Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes.

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.

Ultrastructural characterization of melanosomes of the human pathogenic fungus Fonsecaea pedrosoi.

Melanin is a complex polymer widely distributed in nature and has been described as an important virulence factor in pathogenic fungi. In the majority of fungi, the mechanism of melanin formation remains unclear. In Fonsecaea pedrosoi, the major etiologic agent of chromoblastomycosis, melanin is stored in intracellular vesicles, named melanosomes. This paper details the ultrastructural aspects of melanin formation, its storage and transportation to the cell wall in the human pathogenic fungus F. pedrosoi. In this fungus, melanin synthesis within melanosomes also begins with a fibrillar matrix formation, displaying morphological and structural features similar to melanosomes from amphibian and mammalian cells. Silver precipitation based on Fontana-Masson technique for melanin detection and immunocytochemistry showed that melanosome fuses with fungal cell membrane where the melanin is released and reaches the cell wall. Melanin deposition in the fungal cell wall occurs in concentric layers. Antibodies raised against F. pedrosoi melanin revealed the sites of melanin production and storage in the melanosomes. In addition, a preliminary description of the elemental composition of this organelle by X-ray microanalysis and elemental mapping revealed the presence of calcium, phosphorus and iron concentrated in its matrix, suggesting a new functional role for these organelles as iron storage compartments.

Sub-second cellular dynamics: time-resolved electron microscopy and functional correlation.

Subcellular processes, from molecular events to organellar responses and cell movement, cover a broad scale in time and space. Clearly the extremes, such as ion channel activation are accessible only by electrophysiology, whereas numerous routine methods exist for relatively slow processes. However, many other processes, from a millisecond time scale on, can be "caught" only by methods providing appropriate time resolution. Fast freezing (cryofixation) is the method of choice in that case. In combination with follow-up methodologies appropriate for electron microscopic (EM) analysis, with all its variations, such technologies can also provide high spatial resolution. Such analyses may include, for example, freeze-fracturing for analyzing restructuring of membrane components, scanning EM and other standard EM techniques, as well as analytical EM analyses. The latter encompass energy-dispersive x-ray microanalysis and electron spectroscopic imaging, all applicable, for instance, to the second messenger, calcium. Most importantly, when conducted in parallel, such analyses can provide a structural background to the functional analyses, such as cyclic nucleotide formation or protein de- or rephosphorylation during cell stimulation. In sum, we discuss many examples of how it is practically possible to achieve strict function-structure correlations in the sub-second time range. We complement this review by discussing alternative methods currently available to analyze fast cellular phenomena occurring in the sub-second time range.

Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells.

As amply documented by electrophysiology, depolarisation in Paramecium induces a Ca(2+) influx selectively via ciliary voltage-dependent Ca(2+)-channels, thus inducing ciliary beat reversal. Subsequent downregulation of ciliary Ca(2+) has remained enigmatic. We now analysed this aspect, eventually under overstimulation conditions, by quenched-flow/cryofixation, combined with electron microscope X-ray microanalysis which registers total calcium concentrations, [Ca]. This allows to follow Ca-signals within a time period (> or =30ms) smaller than one ciliary beat ( approximately 50ms) and beyond. Particularly under overstimulation conditions ( approximately 10(-5)M Ca(2+) before, 0.5mM Ca(2+) during stimulation) we find in cilia a [Ca] peak at approximately 80ms and its decay to near-basal levels within 110ms (90%) to 170ms (100% decay). This [Ca] wave is followed, with little delay, by a [Ca] wave into subplasmalemmal Ca-stores (alveolar sacs), culminating at approximately 100ms, with a decay to original levels within 170ms. Also with little delay [Ca] slightly increases in the cytoplasm below. This implies rapid dissipation of Ca(2+) through the ciliary basis, paralleled by a rapid, transient uptake by, and release from cortical stores, suggesting fast exchange mechanisms to be analysed as yet. This novel type of coupling may be relevant for some phenomena described for other cells.

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H+ uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5-6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H+-pyrophosphatase inhibitors. H+ uptake was not promoted by ATP. Addition of 50 microM Ca2+ induced the release of H+, suggesting the presence of a Ca2+/H+ countertransport system in the membranes of the acidic compartments. Na+ was unable to release protons from the organelles. The pyrophosphate-dependent H+ uptake was dependent of ion K+ and inhibited by Na+ Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H+-pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles. Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H+-pyrophosphatase and a Ca2+/H+ antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H+ transport in the acidocalcisomes of this parasite.

One-way calcium spill-over during signal transduction in Paramecium cells: from the cell cortex into cilia, but not in the reverse direction.

We asked to what extent Ca(2+) signals in two different domains of Paramecium cells remain separated during different stimulations. Wild-type (7S) and pawn cells (strain d4-500r, without ciliary voltage-dependent Ca(2+)-channels) were stimulated for trichocyst exocytosis within 80 ms by quenched-flow preparation and analysed by energy-dispersive X-ray microanalysis (EDX), paralleled by fast confocal fluorochrome analysis. We also analysed depolarisation-dependent calcium signalling during ciliary beat rerversal, also by EDX, after 80-ms stimulation in the quenched-flow mode. EDX and fluorochrome analysis enable to register total and free intracellular calcium concentrations, [Ca] and [Ca(2+)], respectively. After exocytosis stimulation we find by both methods that the calcium signal sweeps into the basis of cilia, not only in 7S but also in pawn cells which then also perform ciliary reversal. After depolarisation we see an increase of [Ca] along cilia selectively in 7S, but not in pawn cells. Opposite to exocytosis stimulation, during depolarisation no calcium spill-over into the nearby cytosol and no exocytosis occurs. In sum, we conclude that cilia must contain a very potent Ca(2+) buffering system and that ciliary reversal induction, much more than exocytosis stimulation, involves strict microdomain regulation of Ca(2+) signals.

Microencapsulation of inorganic nanocrystals into PLGA microsphere vaccines enables their intracellular localization in dendritic cells by electron and fluorescence microscopy.

Biodegradable poly-(D,L-lactide-co-glycolide) microspheres (PLGA-MS) are approved as a drug delivery system in humans and represent a promising antigen delivery device for immunotherapy against cancer. Immune responses following PLGA-MS vaccination require cross-presentation of encapsulated antigen by professional antigen presenting cells (APCs). While the potential of PLGA-MS as vaccine formulations is well established, the intracellular pathway of cross-presentation following phagocytosis of PLGA-MS is still under debate. A part of the controversy stems from the difficulty in unambiguously identifying PLGA-MS within cells. Here we show a novel strategy for the efficient encapsulation of inorganic nanocrystals (NCs) into PLGA-MS as a tool to study their intracellular localization. We microencapsulated NCs as an electron dense marker to study the intracellular localization of PLGA-MS by transmission electron microscopy (TEM) and as fluorescent labels for confocal laser scanning microscopy. Using this method, we found PLGA-MS to be rapidly taken up by dendritic cells and macrophages. Co-localization with the lysosomal marker LAMP1 showed a lysosomal storage of PLGA-MS for over two days after uptake, long after the initiation of cross-presentation had occurred. Our data argue against an escape of PLGA-MS from the endosome as has previously been suggested as a mechanism to facilitate cross-presentation of PLGA-MS encapsulated antigen.

Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii.

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+)/H(+) countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

Acidocalcisomes in Apicomplexan parasites.

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidic matrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. In this paper, we review the structural, biochemical and physiological aspects of acidocalcisomes in Apicomplexan parasites and discuss their functional roles in the maintenance of intracellular ion homeostasis.

Quantitative calcium measurements in subcellular compartments of Plasmodium falciparum-infected erythrocytes.

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 anti-malarial 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 Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of approximately 90, approximately 350, and approximately 400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ 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 Ca2+, disfavoring a role as a major intracellular Ca2+ store.

Kinetics of pyrophosphate-driven proton uptake by acidocalcisomes of Leptomonas wallacei.

In this work, we show the kinetics of pyrophosphate-driven H+ uptake by acidocalcisomes in digitonin-permeabilized promastigotes of Leptomonas wallacei. The vacuolar proton pyrophosphatase activity was optimal in the pH range of 7.5-8.0, was inhibited by imidiodiphosphate, and was completely dependent on K+ and PPi. H+ was released with the addition of Ca2+, suggesting the presence of a Ca2+/H+ antiport. In addition, X-ray elemental mapping associated with energy-filtering transmission electron microscopy showed that most of the Ca, Na, Mg, P, K, Fe, and Zn were located in acidocalcisomes. L. wallacei immunolabeled with antibodies against Trypanosoma cruzi pyrophosphatase show intense fluorescence in cytoplasmatic organelles of size and distribution similar to the acidocalcisomes. Altogether, the results show that L. wallacei acidocalcisomes possess a H+-pyrophosphatase with characteristics of type I V-H+-PPase. However, we did not find any evidence, either for the presence of H+-ATPases or for Na+/H+ exchangers in these acidocalcisomes.

Dynamics of polymorphism of acidocalcisomes in Leishmania parasites.

Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 microm diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warren's medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.