Characterization of different forms of yeast acid trehalase in the secretory pathway

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec 18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.     

Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Cortical membrane-trafficking during the meiotic resumption of Xenopus laevis oocytes

Summary The giant mucous cells in the skin of the terrestrial banana slug Ariolimax columbianus secret intact granules containing mucins. Electron microscopy, after ultrarapid freezing and freeze-substitution in osmium, shows that the secreted granules are bounded by two distinct membranes, presumably derived from the Golgi apparatus and the plasmalemma. Relatively stable, intact granules can be obtained in great quantity in our in vitro system. Rapid lysis of the granules was induced by adenosine triphosphate. At much higher concentrations, adenosine diphosphate and 5-adenylimido-diphosphate also caused lysis. Other nucleotides and related compounds, as well as 1,4,5-inositol triphosphate and molluscan neurotransmitters and neuropeptides, had no effect on the granules. The stability of secreted granules varied with the ionic composition of the isosmotic medium in which they were suspended. When the predominant cation in the medium was potassium, and calcium was also present, granules lysed if exposed to shear stress (stirring of the suspension). This did not occur if sodium was the major cation present. None of the other ions in the suspension media had detectable effects on the stability of the granules.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

Effects of osmotic conditions on ultrastructure of rat mammary epithelium

Summary Effects of osmotic conditions on secretion of milk serum were examined using standard transmission electron microscopy. Rat mammary glands were infused with hyper-, iso-, and hypo-osmotic solutions. The intramammary infusion of these agents elicited distinct and repeatable morphological responses from lactating epithelial cells. The response to hyperosmolarity was an increase in compound exocytotic figures and an increase in secretory vesicle size (¯x=1.65 m in diameter). Glands infused with hypo-osmolar solutions exhibited the opposite response; reduction in compound exocytotic figures and reduced vesicle size (¯x=0.34 n in diameter). The response to iso-osmotic solutions was indistinguishable from untreated control tissue. The ratio of vesicular projections to depressions (vesicle membrane/plasma membrane interactions) could be experimentally altered through the intramammary infusion of solutions with different osmotic potentials. These observations support the suggestion that osmotic conditions may influence exocytosis of milk serum.     

Age-related changes of the somatotrophs of the domestic fowl Gallus gallus

Summary The ultrastructure of the somatotrophs of the caudal pituitary of the domestic fowl was studied quantitatively. Two age groups of male chickens were compared: 4–6 weeks and 24–30 weeks post-hatching. With age, somatotrophs decreased from about 40% to about 30% of the pituitary cell population. Their volume density decreased similarly. Mean volume of a somatotroph was the same in young and adult animals. Because the granule volume density of the somatotrophs was unchanged, but the somatotroph volume density of the gland declined, the granule volume density of the caudal pituitary gland dropped in parallel with that of the somatotrophs. Thus the volume of the gland comprised of somatotroph granules fell about 32%: from 6.57% to 4.45%. This lowered pool of stored hormone may be linked to the lowered circulating levels of growth hormone found in older animals by other investigators.The granule volume density of the somatotrophs was unchanged but the numerical density approximately doubled; thus the mean granule size decreased by 47% with age. The relationship of the size reduction of the granules to the lowered plasma growth hormone levels is not understood at present.Supported in part by Hatch and State funds from the New Jersey State Agricultural Experimental Station and NSF grants PCM 8,0227,27 and PCM 8,302197.     

The carbohydrates of secretory granules and the glycocalyx in developing mucoid cells

Summary Complex carbohydrates in secretory granules and at the apical cell surface of mouse gastric mucoid cells were studied during embryogenesis and in the early postnatal period by various cytochemical methods; the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) and tannic acid-uranyl acetate (TA-UA) procedures made neutral mucosubstances (NMS) visible, whereas the hexose residues of glycoconjugates were identified using WGA-, RCA II- and ConA-ferritin. The glycocalyx was stained with ruthenium red (RR). During differentiation of the embryonic mucoid cells the number of secretory granules increased in parallel to the increase in their carbohydrate component. NMS-stainable parts in secretory granules also had binding sites for the conjugates RCA II- and WGA-ferritin, but the binding of ConA could not be identified. The increasing quantity of NMS in secretory granules was correlated with the increased amount of PA-TCH-SP and TA-UA positive substances in the apical glycocalyx only in 14- and 18-day-old embryos. The observed uniform affinity for RR and lectin conjugates in all analysed developmental stages remains to be explained.     

Special secretory cells in the soybean seed coat

Summary The seed coat of soybean (Glycine max L. Merr.) is of physiological interest for synthesis and transport of amino acids and photosynthates during embryo development. A transmission and scanning electron microscopic study to elucidate the structure of the seed coat disclosed a specialized convex area (antipit) appressed to a concave pit in the center of the abaxial surface of the cotyledon. The antipit, which lies on the inner surface of the seed coat at a medial point in the anterior to posterior direction of the seed, contained specialized secretory cells bounded by loose multi-layered cell walls. These cells were rectangular in the developing seed, varied in length, and contributed directly to the convex morphology of the antipit seen on the ventral surface of the seed coat. At maturity these cells assumed the shape of a cone, extending from the aleurone layer in a perpendicular array. The aleurone and cone cells contained numerous Golgi apparatus, laminated rough endoplasmic reticulum, secretory vesicles, and amyloplasts. Secretory vesicles arose directly from tubules of fenestrated trans cisternae of the Golgi apparatus. Mitochondria were clustered with the amyloplasts; stacks of lamellar cisternae of rough endoplasmic reticulum were associated with the nucleus and Golgi apparatus. The cellular contents, the interconnections by plasmodesmata, and the close physical association with the cotyledon suggested that the aleurone and cone cells may be involved in symplastic transport of nutrients for use by the developing embryo.This paper is dedicated to the memory of my parents, Joseph and Theresa Yaklich, who by their example taught me the value of work and the enjoyment of simple things.     

Colocalization of α-lactalbumin and a major casein in secretory vesicles of rat mammary epithelial cells

Summary Whether both casein and noncasein (serum or whey) proteins of milk are contained within the same secretory vesicles of milk secreting mammary epithelial cells was explored. Antibodies to a major casein and to -lactalbumin of rat milk were localized in thin sections with colloidal gold-conjugated second antibodies. Antibodies to the casein component bound to an antigen present within lumina of Golgi apparatus cisternae and within secretory vesicles. This antigen was also recognized in structures within secretory vesicles and within alveolar lumina which were ultrastructurally identified as casein micelles. Antigens recognized by antibodies to -lactalbumin also were present in Golgi apparatus cisternae and within secretory vesicles. Both anti-casein and anti--lactalbumin antibodies recognized antigens within the same secretory vesicles. These observations show that one major noncasein protein of rat's milk is present in casein-containing secretory vesicles.