Acidocalcisomes as calcium- and polyphosphate-storage compartments during embryogenesis of the insect Rhodnius prolixus Stahl

Background

The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus.

Methodology/Principal findings

Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H⁺-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs.

Conclusions

We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells.     

A new model for proton pumping in animal cells: the role of pyrophosphate

The H+-PPase activity was characterized in membrane fractions of ovary and eggs of Rhodnius prolixus. This activity is totally dependent on Mg2+, independent of K+ and strongly inhibited by NaF, IDP and Ca2+. The membrane proteins of eggs were analyzed by western blot using antibodies to the H+-PPase from Arabidopsis thaliana. The immunostain was associated with a single 65-kDa polypeptide. This polypeptide was immunolocalized in yolk granule membranes by optical and transmission electron microscopy. We describe the acidification of yolk granules in the presence of PPi and ATP. This acidification is inhibited in the presence of NAF, Ca2+ and antibodies against H+-PPase. These data show for the first time in animal cells that acidification of yolk granules involves an H+-PPase as well as H+-ATPase.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Polyphosphate polymers during early embryogenesis of Periplaneta americana

Inorganic polyphosphates (PolyP) are linear polymers of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite a wide distribution, their role during insect embryogenesis has not been examined so far. In this study, we show the mobilization of PolyP polymers during the embryogenesis of the cockroach Periplaneta americana. PolyP was detected by enzymatic and fluorimetric assays and found to accumulate in two main sizes by agarose gel electrophoresis. Confocal microscopy showed their presence in small vesicles. In addition, X-ray microanalysis of small vesicles showed considerable amounts of calcium, sodium and magnesium, suggesting an association of PolyP with these elements. Variations of the free Ca⁺², Pi and PolyP levels were observed during the first days of embryogenesis. Our results are consistent with the hypothesis that phosphate ions modulate PolyP variation and that PolyP hydrolysis result in increasing free Ca⁺² levels. This is the first investigation of PolyP metabolism during embryogenesis of an insect and might shed light on the mechanisms involving Pi storage and homeostasis during this period. We suggest that PolyP, mainly stored in small vesicles, might be involved in the functional control of Ca⁺² and Pi homeostasis during early embryogenesis of P. Americana.     

Silencing of Maternal Heme-binding Protein Causes Embryonic Mitochondrial Dysfunction and Impairs Embryogenesis in the Blood Sucking Insect Rhodnius prolixus

The heme molecule is the prosthetic group of many hemeproteins involved in essential physiological processes, such as electron transfer, transport of gases, signal transduction, and gene expression modulation. However, heme is a pro-oxidant molecule capable of propagating reactions leading to the generation of reactive oxygen species. The blood-feeding insect Rhodnius prolixus releases enormous amounts of heme during host blood digestion in the midgut lumen when it is exposed to a physiological oxidative challenge. Additionally, this organism produces a hemolymphatic heme-binding protein (RHBP) that transports heme to pericardial cells for detoxification and to growing oocytes for yolk granules and as a source of heme for embryo development. Here, we show that silencing of RHBP expression in female fat bodies reduced total RHBP circulating in the hemolymph, promoting oxidative damage to hemolymphatic proteins. Moreover, RHBP knockdown did not cause reduction in oviposition but led to the production of heme-depleted eggs (white eggs). A lack of RHBP did not alter oocyte fecundation. However, produced white eggs were nonviable. Embryo development cellularization and vitellin yolk protein degradation, processes that normally occur in early stages of embryogenesis, were compromised in white eggs. Total cytochrome c content, cytochrome c oxidase activity, citrate synthase activity, and oxygen consumption, parameters that indicate mitochondrial function, were significantly reduced in white eggs compared with normal dark red eggs. Our results showed that reduction of heme transport from females to growing oocytes by RHBP leads to embryonic mitochondrial dysfunction and impaired embryogenesis.     

Fine Structures of Oocytes and Accessory Cells of the Ovary in the Starfish Patiria (Asterina) pectinifera at Different Stages of Oogenesis and After 1-Methyladenine-Induced Maturation

Morphological changes in the growing and maturing oocytes of Patiria ( Asterina ) pectinifero were studied by electron microscopy. Oogenesis is of the solitary type. An extensive system of rough endoplasmic reticulum (ER) and Golgi complex (GC) develops in the ooplasm forming the cortical, yolk and secretory granules in its peripheral regions. The contents of the latter granules are released from the oocyte and form the vitelline membrane. At early stages of oogenesis, extensive multiplication of mitochondria results in formation of a large aggregate of these organelles in the perinuclear cytoplasm ("yolk nucleus"). After maturation of full grown oocytes has been induced by 1-methyladenine, the membranous cell structures are rapidly rearranged: vast aggregates of ER cisternae in the surface cytoplasm layer and single ER cisternae among yolk granules are disintegrated to small vesicles; the GC is reduced. These processes are suggested to be somehow related to changes in hydration of the cytoplasm and in rigidity of its surface layer. In maturing oocytes, the yolk granules form characteristic linear rows, trabeculae, traversing the cytoplasm and their boundary membranes fuse in zones of contact. Some granules are converted to multivesicular bodies, thus suggesting the activation of hydrolytic enzymes that form part of the yolk in echinoderms.     

Oocyte-follicle cell differentiation in two species of amphineurans (Mollusca), Mopalia mucosa and Chaetopleura apiculata

Differentiating oocytes and associated follicle cells of two species of amphineurans (Mollusca) Mopalia muscosa and Chaetopleura apiculata have been studied by techniques of light and electron microscopy. In addition to the regularly occurring organelles, the ooplasm of young oocytes contains large, randomly situated, basophilic regions. These regions are not demonstrable in mature eggs. As oocytes differentiate, lipid, pigment and protein-carbohydrate yolk bodies accumulate within the ooplasm. Concomitant with the appearance of pigment and the protein carbohydrate containing yolk bodies, the saccules of the Golgi complex become filled with a dense material. Associated with the Golgi complex are cisternae of the rough endoplasmic reticulum which are filled with an electron opaque substance which is thought to be composed of protein synthesized by this organelle. That portion of the cisternae of the endoplasmic reticulum facing the Golgi complex shows evaginations. These evaginations are thought to finalize into protein containing vesicles that subsequently fuse with the Golgi complex. Thus, the Golgi complex in these oocytes might serve as a center for packaging and concentrating the protein used in the construction of the protein containing pigment or protein-carbohydrate yolk bodies. The suggestion is made that the Golgi complex may also synthesize the carbohydrate portion of the formentioned yolk bodies. In an adnuclear position in young oocytes are some acid mucopolysaccharide containing vacuolar bodies. In mature eggs, these structures are found within the peripheral ooplasm and we have referred to them as cortical granules. There is no alteration of these cortical granules during sperm activation.     

Presence of calcium in polychaete cortical granules demonstrated by X-ray microanalysis on ultrathin cryo-sections of oocytes and eggs

Summary Cortical granules from fertilized eggs, oocytes and nurse cells of Ophryotrocha labronica have been analyzed for the presence of calcium using cryo-ultramicrotomy and X-ray microprobe analysis. All cortical granules showed a significant peak for calcium, but yolk granules were without calcium. These results support the hypothesis that the discharge of cortical granules shortly after fertilization is a self-propagating phenomenon involving the diffusion of Ca²⁺ from bursting granules.     

Fertilization envelope in diapause eggs of Pontella mediterranea (Crustacea, Copepoda).

The sequence and timing of morphological changes during envelope formation was followed in diapause eggs of Pontella mediterranea (Crustacea, Copepoda). The multilayer coat enveloping these eggs resulted from the exocytosis of 4 types of cortical vesicles that sequentially released their contents in the perivitelline space. These included small high-density vesicles (hDV) with electron-dense material, vesicles (V) with dense ring granules and a uniform matrix contained within the same compartment, large high-density (HDV) vesicles, and large moderately dense (MDV) vesicles. All of these cortical vesicles were present in newly spawned, fertilized eggs. Their exocytosis resulted from egg activation. One of these cortical vesicles (V) was similar in morphology to the intracisternal granules precursors of endogenous yolk. Intracisternal granules, characteristic of previtellogenic oocytes of many crustaceans, were present in previtellogenic oocytes of P. mediterranea but disappeared in later stages of oocyte development once yolk formation was completed. We discuss the role of cortical vesicles in the formation of the complex extracellular coat enveloping copepod diapause eggs.     

Sea urchin spectrin in oogenesis and embryogenesis: a multifunctional integrator of membrane-cytoskeletal interactions

Using indirect immunofluorescence microscopy on semithin cryosections of maturing ovarian tissue, eggs, and developing embryos, we have mapped the cellular distribution and dynamic redistribution of spectrin in oogenesis and early embryogenesis. During oogenesis, spectrin is initially found in the cortex of oogonia and previtellogenic oocytes, and later accumulates in the cytoplasm of vitellogenic oocytes on the surfaces of cortical granules, pigment granules/acidic vesicles, and yolk platelets. Following egg activation, spectrin undergoes a rapid redistribution coincident with three major developmental events including: (1) restructuring of the cell surface, (2) translocation of pigment granules/acidic vesicles to the cortex during the first cell cycle, and (3) amplification of the embryo's surface during the rapid cleavage phase of early embryogenesis. The synthesis and storage of spectrin during oogenesis appears to prime the egg with a preestablished pool of membrane-cytoskeletal precursor for use during embryogenesis. Results from this study support the hypothesis that spectrin may function as a key integrator and modulator of multiple membrane-cytoskeletal functions during embryonic growth and cellular differentiation.     

Ultrastructural observations of the early and late stages of gorgonian coral (Junceella juncea) oocytes

The developmental oogenesis of gorgonian coral was investigated at the histological level. The objective of this study was to examine and improve the understanding of Junceella juncea oogenesis using ultrastructural methods, such as histological sectioning and transmission electron microscopy. At least three types of yolk materials were observed in this study: yolk body, lipid granules and cortical alveoli. Some of the complex yolk materials were encompassed by concentric or arched layers of smooth and rough endoplasmic reticulum and the Golgi complex in early stage oocytes. Different types of vesicles were found in both early and late stage oocytes and some granules could be seen inside the empty vesicles. This may be a possible method for elaborating complex yolk materials. Homogeneous yolks from different types of inclusions were abundant and the autosynthesis of yolk may be a major mechanism in J. juncea oocytes. This is the first report of the ultrastructural observation of oogenesis in gorgonian coral species using transmission electron microscopy. Our study obtained relatively detailed information at the ultrastructural level, and it provides an overview of the oocyte ultrastucture of the gorgonian coral J. juncea.     

Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca²⁺-ATPase, V-H⁺-ATPase, H⁺-PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes.     

Dirofilaria immitis: ultrastructural aspects of oocyte development and zygote formation.

Ultrastructural observations of the ovary and uterus of Dirofilaria immitis reveal some characteristics of oogonia, oocytes, and uterine sperm. Oogonia are confined to the distal portion of the ovary including a blind tip, where a morphologically distinct terminal cap cell was not observed. These cells contain a nucleus with a nucleolus, numerous dense bodies, scanty ribosomes, lipid droplets, and an occasional mitochondrion. Endoplasmic reticulum is lacking and Golgi complexes were observed only in fully grown oogonia. Primary oocytes located in the middle portion of the ovary are large, elongate, and have a complete set of organelles including many small mitochondria, fragmentary endoplasmic reticulum, ribosomes, Golgi complexes, and very few dense bodies. These cells are arranged into many rosettes about central cytoplasmic masses, the rachises, to which they maintain cytoplasmic continuity by pseudopodlike processes. The rachises contain no organelle except a few dense granules and are bound by winding membranes. Oocytes from the proximal portion differ from those of the middle portion of the ovary in their larger size, round shape, absence of many organelles, presence of small dense granules, and lacking a rachis. Dense bodies are specific to the oogonia and exhibit DNase susceptibility and a positive reaction for a mitochondrial enzyme. These findings together with their decreased number and a concomitant increase of mitochondria in the oocytes suggest a relationship between these bodies and mitochondria.Uterine sperm of D. immitis are of the amoeboid type and contain several chromatin masses without a nuclear envelope, many mitochondria, and specialized membranous organelles referred to as mesosomelike vesicles. The vesicles are probably originated from the sperm plasma membrane. Upon fertilization, the entire spermatozoon penetrates the oocyte and its contents are gradually dissolved in the ooplasm with a simultaneous appearance of large numbers of ribosomes at the site of dissolution. Ribosomes were later found in the nucleus. A pronucleus was not observed. These findings are basically in agreement with those described for Ascaris but differ in the morphologic features and number of rachises, presence of dense bodies, absence of refringent granules in the oocytes and the absence of a refringent body and presence of several chromatin masses in the sperm.     

凡纳滨对虾卵母细胞卵黄发生的超微结构

利用电镜研究凡纳滨对虾卵母细胞卵黄发生的全过程。结果表明 :凡纳滨对虾卵黄的发生是双源性的。卵黄发生早、中期是内源性卵黄大量合成的阶段 ,卵黄发生中、后期则以外源性卵黄的合成为主。内源性卵黄主要由内质网、线粒体、核糖体、溶酶体、高尔基器等多种胞器活跃参与形成。其中数量众多的囊泡状粗面内质网是形成内源性卵黄粒的最主要的细胞器 ;部分线粒体参与卵黄粒的合成并自身最终演变为卵黄粒 ;丰富的游离核糖体合成了大量致密的蛋白质颗粒并在卵质中直接聚集融合成无膜的卵黄粒 ;溶酶体通过吞噬、消化内含物来形成卵黄粒和脂滴 ,且方式多样 ;高尔基器不直接参与形成卵黄粒。外源性卵黄主要通过卵质膜的微吞饮活动从卵周隙或卵泡细胞中摄取外源物质来形成     

Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner (Lepidoptera: Noctuidae): A role for inorganic polyphosphate towards yolk mobilization

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.     

Interspecific fusion of sea urchin eggs: Surface events and cytoplasmic mixing

Here we describe a new method of fusing fertilized and unfertilized sea urchin eggs. After removal of materials peripheral to the plasma membranes, the plycation polyarginine is used to induce the eggs to adhere to one another. The subsequent incubation of the eggs in an artificial sea water with a slightly lower osmolarity and a higher calcium content than normal sea water results in successful fusion. We demonstrate with scanning electron microscopy (SEM) that our artificial sea water serves to smooth the plasma membranes—a characteristic which we find to be a requirement in order for fusion to occur. We also demonstrate that our fusion procedure can be used to identify and follow the development of hybrids composed of eggs of different histories. Using species whose cytoplasms are distinguishable by light microscopy, Strongylocentrotus purpuratus and Lytechlnus pictus, we consider the mixing of cytoplasms using yolk granules as markers. We find that if two unfertilized eggs are fused, the species-specific yolk particles are free to mix. In contrast, we observe no mixing of the yolk particles in either fertilized-unfertilized or fertilized-fertilized interspecies hybrids until the time of mitosis. At that time, the yolk particles are free to move around the region containing the mitotic figure(s). However, mixing of the cytoplasmic yolk granules is never completed during this brief period. Even after cytokinesis takes place, one can see the segregation of the yolk granules in the cytoplasms of the two species.     

A serine proteinase in Drosophila embryos: Yolk localization and developmental activation

A serine proteinase (molecular mass, about 25,000 Da) has been found in Drosophila mature oocytes. (1) The detected activity increases exponentially during embryogenesis. (2) The subcellular localization changes from the yolk granules, in oocytes, to the soluble fraction, in late embryos. (3) The proteinase cleaves by basic residues, but arginine is preferred (about 7-fold) over lysine.     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.     

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots

A H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H⁺-PPase catalyzed the electrogenic transport of H⁺ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb⁺ = K⁺ > Cs⁺; but it was inhibited by Na⁺ or Li⁺. Maximum rates of transport required at least 20 millimolar K⁺ and the K_m for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K⁺-dependent PPase activity with an I₅₀ of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H⁺-ATPase (e.g. a disulfonic stilbene or NO₃⁻) had no effect on the PPase activity. Vanadate and azide were also ineffective. H⁺-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H⁺-PPase and the H⁺-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H⁺ pumping activity was strongly affected by Mg²⁺ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg²⁺, H⁺ pumping showed a K_maPP for PPi of 15 micromolar. The rate of H⁺ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H⁺-PPase.     

Calcium‐ and polyphosphate‐containing acidocalcisomes in chicken egg yolk

Background information. Poly P (inorganic polyphosphate) is a polymer formed by P_i residues linked by high‐energy phosphoanhydride bonds. The presence of poly P in bacteria, fungi, algae and protists has been widely recognized, but the distribution of poly P in more complex eukaryotes has been poorly studied. Poly P accumulates, together with calcium, in acidic vesicles or acidocalcisomes in a number of organisms and possesses a diverse array of functions, including roles in stress response, blood clotting, inflammation, calcification, cell proliferation and apoptosis. Results. We report here that a considerable amount of phosphorus in the yolk of chicken eggs is in the form of poly P. DAPI (4′,6‐diamidino‐2‐phenylindole) staining showed that poly P is localized mainly in electron‐dense vesicles located inside larger vacuoles (compound organelles) that are randomly distributed in the yolk. These internal vesicles were shown to contain calcium, potassium, sodium, magnesium, phosphorus, chlorine, iron and zinc, as detected by X‐ray microanalysis and elemental mapping. These vesicles stain with the acidophilic dye Acridine Orange. The presence of poly P in organellar fractions of the egg yolk was evident in agarose gels stained with Toluidine Blue and DAPI. Of the total phosphate (P_i) of yolk organelles, 16% is present in the form of poly P. Total poly P content was not altered during the first 4 days of embryogenesis, but poly P chain length decreased after 1 day of development. Conclusions. The results of the present study identify a novel organelle in chicken egg yolk comprising acidic vesicles with a morphology, physiology and composition similar to those of acidocalcisomes, within larger acidic vacuoles. The elemental composition of these acidocalcisomes is proportionally similar to the elemental composition of the yolk, suggesting that most of these elements are located in these organelles, which might be an important storage compartment in eggs.