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.     

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.     

Restriction of secretory granule motion near the plasma membrane of chromaffin cells

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.     

Differentiation of mucilage secretory cells of the Arabidopsis seed coat

In some plant species, including Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type with a unique morphology and containing large quantities of polysaccharide mucilage (pectin). Such seed coat mucilage cells are necessary for neither viability nor germination under normal laboratory conditions. Thus, the Arabidopsis seed coat offers a unique system with which to use genetics to identify genes controlling cell morphogenesis and complex polysaccharide biosynthesis and secretion. As a first step in the application of this system, we have used microscopy to investigate the structure and differentiation of Arabidopsis seed coat mucilage cells, including cell morphogenesis and the synthesis, secretion, and extrusion of mucilage. During seed coat development in Arabidopsis, the epidermal cells of the outer ovule integument grow and differentiate into cells that produce large quantities of mucilage between the primary cell wall and plasma membrane. Concurrent with mucilage production, the cytoplasm is shaped into a column in the center of the cell. Following mucilage secretion the cytoplasmic column is surrounded by a secondary cell wall to form a structure known as the columella. Thus, differentiation of the seed coat mucilage cells involves a highly regulated series of events including growth, morphogenesis, mucilage biosynthesis and secretion, and secondary cell wall synthesis.     

The demonstration of potential-dependent potassium channels in the plasma membrane of secretory cells

Outward current of the salivary gland cells membrane of chironomus larva activated by the displacement of the membrane potential to the region of positive values has been registered by the voltage-clamp method under conditions of intracellular dialysis in the presence of only the potassium transmembrane gradient. Activation threshold of the current is about +10 mV. Subsequent displacement of the membrane potential to the region of positive values causes an increase of the current. Time constant of the current activation is (652 +/- 57) ms. The current decreases with the intracellular potassium concentration, under the influence of tetraethyl-ammonium and 4-aminopyridine. Thus, high threshold potential-dependent potassium channels are presented in the secretory cells membrane.     

Unconventional secretory routes: direct protein export across the plasma membrane of mammalian cells

The vast majority of extracellular proteins are exported from mammalian cells by the endoplasmic reticulum/Golgi-dependent secretory pathway. For poorly understood reasons, however, a heterogenous group of extracellular proteins has been discovered that does not make use of signal peptide-dependent secretory transport. Both the release mechanisms and the molecular identity of the secretory machines involved have remained elusive. Recent studies now have established a subgroup of unconventional secretory proteins capable of translocating from the cytoplasm directly across the plasma membrane to get access to the exterior of eukaryotic cells. This review aims to focus on a detailed comparison of the subcellular site of membrane translocation of various unconventional secretory proteins such as the proangiogenic molecule fibroblast growth factor-2 (FGF-2) and Leishmania hydrophilic acylated surface protein B (HASP B). A potential link between membrane translocation and quality control as an integral part of unconventional secretory processes is discussed.     

Cortical microtubules mark the mucilage secretion domain of the plasma membrane in Arabidopsis seed coat cells

During their differentiation Arabidopsis thaliana seed coat cells undergo a brief but intense period of secretory activity that leads to dramatic morphological changes. Pectic mucilage is secreted to one domain of the plasma membrane and accumulates under the primary cell wall in a ring-shaped moat around an anticlinal cytoplasmic column. Using cryofixation/transmission electron microscopy and immunofluorescence, the cytoskeletal architecture of seed coat cells was explored, with emphasis on its organization, function and the large amount of pectin secretion at 7 days post-anthesis. The specific domain of the plasma membrane where mucilage secretion is targeted was lined by abundant cortical microtubules while the rest of the cortical cytoplasm contained few microtubules. Actin microfilaments, in contrast, were evenly distributed around the cell. Disruption of the microtubules in the temperature-sensitive mor1-1 mutant affected the eventual release of mucilage from mature seeds but did not appear to alter the targeted secretion of vesicles to the mucilage pocket, the shape of seed coat cells or their secondary cell wall deposition. The concentration of cortical microtubules at the site of high vesicle secretion in the seed coat may utilize the same mechanisms required for the formation of preprophase bands or the bands of microtubules associated with spiral secondary cell wall thickening during protoxylem development.     

Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells

Cdc42 is a Rho-family GTPase that in yeast is important in establishing polarized bud growth. Here we show that Cdc42 is also essential in establishing and maintaining polarity in epithelial cells. Functional deletion of Cdc42 in Madin-Darby canine kidney (MDCK) cells results in the selective depolarization of basolateral membrane proteins; the polarity of apical proteins remains unaffected. This phenotype does not reflect major alterations in the actin cytoskeleton, but rather results from the selective inhibition of membrane traffic to the basolateral plasma membrane in both the endocytic and the secretory pathways. Thus, Cdc42 plays a critical part in epithelial-cell polarity, by, unexpectedly, regulating the fidelity of membrane transport.     

Digestive enzymes associated with the glycocalyx,microvillar membranes and secretory vesicles from midgut cells of Tenebrio molitor larvae

Acetylglucosaminidase, amylase, cellobiase and maltase are more active in anterior midgut cells, whereas aminopeptidase, carboxypeptidase and trypsin are more active in posterior midgut cells of Tenebrio molitor larvae. Differential centrifugation of midgut homogenates prepared in saline (or mannitol) isotonic buffered solutions revealed that aminopeptidase is associated with membranes, which occur in subcellular fractions displaying many microvilli. Carboxypeptidase, trypsin and the carbohydrases are mostly found in the soluble fraction, although significant amounts sediment together with cell vesicles. Data on differential calcium precipitation of midgut homogenates and on partial ultrasound disruption of midgut tissue suggest that aminopeptidase is a microvillar enzyme and that the digestive enzymes recovered in the soluble fraction of cells are loosely bound to the cell glycocalyx. About 5% of the non-absorbable dye amaranth fed to T. molitor larvae remains in the midgut tissue after rinsing. Most dye was recovered in the soluble fraction of midgut cells. This provided further support for the hypothesis that the digestive enzymes found in the soluble fraction are actually extracellular and that the true intracellular enzymes are those associated with cell vesicles. The results suggest that the carbohydrases are secreted by exocytosis from the anterior midgut and carboxypeptidase and trypsin from the posterior midgut.     

Protein kinase C-dependent supply of secretory granules to the plasma membrane

To elucidate the mechanism for supplying secretory granules to the cell membrane, chromaffin cells isolated from the bovine adrenal medulla were observed by the evanescent wave microscopy after staining their granules with acridine orange. The secretory granules showed only a very small fluctuation, indicating their docking to the plasma membrane. The rate and range of movement increased greatly by application of botulinum toxin A or C. The number of secretory granules docked to the plasma membrane significantly decreased by botulinum toxin C. Conversely, the number increased greatly by activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu). In the presence of an anti-actin reagent cytochalasin D, no increasing effect of PDBu on the number of docked granules was observed. While in the presence of an anti-mitotic reagent, colchicine, a clear increasing effect of PDBu was observed. The final step for supplying granules to the plasma membrane in endocrine cells is concluded to be mediated by a phosphorylation-dependent and actin-based transport system.     

Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway.

Immunoelectron microscopy of Saccharomyces cerevisiae cells embedded in Lowicryl K4M has been used to localize invertase and plasma membrane (PM) ATPase in secretory organelles. sec mutant cells incubated at 37 degrees C were prepared for electron microscopy, and thin sections were incubated with polyclonal antibodies, followed by decoration with protein A-gold. Specific labeling of invertase was seen in the lumen of the endoplasmic reticulum, Golgi apparatus, and secretory vesicles in mutant cells that exaggerate these organelles. PM ATPase accumulated within the same organelles. Double-immune labeling revealed that invertase and PM ATPase colocalized in secretory vesicles. These results strengthen the view that secretion and plasma membrane assembly are biosynthetically coupled in yeast.     

Defective plasma membrane assembly in yeast secretory mutants.

Yeast mutants that are conditionally blocked at distinctive steps in secretion and export of cell surface proteins have been used to monitor assembly of integral plasma membrane proteins. Mutants blocked in transport from the endoplasmic reticulum (sec18), from the Golgi body (sec7 and sec14), and in transport of secretory vesicles (sec1) show dramatically reduced assembly of galactose and arginine permease activities. Simultaneous induction of galactose permease and alpha-galactosidase (a secreted glycoprotein) in sec mutant cells at the nonpermissive temperature (37 degrees C) shows that both activities accumulate and can be exported coordinately when cells are returned to the permissive temperature (24 degrees C) in the presence or absence of cycloheximide. Plasma membrane fractions isolated from sec mutant cells radiolabeled at 37 degrees C have been analyzed by two-dimensional sodium dodecyl sulfate-gel electrophoresis. Although most of the major protein species seen in plasma membranes from wild-type cells are not efficiently localized in sec18 or sec7, several of these proteins appear in plasma membranes from sec1 cells. These results may be explained by contamination of plasma membrane fractions with precursor vesicles that accumulate in sec1 cells. Alternatively, some proteins may branch off during transport along the secretory pathway and be inserted into the plasma membrane by a different mechanism.     

Localization of plasma membrane and secretory calcium pumps in the mammary gland

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.     

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat

In the initial stage of ascidian fertilization sequential sperm–egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66kDa, 120kDa and 140kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata .     

Studies on the surface coat (glycocalyx) of the dauer larva of Anguina agrostis

Imprints of the surface coat (glycocalyx) from the cuticles of living second stage dauer larvae (DL2) of Anguina agrostis (syn. A. funesta) have been examined using incident light fluorescence microscopy and scanning electron microscopy. These surface coats contain residues of N-acetyl-D-glucosamine which were detected by treatment with wheat germ agglutinin labelled with either fluorescein or rhodamine. They also contain protein which was demonstrated by treatment with either pepsin or trypsin. These enzymes inhibited the attachment of the coryneform bacterium Clavibacter sp. to the surface coat, indicating that proteins play a crucial role in the adhesion of these bacteria to the nematode. This inhibition of attachment was reversed within 18 h after removal of the DL2 from the enzymes, indicating that the nematode was capable of renewing its surface proteins.     

RAGE recycles at the plasma membrane in S100B secretory vesicles and promotes Schwann cells morphological changes

RAGE is a multiligand receptor of the immunoglobulin superfamily involved in regeneration of injured peripheral nerve and cell motility. RAGE is implicated in the development of various chronic diseases, such as neurodegenerative disorders, inflammatory responses, and diabetic complications. The correlation between RAGE endocytic trafficking and RAGE function is still uninvestigated. S100B is one of the ligands of RAGE. The molecular mechanisms responsible of S100B translocation in exocytic vesicles are still poorly investigated. In the present study we elucidate the role of RAGE endocytic trafficking in promoting S100B secretion in Schwann cells. Here we show that RAGE-induced secretion of S100B requires phosphorylated caveolin1-dependent endocytosis of RAGE. Endocytosis of RAGE in response to ligand binding promotes the fusion of endosomes with S100B-positive secretory vesicles. Src promotes the fusion of endosomes with S100B-secretory vesicles. Inhibition of src induces RAGE degradation. RAGE-mediated src activation induces cav1 phosphorylation and relocalization in the perinuclear compartment. RAGE signaling and recycling are required for S100-induced Schwann cells morphological changes and are inhibited by high-glucose, suggesting a possible link between diabetes and peripheral nerve injury. Indeed, high glucose inhibits RAGE-mediated src activation. Src inhibition blocks RAGE recycling, S100B secretion, and morphological changes. In summary, we identified a novel pathway of vesicular trafficking required for the amplification of RAGE signaling and cytoskeleton dynamics that is potentially involved in the regeneration of injured peripheral nerve.     

Biosynthesis of the glycoproteins present in plasma membrane, lysosomes and secretory materials, as visualized by radioautography

Synopsis The three major types of glycoproteins present in animal cells, that is, the secretory, lysosomal and plasma membrane glycoproteins, were examined with regard to the sites of synthesis of their carbohydrate side chains and to their subsequent migration within cells.The site at which a monosaccharide is added to a growing glycoprotein depends on the position of that monosaccharide in the carbohydrate side-chain. Thus, radiauutography of thyroid cells within minutes of the intravenous injection of labelled mannose, a sugar located near the base of the larger side-chains, reveals that it is incorporated in rough endoplasmic reticulum, whereas the more distally located galactose and fucose are incorporated in the Golgi apparatus. Recently [³H]N-acetylmannosamine, a specific precursor for the terminally located sialic acid residues, was shown to be also added in the Golgi apparatus. Presumably synthesis of glycoproteins is completed in this organelle.Radioautographs of animals sacrificed a few hours after injection of [³H]N-acetylmannosamine show that, in many secretory cells, labelled glycoproteins pass into secretory products. In these cells, as well as in non-secretory cells, the label may also appear within lysosomes and at the cell surface. In the latter site, it is presumably included within the plasma membrane glycoproteins whose carbohydrate side-chains form the cell coat. The continual migration of glycoproteins from Golgi apparatus to cell surface implies turnover of plasma membrane glycoproteins. Radioautographic quantitation of [³H]fucose label at the surface of proximal tubule cells in the kidney of singly-injected adult mice have shown that, after an initial peak, cell surface labelling decreases at a rate indicating a half-life of plasma membrane glycoproteins of about three days.