Acidocalcisomes

Abstract

Acidocalcisomes are acidic organelles containing calcium and a high concentration of phosphorus in the form of pyrophosphate (PP(i)) and polyphosphate (poly P). Organelles with these characteristics have been found from bacteria to human cells implying an early appearance and persistence over evolutionary time or their appearance by convergent evolution. Acidification of the organelles is driven by the presence of vacuolar proton pumps, one of which, the vacuolar proton pyrophosphatase, is absent in animals, where it is substituted by a vacuolar proton ATPase. A number of other pumps, antiporters, and channels have been described in acidocalcisomes of different species and are responsible for their internal content. Enzymes involved in the synthesis and degradation of PP(i) and poly P are present within the organelle. Acidocalcisomes function as storage sites for cations and phosphorus, and participate in PP(i) and poly P metabolism, calcium homeostasis, maintenance of intracellular pH, and osmoregulation. Experiments in which the acidocalcisome Ca(2+)-ATPase of different parasites were downregulated or eliminated, or acidocalcisome Ca(2+) was depleted revealed the importance of this store in Ca(2+) signaling needed for host invasion and virulence. Acidocalcisomes interact with other organelles in a number of organisms suggesting their association with the endosomal/lysosomal pathway, and are considered part of the lysosome-related group of organelles.

Fig. 1

Acidocalcisomes in Agrobacterium tumefaciens. (A) electron micrograph of intact bacteria. Arrow shows an electron-dense material in the periphery of an acidocalcisome. White arrowhead shows an electron-dense inclusion. (B) Staining of acidocalcisomes with DAPI (yellow). (C) (D) staining of acidocalcisomes with antibodies against a V-H⁺-PPase as detected by immunofluorescence (C) or immunoelectron microscopy (D). Arrowheads in (C) show the acidocalcisomes in green. Inset is at higher magnification. Arrowheads in (D) show the gold particles labeling the membrane of an acidocalcisome (vg, volutin granule). Inset in (D) shows an immunoblot analysis of a bacterial lysate. PPase, antibody against V-H⁺-PPase showing a band of 72 kDa. C, control treated with preimmune serum. Bars, A = 0.1 μm; B, C = 0.5 μm; D = 40 nm. A, C, and D are reproduced with permission from [10], © The American Society for Biochemistry and Molecular Biology.

Fig. 2

Morphology of acidocalcisomes in whole cells and sections. (A) Ultrathin sections of Toxoplasma gondii showing the acidocalcisomes as empty vesicles or containing electron dense material (arrows). (B) Acridine orange staining of acidocalcisomes (red vesicles) of T. gondii tachyzoites. (C) Visualization of acidocalcisomes in whole unfixed Toxoplasma gondii allowed to adhere to Formvar- and carbon-coated grids and then observed by direct transmission electron microscopy (using an energy filter). Acidocalcisomes (black granules) appear disperse in the cytoplasm. Bars, (A) = 500 nm; (B) = 3 μm; (C) = 1 μm. Reproduced with permission from [15], © Elsevier.

Fig. 3

Schematic representation of an acidocalcisome. A H⁺ gradient is established by a vacuolar ATPase (V-H⁺-ATPase) and a vacuolar pyrophosphatase (V-H⁺-PPase). Ca²⁺ transport is driven by a Ca²⁺-ATPase. Other transporters include Na⁺/H⁺, and Ca²⁺/H⁺ exchangers, and a water channel or aquaporin. A vacuolar transporter chaperone (VTC) complex is involved in synthesis and translocation of poly P. Transporters for basic amino acids, P_i, PP_i, and cations are potentially present (in green). The matrix is rich in PP_i and polyphosphate (poly P) and enzymes involved in their metabolism (exopolyphosphatase (PPX), and pyrophosphatase (PPase). Not all the enzymes and transporters are present in all acidocalcisomes.

Fig. 4

Acidocalcisomes in chicken egg yolk. (A) Compound organelle observed by energy-filtering transmission electron microscopy. The internal vesicles (acidocalcisomes) are electron-dense. (B) acridine orange staining of the compound organelle showing the more acidic internal vesicles (acidocalcisomes). (C-F) Elemental mapping of the compound organelle showed in (C) reveals the localization of phosphorus (P-Ka), calcium (Ca-Ka), and potassium (K-Ka). (G-H) freeze fracture analysis of acidocalcisome fractions showing the E (external face of the inner leaflet) (G) and P (internal face of the outer leaflet) (H) faces of fractured membranes. Intramembrane particles (IMPs) (arrows) are randomly distributed on the E and P of membranes. Scale bars, (A, B) =5 μm; (C-F) = 10 μm; (G-H) = 2 μm. Reproduced with permission from [47], © Portland Press Ltd.

Fig. 5. Proposed Ca²⁺ mobilization pathways in sea urchin eggs

Endoplasmic reticulum (ER), acidocalcisomes, and yolk platelets are major Ca²⁺ storage organelles. IP₃ receptors (IP₃R) and ryanodine receptors (RyR) mediate Ca²⁺ release from the endoplasmic reticulum (ER) in response to increases in inositol 1,4,5-trisphosphate (IP₃) and cyclic ADP ribose (cADPR), respectively. Both receptors types are in addition, activated by Ca²⁺ via the so-called Ca²⁺-induced Ca²⁺ release mechanism (CICR). NAADP triggers Ca²⁺ release from yolk platelets (gray) and this in turn triggers ER Ca²⁺ mobilization through Ca²⁺-induced Ca²⁺ release via IP₃R and ryanodine (RyR) receptors. Na⁺ entry after fertilization leads to alkalinization of the cytosol and stimulates Ca²⁺ release from acidocalcisomes by coupling the activity of Na⁺/H⁺ and Ca²⁺/H⁺ exchangers. GPN is hydrolyzed by a cathepsin C increasing the osmolarity of yolk platelets, attracting water, and leading to osmotic lysis and Ca²⁺ release. Both acidocalcisomes and yolk platelets possess bafylomycin A₁-sensitive vacuolar type H⁺-ATPases to acidify the organelles. Poly P is hydrolyzed after alkalinization of acidocalcisomes. Reproduced with permission from [46], © Portland Press Ltd.

Fig. 6

Electron microscopy of whole platelets (A) or dense granule (acidocalcisome) fraction (B-C). (A) Transmission electron microscopy of an unfixed and unstained platelet preparation. Dense granules are identified by arrows. (C) Direct observation of unfixed and unstained dense granules air-dried directly onto microscope grids. The inset shows at higher magnification the sponge-like structure of the dense granules after exprosure to the electron beam. (C) fixed and sectioned dense granule fraction. Bars, (A) = 1 μm; (B-C) = 0.25 μm. Reproduced with permission from [11], © The American Society for Biochemistry and Molecular Biology.