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Made available in DSpace on 2020-12-12T02:44:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Suicidal behavior in termite workers is an extreme defensive strategy, probably a consequence of having a low number of soldiers available in the colony and there being high predation from enemies. We investigated the suicidal mechanism in workers of the Neotropical termite Neocapritermes opacus, which involves salivary gland autothysis followed by body cuticle rupture and the release of a defensive secretion. Autothysis was triggered by a physical stimulus such as a soldier bite that causes the protrusion of the salivary acini, burst reservoirs, and foregut. Histochemical and ultrastructural analyses showed salivary acini composed of peripheral parietal cells and two types of central cells, types I and II. Type I cells are filled with large electron-lucent secretory vesicles, which reacted positively to bromophenol blue and xylidine-Ponceau tests, indicating the occurrence of proteins. Type II cells are elongated and display smaller apical secretory vesicles. Parietal cells present an intracellular canaliculus with dense microvilli and cytoplasm rich in mitochondria and large electron-dense vesicles, which may participate in the self-destructive mechanism. Worker suicidal behavior was previously reported for N. taracua and N. braziliensis. N. opacus is a new species in which a salivary weapon has been developed and factors contributing to this altruistic response are discussed. Laboratório de Cupins Departamento de Biologia Instituto de Biociências UNESP - Univ Estadual Paulista, Av. 24A, No. 1515 Laboratório de Cupins Departamento de Biologia Instituto de Biociências UNESP - Univ Estadual Paulista, Av. 24A, No. 1515

The AP-1 complex is essential for membrane protein traffic via its role in the pinching-off and sorting of secretory vesicles (SVs) from the trans-Golgi and/or endosomes. While its essentiality is undisputed in metazoa, its role in simpler eukaryotes seems less clear. Here, we dissect the role of AP-1 in the filamentous fungus Aspergillus nidulans and show that it is absolutely essential for growth due to its role in clathrin-dependent maintenance of polar traffic of specific membrane cargoes toward the apex of growing hyphae. We provide evidence that AP-1 is involved in both anterograde sorting of RabERab11-labeled SVs and RabA/BRab5-dependent endosome recycling. Additionally, AP-1 is shown to be critical for microtubule and septin organization, further rationalizing its essentiality in cells that face the challenge of cytoskeleton-dependent polarized cargo traffic. This work also opens a novel issue on how nonpolar cargoes, such as transporters, are sorted to the eukaryotic plasma membrane.

F-actin has been shown to be essential for tip growth in an array of plant models, including Physcomitrella patens One hypothesis is that diffusion can transport secretory vesicles, while actin plays a regulatory role during secretion. Alternatively, it is possible that actin-based transport is necessary to overcome vesicle transport limitations to sustain secretion. Therefore, a quantitative analysis of diffusion, secretion kinetics, and cell geometry is necessary to clarify the role of actin in polarized growth. Using fluorescence recovery after photobleaching analysis, we first show that secretory vesicles move toward and accumulate at the tip in an actin-dependent manner. We then depolymerized F-actin to decouple vesicle diffusion from actin-mediated transport and measured the diffusion coefficient and concentration of vesicles. Using these values, we constructed a theoretical diffusion-based model for growth, demonstrating that with fast-enough vesicle fusion kinetics, diffusion could support normal cell growth rates. We further refined our model to explore how experimentally extrapolated vesicle fusion kinetics and the size of the secretion zone limit diffusion-based growth. This model predicts that diffusion-mediated growth is dependent on the size of the region of exocytosis at the tip and that diffusion-based growth would be significantly slower than normal cell growth. To further explore the size of the secretion zone, we used a cell wall degradation enzyme cocktail and determined that the secretion zone is smaller than 6 μm in diameter at the tip. Taken together, our results highlight the requirement for active transport in polarized growth and provide important insight into vesicle secretion during tip growth.

Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth.

To study the role of late secretion in Candida albicans pathogenesis, we created conditional mutant C. albicans strains in which the t-SNARE-encoding genes SSO2 or SEC9 were placed under the control of a tetracycline-regulated promoter. In repressing conditions, C. albicans tetR-SSO2 and tetR-SEC9 mutant strains were defective in cytokinesis and secretion of aspartyl proteases and lipases. The mutant strains also exhibited a defect in filamentation compared to controls, and thus we followed the fate of the C. albicans Spitzenkörper, an assembly of secretory vesicles thought to act as a vesicle-supply center for the growing hyphae. In the absence of Ca Sso2p, the Spitzenkörper dissipated within 5 hours and thin-section electron microscopy revealed an accumulation of secretory vesicles. Moreover, the hyphal tip developed into a globular yeast-like structure rather than maintaining a typical narrow hyphae. These studies indicate that late secretory t-SNARE proteins in C. albicans are required for fundamental cellular processes and contribute to virulence-related attributes of C. albicans pathogenesis. Moreover, these results provide direct evidence for a key role of SNARE proteins in vesicle-mediated polarized hyphal growth of C. albicans.

Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH-dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters.

Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis.

Plant cell growth requires an increase in surface area which is achieved by the de novo synthesis and delivery of new cell membrane and cell wall components from the interior of the cell to the cell surface. This delivery is accomplished by secretory vesicles that dock to and ultimately fuse with

Zinc plays essential roles in the early secretory pathway for a number of secretory, membrane-bound, and endosome/lysosome-resident enzymes. It enables the enzymes to fold properly and become functional, by binding as a structural or catalytic component. Moreover, zinc secreted from the secretory vesicles/granules into the extracellular space has a pivotal role as a signaling molecule for various physiological functions. Zinc transporters of the Slc30a/ZnT and Slc39a/Zip families play crucial roles in these functions, mediating zinc influx to and efflux from the lumen of the secretory pathway, constitutively or in a cell-specific manner. This paper reviews current knowledge of the ways these two zinc transporters perform these tasks by manipulating zinc homeostasis in the secretory pathway. Recent questions concerning zinc released into the cytoplasm from the secretory pathway, which then functions as an intracellular signaling molecule, are also briefly reviewed, emphasizing zinc transporter functions.

Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

The posterior esophagus of Bulla striata, running from the gizzard to the stomach, was investigated with light and electron microscopy to obtain new data for a comparative analysis of the digestive system in cephalaspidean opisthobranchs. In this species, the posterior esophagus can be divided into two regions. In the first, the epithelium is formed by columnar cells with apical microvilli embedded in a cuticle. Many epithelial and subepithelial secretory cells are present in this region. In both, electron-lucent secretory vesicles containing filaments and a peripheral round mass of secretory material fill the cytoplasm. These acid mucus-secreting cells may also contain a few dense secretory vesicles. In the second part of the posterior esophagus, the cuticle is absent and the epithelium is ciliated. In this region, epithelial cells may contain larger lipid droplets and glycogen reserves. Subepithelial secretory cells are not present, and in epithelial secretory cells the number of dense vesicles increases, but most secretory cells still contain some electron-lucent vesicles. These cells secrete a mixture of proteins and acid polysaccharides and should be considered seromucous. The secretory cells of the posterior esophagus are significantly different from those previously reported in the anterior esophagus of this herbivorous species.

In differentiating B cells, proliferation of endoplasmic reticulum appears as tightly clustered juxtanuclear spherical structures which expand to fill the cytosol without synchronizing to cell expansion. The differentiation of B cells into Ig-secreting plasma cells requires the expansion of secretory organelles to cope with the increased cargo load. To evaluate the timeline of this process, we have quantitated the kinetics of secretory organelle expansion relative to Ig secretion and examined regulatory components of secretory transport following in vitro activation of human B lymphocytes. Unstimulated B cells contain minimal endomembranes. After activation, ER membrane induction appears as tightly packed spherical structures of 0.5–1 μm diameter concentrated in a juxtanuclear position. When the cells differentiate into plasmablasts, there is dramatic cell-size increase, but the ER remains concentrated close to the nucleus and only later fills the entire cell. In sharp contrast, previous studies in other cell types have found that the ER expands in synchrony with increasing cell size during interphase, by extension of ER tubules under the PM. In this study, the Golgi remains consistently as a single juxtanuclear structure but linearly expands sixfold in volume during B cell activation. Furthermore, following active cell proliferation, ER exit sites proliferate rapidly, increasing almost fourfold in number, in parallel with a sharp increase in Ig secretion. These findings demonstrate that the control of organelle biogenesis and expansion in primary human B cells are differentially regulated by cargo flux caused by Ig synthesis.

This work studied the ultrastructure of the midgut cells of Cimex hemipterus Fabricius (Hemiptera: Cimicidae). The midgut of adult insects was analyzed on different days after a bloodmeal, and three anatomical regions with different digestive functions were apparent. In the anterior midgut, the digestive cells had many spherocrystals, lipid inclusions, and glycogen deposits, suggesting a role in water absorption, ion regulation, digestion, and storage of lipids and sugars. The digestive cells in the middle midgut contained secretory granules in the apical cytoplasm, lysosomes, and large amounts of rough endoplasmic reticulum, suggesting that this midgut region was active in digestive processes. The posterior midgut contained digestive cells with secretory vesicles, lysosomes, rough endoplasmic reticulum, and spherocrystals, suggesting digestion and ion/water absorption. Also, there was strong evidence that the posterior midgut may be the major site of nutrient absorption. The hematophagous heteropteran groups share many of these blood digestion mechanisms.

The human FcαRΙ (CD89) is expressed on cells of myeloid lineage and plays an important role in host defense. Neutrophils make up the majority of FcαRΙ-positive cells. Previous reports suggested that FcαR was stored in neutrophil intracellular pools, and it could be mobilized quickly once neutrophils were activated. However, the subcellular localization of FcαR in neutrophils has not been defined yet. In this sudy, we identified that FcαR was stored in secretory vesicles and tertiary granules of neutrophils by flow cytometry analysis, ELISA, confocal microscopy, and Western blotting. The molecular mass of FcαR in secretory vesicles was different from that in tertiary granules. FcαR stored in tertiary granules had a molecular mass of 50–70 kDa, whereas FcαR in secretory vesicles and membranes had a molecular mass of 55–75 kDa. After treatment by peptide-N-glycosidase F, an enzyme that removes N-glycosylation, FcαR from secretory vesicles and tertiary granules revealed a core protein of 32 kDa, which was the same as the backbone of full length of FcαR. A smaller FcαR variant with a core protein of 29–30 kDa was found in tertiary granules but not in secretory vesicles. The nature of the small variant is not clear at present and remains to be investigated further.

Mature human neutrophils contain small amounts of interleukin-8 [CXC chemokine ligand 8 (CXCL-8)], which upon proinflammatory activation, increases significantly. It has been suggested that the CXCL-8 content of resting human neutrophils is stored in the secretory vesicles. Here, we have used a fractionation technique, which allows isolation of these vesicles, and we find that CXCL-8 neither colocalizes with the secretory vesicles nor with markers of any of the classical neutrophil granules. To increase resolution in the system, we induced CXCL-8 production by lipopolysaccharide. After 8 h of stimulation, CXCL-8 was visualized within the cell using immunoelectron microscopy. The images revealed CXCL-8-containing stuctures resembling neutrophil granules, and these were distinct from all known neutrophil organelles, as shown by double immunostaining. Further, the CXCL-8 organelle was present in nonstimulated neutrophil cytoplasts, entities lacking all other known granules and secretory vesicles. Upon fractionation of the cytoplasts, CXCL-8 was found to partly cofractionate with calnexin, a marker for endoplasmic reticulum (ER). Thus, part of CXCL-8 may be localized to the ER or ER-like structures in the neutrophil.

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. It secretes an extracellular cyst wall using a poorly understood process. An encystation-specific secretory vesicle (ESV) was previously described containing cyst wall proteins. The process of release of these vesicles has been suggested to occur after fragmentation of large ESV in small secretory vesicles, followed by exocytosis, but it was not demonstrated. The release of the ESV was studied by transmission electron microscopy. It was observed: (1) the moment of vesicle release; (2) that a large vesicle is exocytosed and does not fragment into small vesicles; (3) membrane fusion is distinct from traditional exocytosis since it is incomplete; (4) the occurrence of membrane fragmentation and that those membranes reseal to form ghosts; (5) these membrane ghosts may be endocytosed, adhered to flagellar surface or/and form empty vesicles in the extracellular medium.

Vesicular integral protein of 36 kDa (VIP36) is an intracellular lectin recognizing high-mannose type glycans and is highly expressed in salivary glands, especially the parotid gland, which secretes alpha-amylase in large quantities. Here immunoelectron microscopy demonstrated that VIP36 was primarily localized to secretory vesicles in the glandula parotis of the rat, where alpha-amylase also resided. A secretory vesicle fraction, prepared by Percoll density gradient centrifugation, contained both VIP36 and alpha-amylase. Moreover, alpha-amylase that was localized to these secretory vesicles contained high-mannose type glycans. In addition, VIP36 coprecipitated with alpha-amylase in an endo H treatment-sensitive manner. These results suggest that VIP36 is involved in the secretion of alpha-amylase in the rat parotid gland.

The subcellular localization of GPI-80, a novel, adhesion-regulating protein, was investigated in human neutrophils. Surface expression of GPI-80 was determined by FACS analysis as well as by the ability for phospholipase C to cleave the protein from the cell surface. Increasing amounts of GPI-80 were exposed on the cell surface after weak stimulation with the chemoattractant fMLF, suggesting that the protein can be translocated to the plasma membrane from intracellular stores. By subcellular fractionation of the neutrophils, GPI-80 was defined as a component of a light membrane fraction, containing secretory vesicles and plasma membranes, and it was absent from the neutrophil granule fractions. Separation of the plasma membranes from the secretory vesicles by flotation gradient fractionation confirmed that the GPI-80 was localized in the mobilizable secretory vesicles by approximately 50%, and the rest was plasma membrane-bound. Thus, we identify secretory vesicles as the reservoir of GPI-80 from which it may translocate to the plasma membrane after weak stimulation of the cells.

This paper examines morphological and ultrastructural changes in foetal mouse cerebellar neurons treated with a culture medium containing diethylcarbamazine (DEC), a widely used anti-parasitic drug known to interfere with vesicle transfer to and from the Golgi apparatus. The effects of DEC were drastic, with the complete disorganization of the Golgi apparatus and the appearance of large vacuoles within the cytoplasm. Quantitative data comparing normal and DEC-treated neurons in culture demonstrated significant differences between the length of Golgi cisternae and numbers of clear vesicles and secretory vesicles. The evidence therefore suggests that the various synthetic and secretory activities involving these organelles are severely impaired.

The mucilage-secreting desmid, Closterium acerosum, is sensitive to the secretory inhibiting drug, brefeldin A (BFA). After 5 min of treatment with 5 μg ml -1 of BFA, the Golgi body displays the following alterations: the number of cisternae decreases from 12-15 to 6-7; peripheries of cisternae from the same Golgi body often fuse to yield unique profiles; secretory vesicles still emerge from the Golgi body; the cisternal stack dissociates to form irregular masses in the valleys of cytoplasm created by the lobes of the chloroplast. Fluoresbrite bead labelling shows that mucilage production ceases in cells treated with BFA even after only 5 min of treatment. Immunogold labelling using anti-mucilage antibody reveals that mucilage production still occurs in the Golgi body and associated vesicles. Helix pomatia lectin-gold labelling shows that wall synthesis still occurs in BFA-treated Golgi bodies and wall precursors accumulate in the perforate cisternal/vesicular masses seen in the TGN region of the Golgi stack.

Mammalian sperm contain a single large secretory vesicle, or acrosome. Exocytosis of this vesicle (the acrosome reaction) occurs as an early step in the gamete interaction process and is an essential prerequisite for late events, including sperm-egg fusion [1]. The control of fertilization is, from this perspective, a question of the regulation of a secretory event. It may be useful to compare sperm to other secretory systems. Typical somatic secretory cells such as mast cells, the neuronal presynaptic terminal, and exocrine cells contain large numbers of secretory vesicles whereas sperm possess a single acrosome. Those familiar somatic secretory models tolerate a low level of basal vesicle release that is augmented upon the arrival of an external stimulus. For example, miniature synaptic potentials are due to the stochastic release of individual vesicles, and stimuli, in the form of membrane depolarization, increase the number of released vesicles [2, 3]. In contrast, it can be argued that spontaneous exocytosis must be strictly controlled in sperm and may require a specialized regulatory mechanism. Key elements in this argument include the observations that 1) sperm that complete the acrosome reaction at a distance from eggs have decreased capacity to penetrate through the cumulus oophorus [4] and to adhere to the egg’s extracellular matrix, or zona pellucida [5, 6], and 2) sperm that fail to initiate acrosome reactions cannot penetrate the zona pellucida and hence are denied access to the egg plasma membrane [1]. Sperm consequently face an ‘‘acrosome reaction problem,’’ in which secretion must be coordinated with egg contact and yet spontaneous secretory rates must be suppressed. It is now understood that the initiation of acrosome reactions during egg contact is accomplished by the presence in the zona pellucida of an acrosome reaction-inducing agonist, the glycoprotein ZP3 [7]. However, individual sperm must still prevent spontaneous secretion until the time of sperm-egg contact. One common theme in the regulation of exocytotic processes is the general role of intracellular Ca21 ([Ca]i) as a mediator of stimulus-secretion coupling. In both sperm and somatic cells, elevations of [Ca]i are necessary and

The baboon oviductal epithelium differentiates into a tall columnar epithelium consisting of ciliated and secretory cells during the follicular phase of the menstrual cycle in response to rising oestradiol levels. The apical tips of these secretory cells are filled with membrane-bound secretory granules. During the luteal phase when progesterone levels are elevated, the epithelium regresses and deciliation occurs. Analysis of secretory proteins obtained from explant culture media by SDS-PAGE followed by fluorography or Western blots has revealed that the baboon oviduct synthesizes and secretes a high molecular weight glycoprotein during the follicular phase of the cycle. Immunocytochemistry demonstrated that this oviductal glycoprotein is localized to the secretory granules of epithelial secretory cells, is oviduct specific, and that following secretion the oviductal glycoprotein binds to the zona pellucida and perivitelline space of ovulated oocytes and embryos within the oviduct. Similar proteins have been characterized in other mammalian species. cDNA data show that the complete coding sequence is 2228 bp for a protein of 623 amino acids. A Genbank search showed that baboon oviductal glycoprotein has high homology to other oviductal glycoprotein sequences at both the nucleotide and amino acid levels. Studies conducted to date probing the biological function of oviductal glycoprotein indicate that this protein plays a role in prefertilization reproductive events (sperm capacitation; sperm-zona binding; zona penetration). Additional experiments are needed to reveal a specific function and mechanism for this molecule.

One of the basic cellular functions of nearly every cell type is the exocytotic release of synthesized molecules, stored and packaged into intracellular vesicles or granules. A variety of approaches has been used to identify and characterize the molecules that mediate vesicular trafficking along the secretory pathway. The findings obtained with these approaches suggest that common mechanisms may underlie a wide variety of vesicle-mediated transport steps. This review presents some of the recent findings regarding the study of the cellular mechanisms which control neurotransmitter and hormone release from neurons and endocrine cells respectively, and focuses on regulation of these mechanisms. The similarities between these two cell types can be seen as evidence to support the hypothesis according to which the regulated exocytosis apparatus could have evolved from a constitutive fusion machinery to which some key modulators have been added. Insight into secretory vesicles will be relevant not only to the understanding of vesicular trafficking or cell polarity but also to the understanding of higher nervous functions resulting from synaptic plasticity.

Epididymosomes are small membrane vesicles that are secreted by epididymal epithelial cells and are involved in posttesticular sperm maturation. Although their role in protein transfer to the sperm membrane is well documented, we report their capacity to transport microRNAs (miRNAs), which are potent regulators of posttranscriptional gene expression. Using a microperfusion technique combined with a global microarray approach, we demonstrated that epididymosomes from two discrete bovine epididymal regions (caput and cauda) possess distinct miRNA signatures. In addition, we also established that miRNA repertoires contained within epididymosomes differ from those of their parent epithelial cells, suggesting that miRNA populations released from the cells may be selectively sorted. Binding of DilC12-labeled epididymosomes to primary cultured epididymal cells was measured by flow cytometry, and the results indicated that epididymosomes from the median caput and their miRNA content may be incorporated into distal caput epithelial cells. Overall, these findings reveal that distinct miRNA repertoires are released into the intraluminal fluid in a region-specific manner and could be involved in a novel mechanism of intercellular communication throughout the epididymis via epididymosomes.

TMEM230 is a newly identified Parkinson's disease (PD) gene encoding a transmembrane protein whose cellular and pathogenic roles remain largely unknown. Here, we demonstrate that loss of TMEM230 disrupts retromer cargo CI-M6PR (cation-independent mannose 6-phosphate receptor) trafficking and autophagic cargo degradation rates. TMEM230 depletion further inhibits extracellular secretion of the autophagic cargo p62 and immature lysosomal hydrolases in Golgi-derived vesicles leading to their intracellular accumulation, and is specifically mediated by loss of the small GTPase Rab8a. Importantly, PD-linked TMEM230 variants also induce retromer mislocalization, defective cargo trafficking, and impaired autophagy. Finally, we show that knockdown of another PD gene, LRRK2, which phosphorylates Rab8a, similarly impairs retromer trafficking, secretory autophagy and Golgi-derived vesicle secretion, thus demonstrating converging roles of two PD genes TMEM230 and LRRK2 on Rab8a function, and suggesting that retromer and secretory dysfunction play an important role in PD pathogenesis.

The Fallopian tube has been reported to undergo cyclical changes. However, many studies of tubal ultrastructure have either examined one segment of the tube only or studied animal oviducts. The aim of this study was to document in detail the combined morphological and ultrastructural features of the epithelial lining along the length of the tube in women at different stages of the menstrual cycle. We report an increase in the proportion of ciliated cells along the tube, being highest in the fimbriae, but no substantial difference between the follicular and luteal phases of the menstrual cycle. In the late follicular phase, fragments of cytoplasmic and cellular material were seen in the isthmic lumen but not in the outer tubal segments. Similarly, surface domes and secretory granules were more prominent in the mid-tube and ampullary sections than in the fimbriae. This surface activity was followed by relative quiescence in the early/mid luteal phase with reversion to a more active surface but with little secretory activity in the late luteal phase. These findings along the Fallopian tube substantiate the concept of functional differentiation between the different segments and necessitate further studies to determine its clinical relevance.

The fine structure of epidermal cells, particularly in relation to dictyosomes, has been examined in different regions of dark-grown cucumber hypocotyls and in response to auxin treatment, using both dot overlay and image analysis techniques. The most noticeable change in cell structure along the hypocotyls is the increase in vacuolar volume. The volume fraction occupied by dictyosomes and secretory vesicles also increased, whereas that for mitochondria remained relatively constant. During auxin treatment, the volume fraction for dictyosomes showed an increase after 30 min followed by a fall, whereas that occupied by secretory vesicles fell steadily over 90 min. The number of cisternae per dictyosome showed some increase after 2 h of auxin treatment, although the increase in dictyosomal material with cell expansion was largely accounted for by an increase in the number of dictyosomes. Auxin-stimulated elongation growth of the hypocotyls was inhibited by a range of calcium antagonists, chelators and ionophores. The most marked inhibitions were observed with calcium chloride, the chelator chlortetracycline and the ionophores verapamil, nigericin and monensin. Linear transducer experiments showed that these compounds generally caused an immediate reduction in the rate of growth. Fine structural observations carried out on epidermal cells showed the most obvious effects with monensin and nigericin which caused dictyosomes and secretory vesicles to swell. EGTA and LaCl 3 caused secretory vesicles to accumulate around dictyosomes, while the ionophore A23187 had little effect. The results suggest that the concentration of Ca 2+ in the cytoplasm may be critical for cell elongation. Compounds which chelate Ca 2+ appear to be more effective inhibitors of growth in the initial acid-induced phase, whereas those which affect ionic gradients are more disruptive in the second phase.

To elucidate the intracellular localization of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human cardiac myocytes, an immunocytochemical study was carried out by a double immunogold technique using antisera highly specific for ANP and BNP. Surgical and autoptic tissue specimens of human heart were studied. In the atrial myocytes, ANP was localized in almost all of the secretory granules, whereas BNP was colocalized with ANP in some of the granules. Although very few secretory granules were observed in ventricular myocytes, colocalization of ANP and BNP was basically the same as in atrial myocytes. No immunoreactive products were found in the control studies. These results suggest that secretion of BNP is under a regulatory mechanism similar to that of ANP.

Kallikrein-positive cells in the anterior pituitary of female rats were identified to be the same as prolactin-producing cells by using an immunoelectron microscopic method. The kallikrein immunoreactivity was localized at the Golgi apparatus, the rough endoplasmic reticulum, and secretory granules, suggesting that kallikrein is synthesized in the prolactin-producing cells and also may be secreted into the blood vessels.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Copper (Cu) is an essential biometal involved in a number of cell functions. Abnormal Cu homeostasis has been identified as a major factor in a number of neurodegenerative disorders. However, little is known about how cells of brain origin maintain Cu homeostasis and in particular, how they respond to an elevated Cu environment. Understanding these processes is essential to obtaining a greater insight into the pathological changes in neurodegeneration and ageing. Although previous studies have shown that Cu in neurons can be associated with synaptic function, there is little understanding of how Cu modulates the regulated secretory vesicle pathways in these cells. In this study, we examined the effect of elevated intracellular Cu on proteins associated with the regulated secretory vesicle pathway in NGF-differentiated PC12 cells that exhibit neuronal-like properties. Increasing intracellular Cu with a cell-permeable Cu-complex (Cu(II)(gtsm)) resulted in increased expression of synaptophysin and robust translocation of this and additional vesicular proteins from synaptic-like microvesicle (SLMV) fractions to chromogranin-containing putative large dense core vesicle (LDCV) fractions in density gradient preparations. The LDCV fractions also contained substantially elevated Cu levels upon treatment of cells with Cu(II)(gtsm). Expression of the H(+) pump, V-ATPase, which is essential for vesicle maturation, was increased in Cu-treated cells while inhibition of V-ATPase prevented translocation of synaptophysin to LDCV fractions. Cu treatment was found to inhibit release of LDCVs in chromaffin cells due to reduced Ca(2+)-mediated vesicle exocytosis. Our findings demonstrate that elevated Cu can modulate LDCV metabolism potentially resulting in sequestration of Cu in this vesicle pool.

An undifferentiated small cell tumor of the colon that was found by light- and electron-microscopic study to possess features of a malignant carcinoid merged into keratinizing squamous cell carcinoma. Single cells contained both dense-core secretory granules and tonofilaments. The implications of this combination in relation to theories of tumor origin and differentiation are briefly discussed.

Maize (Zea mays) seedlings were grown under standard conditions and the time for reformation of a slime droplet of standard size was determined. The structure of the secretory cells was examined by light and electron microscopy. Osmium-zinc iodide (OZI) impregnation was used to provide contrast enhancement of the dictyosome forming-face cisternae and the endoplasmic reticulum (ER). The three-dimensional structure and relationships of these membrane systems were established from examination of 300 nm thick sections. The presence of a marginal cisternal network at the forming-face was confirmed, but no specific orientation of the ER to this face, or connection of ER to any part of the dictyosome could be demonstrated. Neither observation of reforming slime droplets, nor quantification of secretory vesicle numbers gave any support to the suggestion that slime formation is a phasic, or cyclic, process. Dilute solutions of solvents (1% ethanol, 1% and 01% DMSO) commonly used to solubilize potential inhibitors of Golgi activity were found to delay significantly the time taken for reformation of a slime droplet of standard size. These solutions also had slight effects on the numerical density of secretory vesicles. Cytochalasin D-induced accumulation of secretory vesicles was used to determine rates of vesicle formation (0-39 dictyosome-1 min-1) and hence turnover times for cisternal (6 5 min) and total dictyosome (26-39 min) membranes and the plasma membrane (9-7 min). The volume contribution of the secretory vesicles to the slime droplet was shown to be exceedingly small (0-1 %), throwing doubt on the value of using droplet reformation rates as an indicator of secretory activity.