Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Vacuolar system of ungerminated Colletotrichum graminicola conidia: convergence of autophagic and endocytic pathways

Vacuoles of ungerminated Colletotrichum graminicola conidia engulf cytoplasmic structures by a process analogous to microautophagy, demonstrated by using a vacuolar membrane acid phosphatase marker. Fusion of vesicles with vacuoles, without deposition of the acid phosphatase reaction product has been observed, suggesting other pathways of material delivery to vacuoles than microautophagy. Plasma membrane invaginations, multivesicular bodies and retention of neutral red into small vesicles, which were internalized by the vacuole, were verified. These results provided evidence for endocytosis and an active endosomal system. Together, our findings with C. graminicola demonstrated that vacuoles are very dynamic compartments, playing roles in autophagy and endocytic processes.     

Morphogenesis and Ultrastructure of Claviceps purpurea During Submerged Alkaloid Formation

Criteria for morphogenetic and ultrastructural distinction between conidia and chlamydospores of a submerged culture of Claviceps purpurea (Fr.) Tul. are described. Both the hyphae of the sphacelia (asexual) stage and the conidia contained granular cytoplasm. Cytoplasmic invaginations in vacuoles were transformed to electron-opaque bodies and disintegrated prior to germination. The budding of conidia had basipetal succession. The chlamydospores were formed by rounding up the terminal cells of filamentous hyphae. Homogeneous nonvacuolized cytoplasm with lipid droplets and lipid-forming bodies was characteristic of young chlamydospores. Cristate mitochondria did not appear in the chlamydospores before the alkaloid production phase. Simultaneously a specific organelle in the chlamydospores, a dense body, appeared to absorb intracellular lipids and form large deposits of phospholipid material. No germination of chlamydospores was observed. The ultrastructural pattern described for chlamydospores was also observed in hyphae with reduced proliferation during the alkaloid production phase.     

Fine structure of the formation and fate of the residual bodies of mouse spermatozoa with evidence for the participation of lysosomes

Routine electron microscopy in combination with subcellular localization of acid phosphatase has been employed to study the formation and fate of residual cytoplasmic bodies extruded into the tubular lumen shortly before spermiation. Prior to extrusion the spermatid cytoplasm contains lipid droplets, mitochondria, ribosomes, endoplasmic reticulum, the caudally migrated Golgi apparatus, and numerous multivesicular and multigranular bodies. These membrane-limited bodies and the Golgi zone stain heavily for acid phosphatase. Following extrusion the residual bodies undergo a series of alterations: (1) disruption of multigranular bodies with release of free granules; (2) sequestration of granules, ribosomes, and reticulum inside double-membrane-limited vacuoles derived from Golgi lamellae; (3) appearance of numerous, single-membrane-bound, cytoplasmic vacuoles; (4) fragmentation; (5) peripheral migration toward the tubular wall; and (6) phagocytosis of these migrating fragments by the Sertoli cells. The demonstration of acid phosphatase activity within free granules, the sequestering Golgi lamellae, and both classes of vacuoles suggests that initial residual body degradation occurs through lysosomal cytoplasmic autophagy.     

Subcellular Localization Studies Indicate That Lipoxygenases 1 to 6 Are Not Involved in Lipid Mobilization during Soybean Germination

Soybean (Glycine max) lipoxygenase (LOX) has been proposed to be involved in reserve lipid mobilization during germination. Here, subcellular fractionation studies show that LOX1, -2, -3, -4, -5, and -6 isozymes were associated with the soluble fraction but not with purified oil bodies. The purified oil bodies contained small amounts of LOX1 (<0.01% total activity), which apparently is an artifact of the purification process. Immunogold labeling indicated that, in cotyledon parenchyma cells of LOX wild-type seeds that had soaked and germinated for 4 d, the majority of LOX protein was present in the cytoplasm. In 4-d-germinated cotyledons of a LOX1/2/3 triple null mutant (L0), a small amount of label was found in the cytoplasm. In epidermal cells, LOX appeared in vacuoles of both wild-type and L0 germinated seeds. No LOXs cross-reacting with seed LOX antibodies were found to be associated with the cell wall, plasma membrane, oil bodies, or mitochondria. Lipid analysis showed that degradation rates of total lipids and triacylglycerols between the wild type and L0 were not significantly different. These results suggest that LOX1, -2, -3, -4, -5, and -6 are not directly involved in reserve lipid mobilization during soybean germination.     

豌豆根瘤脂质体的分布及形态特征

豌豆根瘤中有大量的脂质体,广泛分布于非侵染细胞和侵染细胞内,它既可以存在细胞质中,也可位于液泡里面。有时单个存在,有时又多个聚集在一起。非侵染细胞与侵染细胞相比,前者中的脂质体明显多于后者。这些脂质体近似圆形或椭圆形,表面无膜,电子密度较高,内部无固定的结构,常有一些细胞器位于它的附近。讨论了脂质体的细胞发育及细胞种类的关系。     

Acid phosphatase activity in the wild-type and B-mutant hyphae of Schizophyllum commune.

In the wild-type and B-mutant hyphae of Schizophyllum commune, acid phosphatase activity was found in association with vacuoles, lipid bodies, and endoplasmic reticulum. Small granules containing acid phosphatase also occurred in mitochondria and along the nuclear envelope. Both ultrastructural and biochemical studies indicated greater acid phosphatase activity in the B-mutant than in the wild-type hyphae, which suggests that the mutation in the B incompatibility factor increases the production of the acid phosphatase in the mutant hyphae.     

Biochemical and Ultrastructural Changes in Tetrahymena pyriformis During Starvation*

SYNOPSIS. Certain of the ultrastructural and biochemical changes occurring during the first 25 hr of starvation in Tetrahymena pyriformis were studied. Ultrastructurally, numerous profiles of degenerating mitochondria were seen in the early stages of starvation. The presence of oxidizable substrate such as glucose and acetate did not prevent this degeneration. Numerous large nucleoli were formed, many of which seemed to be passing into the cytoplasm as forming autophagic vacuoles. There was a transient increase in Oil Red O-positive bodies, presumably lipid (triglycerides). The extent and duration of this increase were pronounced in the presence of acetate. The lipid droplets appeared to arise within the cisternae of the endoplasmic reticulum. Lipid reserves were apparently utilized prior to carbohydrates, as the disappearance of lipid droplets preceded glycogen utilization, both in the presence of acetate and in the absence of exogenous substrate. A considerable loss of cellular protein also occurred. In cells from inorganic medium supplemented with glucose, glycogen occupied much of the cell, leaving only islands of cell organelles. Acid phosphatase was localized, ultrastructurally, mainly in autophagic vacuoles which contained mitochondria and other cell organelles, and in association with small, double-membraned structures which seemed to be sequestering small areas of cytoplasm. Such sequestered areas also appeared within larger autophagic vacuoles. Residual bodies containing concentric whorls of myelin-like membranes surrounding a more solid core accumulated during starvation. Acid phosphatase activity decreased in amount but not in specific activity. The specific activity of cathespin doubled or tripled, but there was little change in total enzyme.     

Structure and cytochemistry of the procambium in Salix buds during dormancy and dormancy breaking

This report presents a combined investigation of ultrastructural and enzymatic changes in the procambium from late winter to early spring. In January the procambial cells of dormant Salix buds have a convoluted plasma membrane with many plasmalemmasomes, numerous lipid bodies, large stacks of rough ER and plastids surrounded by smooth ER profiles. Several small lysosomes show activity of ATPase and acid phosphatases. In addition ER, nuclear envelopes, dictyosomes, and thylakoids have ATPase activity, and ER and plasmalemma, and nuclei also show acid phosphatase activity. In February metabolism seems to increase as indicated by lysosomes with membranous formations, dilated ER, nuclear envelopes, spiny vesicles, and polysomes. ATPase activity occurs in plasmalemma and vacuoles, and acid phosphatases in the middle lamella region of walls, in plasmalemma, vacuoles, ER, and nuclei. At the end of March, when growth starts inside the buds, but before they break, the stacks of rough ER disappear, and the vacuoles coalesce. Most of the lipid bodies have disappeared and the plastids have accumulated starch. Cell division and differentiation of procambial cells to protophloem and protoxylem have started. The distribution of ATPase increases; activity is found in walls and plasmalemma, and only a few small vacuoles still have ATPase and acid phosphatase activity. Notable is the appearance of ATPase in mitochondrial cristae and nucleoli and the occurrence of rather high levels also in endomembranes and dictyosomes.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

Ultrastructure and Lipase Activity of Cotyledon cell During Pod Development in Peanut

A series of significant changes of the ultrastructure and lipase activity of cotyledon cell were found in peanut (Arachis hypogaea) during pod development. In he initial stage of cotyledon development there were many plastids which kept producing starch grain and there were low lipase activity and very few lipid and protein bodies in the cell. In the middle stage of cotyledon development, a great number of larger lipid bodies were seen in the cell and a lot of protein bodies formed in the vacuoles and continued to increase in size. Lipase activity increased in the cytoplasm, endoplasmic reticulum, protein bodies, plasmalemma and intercellular space. In the later stage of cotyledon development, the lipid bodies did not increase in number but became slightly larger. The protein bodies continued to increase both in number and in size. Lipase acttvity was even hegher in the cytoplasm. In the final stage the protein bodies became irregular in shape and some of them tended to disintegrate with their content entered into the space around the lipid bodies. The lipase activity in the cell declined. The results indicated that the lipid body originated in the cytoplasm and the protein body originated in the vacuole; that the accumulation of oil and protein in peanut cotyledon resulted from the formation and development of lipid and protein bodies in the cell, and that the changes of plasmid and lipase activity in the cell played a role in the development of lipid body during the development of cotyledon.     

Changes in the Plantago lanceolata mature pollen ultrastructure and Pla l 1 allergen localisation after grain hydration

Abstract

We have conducted the study of ultrastructural changes of wall and cytoplasm of Plantago lanceolata (English plantain) pollen grains during the first 10 min of hydration and activation processes, prior to germination, and localisation of Pla l 1, the major allergen of these pollen grains with immunocytochemical methods. The samples were fixed using conventional and freezing protocols for transmission electron microscopy. During the activation process, the intine is thickened in the apertural region and cytoplasm undergoes changes in the number of lipid bodies, amyloplasts, vacuoles and ribosomes. Also, we observed an association between lipid bodies, cisternae of rough endoplasmic reticulum, dictyosomes and vacuoles. An increase in the presence of allergenic particles (Pla l 1) in the exine, intine and the cytoplasm in activated pollen grains was detected, whereas this presence is not significant in mature pollen grains. The increase in the production and release of this allergen when pollen grains are activated suggest that Pla l 1 has an important role in pollen–stigma recognition and in the subsequent development of the pollen tube.     

A role for caleosin in degradation of oil-body storage lipid during seed germination

Caleosin is a Ca(2+)-binding oil-body surface protein. To assess its role in the degradation of oil-bodies, two independent insertion mutants lacking caleosin were studied. Both mutants demonstrated significant delay of breakdown of the 20:1 storage lipid at 48 and 60 h of germination. Additionally, although germination rates for seeds were not affected by the mutations, mutant seedlings grew more slowly than wild type when measured at 48 h of germination, a defect that was corrected with continued growth for 72 and 96 h in the light. After 48 h of germination, wild-type central vacuoles had smooth contours and demonstrated internalization of oil bodies and of membrane containing alpha- and delta-tonoplast intrinsic proteins (TIPs), markers for protein storage vacuoles. In contrast, mutant central vacuoles had distorted limiting membranes displaying domains with clumps of the two TIPs, and they contained fewer oil bodies. Thus, during germination caleosin plays a role in the degradation of storage lipid in oil bodies. Its role involves both the normal modification of storage vacuole membrane and the interaction of oil bodies with vacuoles. The results indicate that interaction of oil bodies with vacuoles is one mechanism that contributes to the degradation of storage lipid.     

Electron microscopic histochemistry of lysosomes in neurosecretory nerve endings and pituicytes of rat posterior pituitary

Summary Electron microscopic localization of acid phosphatase (AcPase) was carried out on posterior pituitary glands from rats. An estimated 5% of the neurosecretory nerve terminals contained structures which showed reaction product. Most of the lysosomes were small dense bodies, often with a membranous substructure. Other lysosomes were larger in size or were found within vacuoles. AcPase was also localized to lysosomes and the Golgi apparatus of pituicytes. Evidence is presented which would associate the large lipid droplets characteristic of pituicytes with AcPase-positive dense bodies. The present study indicates that hydrolytic activity by lysosomes occurs within the terminals of neurosecretory cells, and adds further support to the concept that this process represents a normal phenomenon of cells and their extensions in general.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

Ultracytochemical localization of the vacuolar marker enzymes alkaline phosphatase, adenosine triphosphatase, carboxypeptidase Y and aminopeptidase reveal new concept of vacuole biogenesis in Saccharomyces cerevisiae

Logarithmic cultures of Saccharomyces cerevisiae strains LBG H 1022, FL-100, X 2180 1A and 1B were studied together with the mutants pep4-3, sec18-1 and sec7-1. The necessary ultrastructural observations showed that, as a rule, juvenile vacuoles were formed de novo from perinuclear endoplasmic reticulum cisternae (ER) packed and inflated with electron-dense (polyanionic) matrix material. This process was disturbed solely in the sec18-1 mutant under non-permissive conditions. The vacuolar marker enzymes adenosine triphosphatase (ATPase) and alkaline phosphohydrolase (ALPase) were assayed by the ultracytochemical cerium precipitation technique. The neutral ATPase was active in vacuolar membranes and in the previously shown (coated) microglobules nearby. ALPase activity was detected in microglobules inside juvenile vacuoles, inside nucleus and in the cytoplasm as well as in the membrane vesicles and in the periplasm. The sites of vacuolar protease carboxypeptidase Y (CPY) activity were assayed using N-CBZ-L-tyrosine-4-methoxy-2-naphthyl-amide (CBZ-Tyr-MNA) as substrate and sites of the amino-peptidase M activity using Leu-MNA as substrate. Hexazotized p-rosaniline served as a coupler for the primary reaction product of both the above proteases (MNA) and the resulting azo-dye was osmicated during postfixation. The CPY reaction product was found in both polar layers of vacuolar membranes (homologous to ER) and in ER membranes enclosing condensed lipoprotein bodies which were taken up by the vacuoles of late logarithmic yeast. Both before and after the uptake into the vacuoles the bodies contained the CPY reaction product in concentric layers or in cavities. Microglobules with CPY activity were also observed. Aminopeptidase was localized in microglobules inside the juvenile vacuoles. These findings combined with the previous cytochemical localizations of polyphosphates and X-prolyl-dipeptidyl (amino)peptidase in S. cerevisiae suggest the following cytologic mechanism for the biosynthetic protein transport: coated microglobules convey metabolites and enzymes either to the cell surface for secretion or enter the vacuoles in all phases of the cell cycle. The membrane vesicles represent an alternative secretory mechanism present in yeast cells only during budding. The homology of the ER with the vacuolar membranes and with the surface membranes of the lipoprotein condensates (bodies) indicates a cotranslational entry of the CPY into these membranes. The secondary transfer of a portion of CPY into vacuoles is probably mediated by the lipoprotein uptake process.     

花生荚果发育过程中子叶细胞的超微结构和脂酶活性的研究

在花生(Arachis hypogaea)荚果发育过程中,子叶细胞的超微结构和脂酶活性皆发生了显著变化。子叶生长初期,缅胞中质体较多,并不断形成淀粉粒;脂酶活性低,脂体和蛋白体很少。子叶发育中期,子叶细胞质中出现大量体积较大的脂体,液泡中的蛋白体不断形成和增大,而且细胞质、内质网、蛋白体外膜、细胞质膜和细胞间隙上皆显示较强的脂酶活性。子叶发育后期,脂体数量不再增加,但体积略有增大,间质透明度也有提高;蛋白体增大较小,但数量却进一步增多;细胞质中仍显示较强的脂酶活性。至末期时,蛋白体形态变得不规则,甚至出现部分解体,其基质充挤脂体间隙;细咆中的脂酶活性减弱。研究表明,花生脂体起源于细胞质,蛋白体起源于液泡,子叶油分和蛋白质的积累足体内脂体和蛋白体不断发育的结果,细胞中脂酶活性的变化可能与脂体发育有关。     

Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.     

LOCALIZATION OF ACID PHOSPHATASE IN LIPOFUSCIN GRANULES AND POSSIBLE AUTOPHAGIC VACUOLES IN INTERSTITIAL CELLS OF THE GUINEA PIG TESTIS

The intracellular localization of acid phosphatase in guinea pig testicular interstitial cells was investigated by incubating nonfrozen thick sections of glutaraldehyde-perfused testis in a modified Gomori medium and preparing the tissue for electron microscopy. Lipofuscin pigment granules in these cells contain dense pigment, granular matrix, and often a lipid droplet. Reaction product is seen in the matrix of the pigment granules, and they may therefore be called residual bodies. At least some of the dense pigment appears to be derived from myelin figures and membrane whorls, since suitable intermediates can be seen. Lipid droplets found free in the cytoplasm are another possible source of pigment. In both cases the chemical mechanism is presumed to be autoxidation of unsaturated lipid. Acid phosphatase is present in the inner cisterna of Golgi elements. Enzyme activity also appears in possible autophagic vacuoles bounded by double membranes; the reaction product lies between the membranes. Consideration of the enzyme as a tracer suggests that the autophagic vacuoles are derived from the Golgi complex. Possible stages in the formation of these vacuoles by the inner Golgi cisternae are observed.     

Acid phosphatase localization in the fungus Whetzelinia sclerotiorum

Acid phosphatase was localized by light and electron microscopy in chains of vacuoles in hyphal tip cells of Whetzelinia sclerotiorum. The Enzyme was present in these vacuoles whether or not conditions favored extracellular acid phosphatase secretion. Apical vesicles, microbodies, woronin bodies, and lipid bodies did not contain acid phosphatase. The implications regarding terminology of organelles in filamentous fungi are discussed with special reference to the fungal spherosome concept.Abbreviations AP acid phosphatase     

Exocytosis couples to endocytosis of ferritin in parotid acinar cells from isoprenalin stimulated rats

Distribution of acid phosphatase as a marker enzyme for lysosomes was investigated in the isoprenalin stimulated rat parotid gland. The enzyme was localized in lipofuscin-like bodies as well as in non-discharged granules. The appearance of these bodies was correlated in time to the appearance of smooth vesicles and reduction of the acinar lumen. Ferritin, used as a tracer and introduced into the stimulated gland via cannulated parotid ducts, was found in smooth vesicles, vacuoles and lipofuscin-like bodies throughout the cytoplasm of the acinar cells. Very often ferritin-containing vesicles were found in the vicinity of the Golgi complex. In most cases the vesicles containing ferritin also showed acid phosphatase reaction product. A possible correlation between the lysosomal system and the process of recycling and degradation of membranes in the stimulated gland is discussed.