Distribution of CRCs in the P. tetraurelia cell (PtCRC), subfamilies I to VI (and subtypes analyzed in some detail given in arabic numbers), as localized by immuno-fluorescence. Additonal sites of PtCRC localization are not included in the scheme. These are PtCRC-V-4, occurring along the oral cavity (together with PtCRC-VI-3) and along the cleavage furrow; remarkably PtCRC-V-4 also occurs in the parasomal sacs (clathrin coated pits at the cilairy basis) membranes. Beyond structures identified below the scheme, the following abbreviations are used: in the contractile vacuole (cv) system, a marks ampullae (with extending radial/collecting canals), ds = decorated spongiome and ss = smooth spongiome. Other structures are: ac = acidosomes (vesicles of endosomal origin contributing to phagosome/food vacuole formation), cf = cytopharyngeal fibers; ci = cilia; cp = cytoproct, dv = discoidal vesicles, rv = recycling vesicles, mac = macronucleus, oc = oral cavity, pof = postoral fibers, ps = parasomal sacs (clathrin-coated pits), tr = trichocysts and their “ghosts” (gh) occurring after contents release. Compiled according to ().

Examples of immuno-localization of different PtCRCs. (A) PtCRC-II (IP₃R) is localized to the star-shaped contractile vacuole complex. (B) PtCRC-III-4 is localized to the phagocytic pathway, including recycling vesicles. Here, phagosomes (food vacuoles) show up in dark due to the addition of Indian ink in the medium. For better identification of structures labeled with antibodies against PtCRC-III-4 (green) red antibodies have been applied to stain microtubules. Here, PtCRC-III-4 traffic with recycling vesicles along microtubules that are associated with the postoral fibers (pof) which originate from the oral apparatus (oa). By contrast, the contractile vacuole complex (cvc) is stained only for tubulin, but not for PtCRC-III-4. Also indicated is the number of z-stacks used for a pile-up image in (B). See for further details. (C) Immunolocalization of PtCRC-IV-1 (RyR-LP) by green fluorescence. Red staining comes from labeling of protein disulfide isomerase (PDI), an ER-specific marker. Note the localization of PtCRC-IV-1 preferably to the alveolar sacs (Ca²⁺-stores, represented by the green patches in the cell cortex) and less to the ER. Abbreviations are: oc = oral cavity, cp = cytoproct. (D) PtCRC-V-4 localizes to micronuclei (mic), i.e. their envelopes, as well as to cell surface components (see for details). From (). Scale bars = 10 μm.

(A) Molecular modeling of the inositol 1,4,5-trisphosphate-BD of PtCRC-II (designated PtIP₃R_N) in comparison to type 1 IP₃R of the mouse (MmIP₃R1). The IP₃-BD of the Paramecium molecule has been modeled by comparison with that of MmIP₃R1. Numbers in parenthesis indicate the amino acid sequence included in the modeling procedure. Note considerable coincidence of the motifs in both IP₃Rs, with some additional loops (of yet unexplained significance) in the IP₃-BD of Paramecium. From (). (B) Exocytosis performance (percent of cells performing exocytosis [ordinate] to a different extent indicated in the abscissa) and the effect of PtCRC-IV-1 (RyR-LP) silencing. Two types of control cells have been evaluated: (i) Cells mock-silenced with the vector (pPD) containing only a GFP sequence served as a negative control; (ii) cells silenced in the exocytosis-relevant ND7 gene served as a positive control. The relevant experiment was carried out with a vector containing a sequence appropriate to silence PtCRC-IV. Note considerable depression of AED-stimulated exocytosis after ND7 and PtCRC-IV silencing, respectively. From (). (C) Effect of silencing of PtCRC-IV-1 (RyR-LP) on Ca²⁺-signaling. Cells were exceptionally contained in a medium with [Ca²⁺]_o reduced to a calculated value of ~30 nM, i.e. slightly below internal resting level ([Ca²⁺]_i ~ 50 nM, thus excluding signals from Ca²⁺-influx. The Ca²⁺ signal generated during exocytosis stimulation was evaluated by ratio imaging (f/f_o). Black lines: non-silenced cells, red lines: silenced cells. Exocytosis was stimulated by aminoethyldextrane (AED), 4-chloro-m-cresol (4CmC) and caffeine, respectively. For further details, see text. From ().

Localization of TbIP₃R in procyclic trypomastigotes. (A) TbIP₃R partially co-localizes with TbVP1 in acidocalcisomes (Pearson’s correlation coefficient of 0.874), as shown by immunofluorescence microscopy analysis. The merge images show the co-localization in yellow. (B) Lack of co-localization of TbIP₃R with TbBiP in the endoplasmic reticulum (Pearson’s correlation coefficient of 0.156). Scale bars, 10 μm. DIC, differential interference contrast. From ().

Caged IP₃-dependent Ca²⁺ release in T. brucei. (A–B) Representative traces of Ca²⁺ responses to UV flash in control cells in the absence (A) and presence (B) of caged IP₃, respectively (first flash 3 pulses, second flash 6 pulses). Nigericin (Nig; 5 μM) and Ionomycin (IO; 5μM) were added where indicated. From ().

Schematic representation of the distribution of Ca²⁺ in a T. gondii tachyzoite. Ca²⁺ entry is probably through Ca²⁺ channels (a). Once inside the cells, Ca²⁺ can be translocated back to the extracellular environment, primarily by the action of the PMCA (b). In addition, Ca²⁺ will interact with binding proteins (CBPs) or become sequestered by the ER by the action of the SERCA-Ca²⁺-ATPase (c), passively sequestered by the mitochondrion (d), or sequestered by the acidocalcisome (e) or the PLV (f) by the action of a Ca²⁺-ATPase (TgA1). Ca²⁺ appears to diffuse freely into the nucleus. Calcium could also be released into the cytoplasm from the internal stores, such as the ER, through an uncharacterized channel, which appears to respond to ryanodine and caffeine (g). It may also be released from the PLV and the acidocalcisome (AC) through a Ca²⁺/H⁺ exchanger (h). Acidic compartments such as the acidocalcisome and the PLV contain enzymes involved in their acidification e.g. the H⁺- ATPase (i) and the vacuolar-H⁺-pyrophosphatase (j). Mitochondrial Ca²⁺ release is through a Ca²⁺/H⁺ exchanger (k).

Scheme of the potential contact between acidocalcisomes and mitochondrion in trypanosomes. The scheme depicts the molecules mediating Ca²⁺ influx (MCU, MICU1) and efflux (Ca²⁺/H⁺ exchanger, CAX) across the inner mitochondrial membrane (IMM) at an area of acidocalcisome (ACCSOME)-mitochondrial association. The shades of gray represent the [Ca²⁺]: dark gray: > 500 μM; white, 100 nM; PMCA, plasma membrane-type Ca²⁺-ATPase; OMM, outer mitochondrial membrane; VDAC, voltage-dependent anion-selective channel; IP₃R, inositol 1,4,5-trisphosphate receptor.