeostatic mechanisms of T. gondii.

10.4.4 Acidocalcisomes

The largest store of Ca2+ in T. gondii is found in the acidocalcisomes (Moreno and Zhong, 1996; Bouchot et al., 1999; Luo et al., 2001). These are

FIGURE 10.2 Schematic representation of the distribution of Ca2+ in T. gondii. Ca2+ entry is probably through Ca2+ channels (1). Once inside the cells, Ca2+ can be translocated back to the extracellular environment by a PMCA (plasma membranetype Ca2+-ATPase) (2). In addition, Ca2+ will interact with Ca2+-binding proteins or become sequestered by the endoplasmic reticulum (3), mitochondrion (4), acidocalcisome (5), or nucleus (6). The endoplasmic reticulum contains the SERCA (sarcoplasmic-endoplasmic reticulum Ca2+-ATPase) while the mitochondrion takes up Ca2+ through its uniport. Acidocalcisomes have a PMCA, a vacuolar H+-PPase, and a V-H+-ATPase. Ca2+ appears to diffuse freely into the nucleus. Further details are discussed in the text. ER, endoplasmic reticulum; M, mitochondrion; N, nucleus; AY, mitochondrial membrane potential. Illustration by Cheryl E. Reese. This figure is reproduced in color in the color plate section.

acidic calcium-storage organelles found in a diverse range of microorganisms from bacteria to man (Docampo et al., 2005). They are characterized by their acidic nature, high density (both in weight and by electron microscopy), and high content of pyrophosphate, polyphosphate (poly P), calcium, magnesium, and other elements (Docampo et al., 2005). Acidocalcisomes are similar to the volutin or metachromatic granules first described almost 100 years ago (Kunze, 1907) in coccidians, and detected in 1966 in T. gondii for their ability to stain red when treated with tolui-dine blue (metachromasia) (Mira Gutierrez and Del Ray Calero, 1966). More recently, they were also named 'black granules' (Bonhomme et al., 1993).

The acidity of acidocalcisomes of T. gondii is easily demonstrated through the incubation of tachyzoites with the weak base acridine orange (AO) and subsequent observation by fluorescence microscopy. Cells incubated in the presence of AO show orange labeling of several acidocalcisomes, which are the most acidic compartments in the cell (Figure 10.3). Shaw and colleagues reported that when using DAMP (3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine), which differentially accumulates in acidic compartments, the only acidic compartments at the electron microscope level were mature and forming rhoptries (Shaw et al., 1998). No labeling of other organelles (micronemes, dense granules, endoplasmic retic-ulum, Golgi, or any other membrane-bounded organelles or anything resembling a lysosomal system) was observed. However, no orange staining of the rhoptries is observed by fluorescence microscopy (Figure 10.3). The reasons for this discrepancy are, first, that treatment of the cells for electron microscopy empties the acidocalcisome content (see below) and therefore it would be impossible to see any labeling by electron microscopy techniques that do not preserve its content; and second, even if the rhoptries are acidic, they are not as acidic as the acidocalci-somes. Figure 10.4 shows the lack of co-localization of acidocalcisome and rhoptry markers.

In thin sections, the acidocalcisomes of T. gondii appear as empty vesicles occasionally bearing an electron-dense material that sticks to the inner

FIGURE 10.3 Detection of acidocalcisomes with acridine orange. Isolated tachyzoites were incubated with 25 pM acridine orange in Dulbecco's phosphate buffer saline (PBS) for 10 minutes, washed twice in PBS and mounted in a light microscope slide. Images were obtained using a Zeiss Axioplan fluorescence microscope equipped with a 488-nm excitation filter, a Hamamatzu digital CCD camera (model C5810) and an image analysis system. A final composition of images of cells from different fields was obtained through the clone tool of the Adobe Photoshop program. Bars = 2 pm. This figure is reproduced in color in the color plate section.

FIGURE 10.3 Detection of acidocalcisomes with acridine orange. Isolated tachyzoites were incubated with 25 pM acridine orange in Dulbecco's phosphate buffer saline (PBS) for 10 minutes, washed twice in PBS and mounted in a light microscope slide. Images were obtained using a Zeiss Axioplan fluorescence microscope equipped with a 488-nm excitation filter, a Hamamatzu digital CCD camera (model C5810) and an image analysis system. A final composition of images of cells from different fields was obtained through the clone tool of the Adobe Photoshop program. Bars = 2 pm. This figure is reproduced in color in the color plate section.

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