Roles of Myosin Va and Rab3D in Membrane Remodeling of Immature Secretory Granules

Neuroendocrine secretory granules (SGs) are formed at the trans-Golgi network (TGN) as immature intermediates. In PC12 cells, these immature SGs (ISGs) are transported within seconds to the cell cortex, where they move along actin filaments and complete maturation. This maturation process comprises acidification-dependent processing of cargo proteins, condensation of the SG matrix, and removal of membrane and proteins not destined to mature SGs (MSGs) into ISG-derived vesicles (IDVs). We investigated the roles of myosin Va and Rab3 isoforms in the maturation of ISGs in neuroendocrine PC12 cells. The expression of dominant-negative mutants of myosin Va or Rab3D blocked the removal of the endoprotease furin from ISGs. Furthermore, expression of mutant Rab3D, but not of mutant myosin Va, impaired cargo processing of SGs. In conclusion, our data suggest an implication of myosin Va and Rab3D in the maturation of SGs where they participate in overlapping but not identical tasks.     

Adriamycin Binds to the Matrix of Secretory Granules during Mast Cell Exocytosis

The antineoplastic drug adriamycin induces exocytosis in rat peritoneal mast cells followed by a significant uptake of the drug into the secretory granules. The drug is fluorescent, allowing visualization of its accumulation and binding to mast cell granules by fluorescence microscopy. At the same time, the well known inorganic dye ruthenium red was used as a probe because of its great affinity for heparin in the mast cell secretory granules as visualized by bright field microscopy. Competition between adriamycin and ruthenium red for binding to the negatively charged matrix of granules was demonstrated. Biochemical studies were also performed to confirm microscopic observations. Adriamycin may be of interest for studying mast cell secretion; it is not only a strong fluorescent dye for mast cell granules that are in communication with the extracellular space, but it also induces mast cell exocytosis.     

Antibacterial Peptides Are Present in Chromaffin Cell Secretory Granules

1. Antibacterial activity has recently been associated with the soluble matrix of bovine chromaffin granules. Furthermore, this activity was detected in the contents secreted from cultured chromaffin cells following stimulation.2. The agents responsible for the inhibition of Gram+ and Gram– bacteria growth are granular peptides acting in the micromolar range or below. In secretory granules, these peptides are generated from cleavage of chromogranins and proenkephalin A and are released together with catecholamines into the circulation.3. Secretolytin and enkelytin are the best characterized; these two peptides share sequence homology and similar antibacterial activity with insect cecropins and intestinal diazepam-binding inhibitor. For some of the peptides derived from chromogranin A, posttranslational modifications were essential since antibacterial activity was expressed only when peptides were phosphorylated and/or glycosylated.4. The significance of this activity is not yet understood. It may be reminiscent of some primitive defense mechanism or may serve as a first barrier to bacteria infection during stress, as these peptides are secreted along with catecholamines.     

PC12活细胞中单个分泌囊泡的动态成像

囊泡的荧光标记和动态显微成像观察是研究蛋白质和膜转运机制的重要手段。采用EGFP hpNPY融合荧光蛋白标记PC12细胞的致密大囊泡 ,用全内反射和宽场荧光显微镜对PC12细胞进行成像研究。结果发现 :普通的宽场荧光成像模糊不清 ,难以观察到单个囊泡 ;而全内反射荧光成像则可清晰地分辨出呈现为离散荧光点的单个囊泡 ;并且进一步利用全内反射荧光成像直接观察到了活的PC12细胞中单个囊泡的转运、锚定及与细胞膜的融合过程 ,证实了囊泡的锚定过程是可逆的。     

Molecular mechanism of myosin Va recruitment to dense core secretory granules

The brain-spliced isoform of Myosin Va (BR-MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide-producing cells. The molecular composition of the protein complex that recruits BR-MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG-associated proteins granuphilin-a/b (Gran-a/b), BR-MyoVa and Rab27a, a member of the Rab family of GTPases. Gran-a/b-BR-MyoVa interaction is direct, involves regions downstream of the Rab27-binding domain, and the C-terminal part of Gran-a determines exon specificity. MyoVa and Gran-a/b are partially colocalised on SGs and disruption of Gran-a/b-BR-MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient-stimulated hormone secretion in pancreatic beta-cells. These results indicate the existence of at least another binding partner of BR-MyoVa that was identified as rabphilin-3A (Rph-3A). BR-MyoVa-Rph-3A interaction is also direct and enhanced when secretion is activated. The BR-MyoVa-Rph-3A and BR-MyoVa-Gran-a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR-MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.     

Expression of the Dominant-Negative Tail of Myosin Va Enhances Exocytosis of Large Dense Core Vesicles in Neurons

Regulated exocytosis of secretory vesicles is a fundamental process in neurotransmission and the release of hormones and growth factors. The F-actin-binding motor protein myosin Va was recently shown to be involved in exocytosis of peptide-containing large dense core vesicles of neuroendocrine cells. It has not previously been discussed whether it plays a similar role in neurons. We performed live-cell imaging of cultured hippocampal neurons to measure the exocytosis of large dense core vesicles containing fluorescently labelled neuropeptide Y. To address the role of myosin Va in this process, neurons were transfected with the dominant-negative tail domain of myosin Va (myosinVa-tail). Under control conditions, about 0.75% of the labelled large dense core vesicles underwent exocytosis during 5 min of stimulation. This value was doubled to 1.80% of the vesicles when myosinVa-tail was expressed. Depolymerization of F-actin using latrunculin B resulted in a similar increase in exocytosis in both control and myosinVa-tail expressing cells. Interestingly, the increase in exocytosis caused by myosinVa-tail expression was completely abolished in the presence of KN-62, an inhibitor of calcium–calmodulin-dependent kinase II. We suggest that myosinVa-tail causes the liberation of large dense core vesicles from the actin cytoskeleton, leading to an increase in exocytosis in the cultured hippocampal neurons. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Light-induced exocytosis in cell development and differentiation

Calcium-dependent exocytosis of fluorescently labeled single secretory vesicles in PC12 cells and primary embryonic telencephalon cells can be triggered by illumination with visible light and imaged by TIRF or epifluorescence microscopy. Opsin 3 was identified by quantitative PCR expression analysis as the putative light receptor molecule for light-induced exocytosis. In primary chicken telencephalon cells, light-induced exocytosis is restricted to a specific period during embryonic development, and involves fusion of rather large vesicles. Strictly calcium-dependent exocytosis starts after a delay of a few seconds of illumination and lasts for up to 2 min. We analyzed the frequency, time course and spatial distribution of exocytotic events. Exocytosis in PC12 cells and telencephalon cells occurs at the periphery or the interface between dividing cells, and the duration of single secretion events varies considerably. Our observation strongly supports the idea that light induced exocytosis is most likely a mechanism for building plasma membrane during differentiation, development and proliferation rather than for calcium-dependent neurotransmitter release.     

Myosin Va movements in normal and dilute-lethal axons provide support for a dual filament motor complex.

To investigate the role that myosin Va plays in axonal transport of organelles, myosin Va-associated organelle movements were monitored in living neurons using microinjected fluorescently labeled antibodies to myosin Va or expression of a green fluorescent protein-myosin Va tail construct. Myosin Va-associated organelles made rapid bi-directional movements in both normal and dilute-lethal (myosin Va null) neurites. In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement. In contrast, depolymerization of microtubules in dilute-lethal neurons stopped movement. Myosin Va or synaptic vesicle protein 2 (SV2), which partially colocalizes with myosin Va on organelles, did not accumulate in dilute-lethal neuronal cell bodies because of an anterograde bias associated with organelle transport. However, SV2 showed peripheral accumulations in axon regions of dilute-lethal neurons rich in tyrosinated tubulin. This suggests that myosin Va-associated organelles become stranded in regions rich in dynamic microtubule endings. Consistent with these observations, presynaptic terminals of cerebellar granule cells in dilute-lethal mice showed increased cross-sectional area, and had greater numbers of both synaptic and larger SV2 positive vesicles. Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules. This is consistent with both actin- and microtubule-based motors being present on these organelles. Although myosin V activity is not necessary for long-range transport in axons, myosin Va activity is necessary for local movement or processing of organelles in regions, such as presynaptic terminals that lack microtubules.     

Morphological Analysis of an Isolated Fraction Containing Secretory Granules from the Submandibular Gland of Trypanosoma cruzi Infected and Normal Rats

Normal and Trypanosoma cruzi infected submandibular glands from 42 male Holtzman rats were homogenized in 340 mM sucrose and 0.5 mM EDTA in 10 mM HEPES buffer at pH 7.4. The extract was centrifuged and filtered to prepare a granule rich fraction. Isolated secretory granules are similar morphologically to granules observed in whole submandibular gland preparations. Our results suggest that our isolation technic can be used for morphological and biochemical studies.     

Membrane Dipeptidase and Glutathione Are Major Components of Pig Pancreatic Zymogen Granules

Membrane proteins of highly purified porcine zymogen granules were separated by two-dimensional gel electrophoresis in order to isolate proteins which are involved in intracellular trafficking of digestive enzymes in the exocrine pancreas. A 48-kDa glycoprotein was a major component in membrane preparations washed with 0.1 M Na₂CO₃and 0.5 M NaCl. By N-terminal amino acid sequencing this protein was identified as membrane dipeptidase (MDP; EC 3.4.13.19). MDP mRNA levels in rat pancreas were increased threefold by feeding rats with FOY-305, which is a known stimulus of endogenous cholecystokinin release from the gut. Cholecystokinin then stimulates secretion in pancreatic acinar cells. In another set of experiments treatment of the rat pancreatic acinar tumor cell line AR42J with dexamethasone led to an eightfold increase in the expression of MDP. Thus, the expression pattern of the MDP gene in response to hormonal stimulationin vivoandin vitroresembles those found for most of the enzymes and proteins which are involved in secretion. Since MDP has been thought to have a role in glutathione (GSH) metabolism, we also measured GSH concentration in zymogen granules and found high levels of GSH. Based on our data we propose a working model for the function of MDP. According to this model, MDP might play a pivotal role in maintaining the oxidizing conditions in the ER, which are required for the correct folding of secretory proteins.     

Secretory Vesicle Pools and Rate and Kinetics of Single Vesicle Exocytosis in Neurosecretory Cells

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca²⁺ and the cortical F-actin network. Cortical F-actin disassembly, a Ca²⁺-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.     

Aspartyl Aminopeptidase,Encoded by an Evolutionarily Conserved Syntenic Gene,Is Colocalized with Its Cluster in Secretory Granules of Pancreatic Islet Cells

Aspartyl aminopeptidase (DAP), encoded by the DNPEP gene, is believed to be a cytosolic protein with high enzymatic activity in the neuroendocrine tissues. Bioinformatic analysis revealed that the genomic segment spanning the DNPEP gene is evolutionarily conserved from Caenorhabditis elegans to humans. In the present study, we sought to determine whether the expression of DAP is associated with its clustered genes when expressed in pancreatic islet cells. Using anti-DAP specific antibody in immunofluorescent stainings, we found that DAP was specifically expressed in islet alpha cells but not in exocrine acinar cells. Moreover, using electron microscopy, we found that DAP was associated with a lysosomal-like structure and secretory granules, suggesting that it plays an important role in post-translational processing and the secretion of hormones in islet cells. The identification and characterization of DNPEP syntenic genes confirm that conserved clustered genes can preferentially be expressed in the same signaling pathway.     

Examination of the Role of ADP-Ribosylation Factor and Phospholipase D Activation in Regulated Exocytosis in Chromaffin and PC12 Cells

Abstract: The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca²⁺ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.     

The Arg617-Arg618 cleavage site in the C-terminal domain of PC1 plays a major role in the processing and targeting of the enzyme within the regulated secretory pathway

The C-terminal domain of the prohormone convertase PC1 is involved in targeting of the enzyme to secretory granules in neuroendocrine cells and is subsequently processed in this compartment at an Arg617-Arg618 site. Three other dibasics are found in the C-terminal domain of mouse PC1. Here, we examined the role of the four dibasics in targeting PC1 to secretory granules. All 15 possible combinations of dibasic mutations were performed. Wild-type (WT) and mutant PC1 were stably expressed in neuroendocrine PC12 cells that lacked endogenous PC1. Processing, secretion and intracellular localization of PC1 and its mutants were analyzed. Leaving intact Arg617-Arg618 and mutating any combination of the three other dibasics yielded proteins that were stored and processed in secretory granules, similarly to WT PC1. Mutating Arg617-Arg618 alone or with any one of the three remaining dibasics generated proteins that were efficiently stored in secretory granules but were not processed further. Mutating Arg617-Arg618 with more than one of the remaining dibasics produced proteins that reached the TGN but were not stored in secretory granules and exited the cells through the constitutive secretory pathway. These data demonstrate that the Arg617-Arg618 plays a prominent role in targeting PC1 to secretory granules.     

Regulation of fusion pore closure and compound exocytosis in neuroendocrine PC12 cells by SCAMP1

During exocytosis, neuroendocrine cells can achieve partial release of stored secretory products from dense core vesicles (DCVs) by coupling endocytosis directly at fusion sites and without full discharge. The physiological role of partial secretion is of substantial interest. Much is known about SNARE-mediated initiation of exocytosis and dynamin-mediated completion of endocytosis, but little is known about coupling events. We have used real-time microscopy to examine the role of secretory carrier membrane protein SCAMP1 in exo-endocytic coupling in PC12 cells. While reduced SCAMP1 expression is known to impede dilation of newly opened fusion pores during onset of DCV exocytosis, we now show that SCAMP1 deficiency also inhibits closure of fusion pores after they have opened. Inhibition causes accumulation of fusion figures at the plasma membrane. Closure is recovered by restoring expression and accelerated slightly by overexpression. Interestingly, inhibited pore closure resulting from loss of SCAMP1 appears to increase secondary fusion of DCVs to already-fused DCVs (compound exocytosis). Unexpectedly, reinternalization of expanded DCV membranes following compound exocytosis appears to proceed normally in SCAMP1-deficient cells. SCAMP1's apparent dual role in facilitating dilation and closure of fusion pores implicates its function in exo-endocytic coupling and in the regulation of partial secretion. Secondarily, SCAMP1 may serve to limit the extent of compound exocytosis.     

SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs

Skeletal muscles display a remarkable diversity in their arrangement of fibers into fascicles and in their patterns of innervation, depending on functional requirements and species differences. Most human muscle fascicles, despite their great length, consist of fibers that extend continuously from one tendon to the other with a single nerve endplate band. Other mammalian muscles have multiple endplate bands and fibers that do not insert into both tendons but terminate intrafascicularly. We investigated whether these alternate structural features may dictate different modes of cell hypertrophy in two mouse gracilis muscles, in response to expression of a muscle-specific insulin-like growth factor (IGF)-1 transgene (mIGF-1) or to chronic exercise. Both hypertrophic stimuli independently activated GATA-2 expression and increased muscle cross-sectional area in both muscle types, with additive effects in exercising myosin light chain/mIGF transgenic mice, but without increasing fiber number. In singly innervated gracilis posterior muscle, hypertrophy was characterized by a greater average diameter of individual fibers, and centralized nuclei. In contrast, hypertrophic gracilis anterior muscle, which is multiply innervated, contained longer muscle fibers, with no increase in average diameter, or in centralized nuclei. Different modes of muscle hypertrophy in domestic and laboratory animals have important implications for building appropriate models of human neuromuscular disease.     

Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters

Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20-fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5-h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10-20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase-deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co-transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.     

Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies

Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.     

The Latency of Exocytosis Varies with the Mechanism of Stimulated Release in PC12 Cells

Abstract: To compare the time course of different mechanisms of chemically stimulated release, amperometric detection of dopamine was carried out at single PC12 cells. The rapid response of carbon fiber microelectrodes allowed the detection of single exocytotic events, thus providing time-resolved information about the dynamics of stimulated release, in particular the latency between the stimulation of a cell and the secretion of catecholamines. On rapid depolarization of the cell membrane caused by application of 105 m M K⁺, almost immediate (6 ± 1 s) release of dopamine was observed. Stimulation with 1 m M nicotine, involving the stimulant binding to a ligand-gated ion channel, resulted in a short (37 ± 5 s) delay between stimulation and secretion. Application of 1 m M muscarine to the cells caused a long (103 ± 11 s) latency before exocytosis was detected. A biphasic response that appeared to be similar to a combination of nicotine- and muscarine-stimulated release was observed when cells were stimulated with 10 m M acetylcholine. Thus, it appears that the dynamics of stimulated release at single PC12 cells is significantly affected by the mechanism leading to exocytosis.     

Loaded dopamine is preferentially stored in the halo portion of PC12 cell dense core vesicles

Large dense core vesicles in rat pheochromocytoma cells are morphologically distinct from dense core vesicles in mast and chromaffin cells in that the dense core occupies a much smaller fraction of the vesicular volume, allowing for a much larger vesicular clear space, or halo. In this work, we present evidence indicating that upon treatment with L-DOPA the majority of the dopamine loaded into these vesicles is preferentially compartmentalized into the halo portion of the vesicle. Amperometry was used to monitor release of loaded neurotransmitter from cells in both isotonic and hypertonic extracellular conditions, with the latter condition causing inhibition of dense core dissociation. In combination with this we have used transmission electron microscopy to determine the morphological characteristics of dense core vesicles before and after treatment with L-DOPA in solutions of varied osmolarity. The results provide a more complete understanding of the complex interaction of molecules within dense core vesicles, suggesting that newly loaded dopamine is located in the halo of the vesicle. This finding has fundamental significance for studies of neurotransmitter release from dense core vesicles, as the core appears to have a function involving more than simple storage of neurotransmitter and associated molecules, and the often overlooked vesicular halo appears to be an important storage compartment for neurotransmitter.