Movement Of Secretory Granules Research Articles

Decreased intracellular granule movement and glucagon secretion in pancreatic α cells attached to superior cervical ganglion neurites

Autonomic neurons innervate pancreatic islets of Langerhans and participate in the maintenance of blood glucose concentrations by controlling hormone levels through attachment with islet cells. We previously found that stimulated superior cervical ganglia (SCG) could induce Ca2+ oscillation in α cells via neuropeptide substance P using an in vitro co-culture model. In this study, we studied the effect of SCG neurite adhesion on intracellular secretory granule movement and glucagon secretion in α cells stimulated by low glucose concentration. Spinning disk microscopic analysis revealed that the mean velocity of intracellular granules was significantly lower in α cells attached to SCG neurites than that in those without neurites under low (2mM), middle (10mM), and high (20mM) glucose concentrations. Stimulation by a low (2mM) glucose concentration significantly increased glucagon secretion in α cells lacking neurites but not in those bound to neurites. These results suggest that adhesion to SCG neurites decreases low glucose-induced glucagon secretion in pancreatic α cells by attenuating intracellular granule movement activity.

A Role for Rab7 in the Movement of Secretory Granules in Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP-bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over-expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus-end motor, dynein.

The P2X1 receptor and platelet function

Extracellular nucleotides are ubiquitous signalling molecules, acting via the P2 class of surface receptors. Platelets express three P2 receptor subtypes, ADP-dependent P2Y1 and P2Y12 G-protein-coupled receptors and the ATP-gated P2X1 non-selective cation channel. Platelet P2X1 receptors can generate significant increases in intracellular Ca2+, leading to shape change, movement of secretory granules and low levels of αIIbβ3 integrin activation. P2X1 can also synergise with several other receptors to amplify signalling and functional events in the platelet. In particular, activation of P2X1 receptors by ATP released from dense granules amplifies the aggregation responses to low levels of the major agonists, collagen and thrombin. In vivo studies using transgenic murine models show that P2X1 receptors amplify localised thrombosis following damage of small arteries and arterioles and also contribute to thromboembolism induced by intravenous co-injection of collagen and adrenaline. In vitro, under flow conditions, P2X1 receptors contribute more to aggregate formation on collagen-coated surfaces as the shear rate is increased, which may explain their greater contribution to localised thrombosis in arterioles compared to venules within in vivo models. Since shear increases substantially near sites of stenosis, anti-P2X1 therapy represents a potential means of reducing thrombotic events at atherosclerotic plaques.

Observation of Insulin Exocytosis by a Pancreatic β Cell Line with Total Internal Reflection Fluorescence Microscopy

NSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence (TIRF) microscopy has been employed to study insulin secretion. This imaging technique can explore events taking place near or on live cell membrane, such as secretory granule movement, exocytosis, vesicle content release, and membrane fusion. In the present paper, we applied TIRF microscopy to the observation of insulin exocytosis by the pancreatic β cell line Ins-1.

GENERATION OF TRANSGENIC MICE FOR IN VIVO DETECTION OF INSULIN-CONTAINING GRANULE EXOCYTOSIS AND QUANTIFICATION OF INSULIN SECRETION

Insulin secretion is a complex and highly regulated process. Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation, it remains unclear how conclusions from these studies apply to living animals. That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo. When combined with genetically encoded biosensor, optical imaging is a powerful tool for visualization of molecular events in vivo. In this study, we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins, a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol. Upon exocytosis of secretory granules, the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH, while mCherry fluorescence remained constant in the process, thus allowing ratiometric quantification of insulin secretory events. Furthermore, mCherry fluorescence enabled tracking the movement of secretory granules in living cells. We validated this approach in insulin-secreting cells, and generated a transgenic mouse line expressing the optical sensor specifically in pancreatic β-cells. The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.

Actin Cytoskeleton Controls Movement Of Intracellular Organelles In Epithelia

Background: Intracellular organelles move along microtubules and filamentous actin (F-actin) using molecular motors. Previously we reported that movement of secretory granules was hindered by Ca2+-dependent formation of a dense cytoplasmic F-actin meshwork in pancreatic duct epithelial cells (PDEC). Aim: Here we addressed whether other organelles such as mitochondria and lysosomes also are controlled by the same mechanism. Methods: Mitochondria and lysosomes were labeled with fluorescent MitoTracker Red and LysoTracker Red, respectively. Their movements were monitored every 1.2 s with high resolution fluorescence microscopy and measured by comparing subsequent images. Cells were stimulated with UTP to activate an endogenous P2Y2 G-protein coupled receptor to increase intracellular Ca2+. Results: Mitochondria and lysosomes moving actively at rest stopped rapidly (τ ∼ 2.6 s) after Ca2+ rise. This organelle ‘freezing’ was accompanied by the formation of F-actin in the whole cytoplasm as stained with phalloidin-Alexa 488. EGFP-actin binding domain 2 (ABD2) expressed in PDEC, indicated a rapid formation of cytoplasmic F-actin (τ ∼ 7.8 s). In addition, the freezing of the organelles was blocked by latrunculin B, an inhibitor of F-actin formation. Conclusion: Ca2+-dependent formation of a fine F-actin meshwork reduces the movement of intracellular organelles physically in PDEC. The freezing mechanism controls exocytosis of secretory granules and may affect local ATP supply by controlling mitochondrial distribution in PDEC.Supported by UW Royalty Research Fund and an NIH grant (GM083913).

TCF7L2 Regulates Late Events in Insulin Secretion From Pancreatic Islet β-Cells

OBJECTIVEPolymorphisms in the human TCF7L2 gene are associated with reduced insulin secretion and an increased risk of type 2 diabetes. However, the mechanisms by which TCF7L2 affect insulin secretion are still unclear. We define the effects of TCF7L2 expression level on mature β-cell function and suggest a potential mechanism for its actions.RESEARCH DESIGN AND METHODSTCF7L2 expression in rodent islets and β-cell lines was altered using RNAi or adenoviral transduction. β-Cell gene profiles were measured by quantitative real-time PCR and the effects on intracellular signaling and exocytosis by live cell imaging, electron microscopy, and patch clamp electrophysiology.RESULTSReducing TCF7L2 expression levels by RNAi decreased glucose- but not KCl-induced insulin secretion. The glucose-induced increments in both ATP/ADP ratio and cytosolic free Ca2+ concentration ([Ca2+]i) were increased compared with controls. Overexpression of TCF7L2 exerted minor inhibitory effects on glucose-regulated changes in [Ca2+]i and insulin release. Gene expression profiling in TCF7L2-silenced cells revealed increased levels of mRNA encoding syntaxin 1A but decreased Munc18–1 and ZnT8 mRNA. Whereas the number of morphologically docked vesicles was unchanged by TCF7L2 suppression, secretory granule movement increased and capacitance changes decreased, indicative of defective vesicle fusion.CONCLUSION—TCF7L2 is involved in maintaining expression of β-cell genes regulating secretory granule fusion. Defective insulin exocytosis may thus underlie increased diabetes incidence in carriers of the at-risk TCF7L2 alleles.

Monitoring of glucose-regulated single insulin secretory granule movement by selective photoactivation

Fluorescence microscopy opens new perspectives for the analysis of insulin secretory granule movement. In this study, we examined whether recently developed photoactivatable/photoconvertible proteins are a useful tool for studying this process at the single granule level in insulin-secreting cells after glucose stimulation. Plasmids were generated for expression of fusion proteins of the granule membrane phosphatase phogrin or the granule cargo protein neuropeptide Y (NPY) with the photoactivatable green fluorescent protein mutant A206K (PA-GFP-A206K), the photoconvertible protein Dendra2 and the fluorescent protein mCherry. Transfected insulin-secreting MIN6 cells were analysed by fluorescence microscopy. Point-resolved 405 nm light exposure during image acquisition of MIN6 cells transiently transfected with Phogrin-PA-GFP-A206K or NPY-PA-GFP-A206K as well as of stable MIN6-Phogrin-Dendra2 cells resulted in selective visualisation of few granules by green or red fluorescence, respectively. Movement of these granules was analysed by an automated tracking method from confocal 3D image series. The high spatiotemporal resolution facilitated an elongated tracking of single granules. Interestingly, the track speed and track displacement of granules after 1 h starvation and subsequent glucose stimulation was lower in cells pre-cultured for 48 h at 3 mmol/l glucose than in cells pre-cultured at 25 mmol/l glucose. Targeting of the granule membrane or its cargo with a photoactivatable/photoconvertible protein allows in-depth visualisation and tracking of single insulin granules in dependence upon glucose. This technique may also open the way to elucidating the regulation of granule movement velocity within the pancreatic beta cell with respect to secretory defects in type 2 diabetes.

The role of Rab27A in human eosinophil exocytosis (38.3)

Abstract Eosinophils play an effector role in airway damage in asthma and related disorders, since their secreted granule-stored products can induce many of the clinical features of asthma. Degranulation of eosinophils requires the activity of several GTP-binding proteins, many of which remain unidentified. We hypothesized that Rab27A is one of these GTP-binding proteins because, in other cells, it interacts with effectors that regulate vesicle motility, docking and fusion. Humans with Rab27A-deficiency develop Griscelli syndrome which is characterized by immunodeficiency and partial albinism due to secretory defects in various cell types. We have established that Rab27A is expressed in human peripheral blood eosinophils as determined by RT-PCR and Western blotting. To asses Rab27A activation we developed a novel assay allowing the pulldown of the active, GTP-bound form. Rab27A was activated in several leukocyte populations upon cell stimulation with degranulatory stimuli. In human eosinophils, Rab27A was transiently activated by secretory-IgA stimulation. Rab27A activation followed a temporal pattern similar to that of degranulation, suggesting that Rab27A activation induces secretory granule movement and exocytosis. A clearer understanding of granulocyte exocytosis may contribute to novel therapeutic strategies in asthma, allergic inflammation and related conditions. Funding provided by the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research

Novel Insights Into the Regulation of the Bound and Diffusible Glucokinase in MIN6 β-Cells

A stable MIN6 beta-cell clone overexpressing glucokinase as an enhanced cyan fluorescent protein (ECFP) fusion construct was generated for analysis of glucokinase regulation in these glucose-responsive insulin-secreting cells. A higher glucokinase enzyme activity accompanied by an improved glucose-induced insulin secretion indicated the integration of ECFP-glucokinase into the functional pool of glucokinase protein in MIN6-ECFP-glucokinase cells. Fluorescence recovery after photobleaching experiments of MIN6-ECFP-glucokinase cells and photoactivation of a transiently transfected photoswitchable cyan fluorescent protein (PS-CFP)-glucokinase construct in MIN6 cells indicate a higher motility of the diffusible glucokinase fraction at high glucose concentrations. In agreement with previous studies, we observed significant binding of ECFP-glucokinase to insulin secretory granules. Using fluorescence lifetime imaging, we obtained evidence for an association between glucokinase and alpha-tubulin in MIN6-ECFP-glucokinase cells. Furthermore, immunohistochemistry and fluorescence resonance energy transfer analysis by acceptor photobleaching showed distinct association between endogenous glucokinase and alpha-tubulin as well as beta-tubulin in MIN6 cells. Interestingly, glucokinase was also colocalized with kinesin, a motor protein involved in insulin secretory granule movement. Therefore, we suggest a role of a bound glucokinase protein fraction in the regulation of insulin granule movement along tubulin filaments.

Confocal Laser Scanning Microscopy to Study Molecular Mechanism of Mast Cell Activation

AbstractFor Abstract see ChemInform Abstract in Full Text.

Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica

Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments have been localised in the tegument of the 3-week-old juvenile and adult Fasciola gigantica by means of immunofluorescence and immunoperoxidase techniques, using mouse monoclonal anti-alpha-tubulin, anti-cytokeratin antibodies and biotinylated-phalloidin, respectively. The immunostainings with the above probes were also performed in adult Schistosoma mansoni for comparison. The presence of tubulin, indicative of microtubules, was demonstrated in the tegumental cell bodies, their cytoplasmic processes, and the basal layer of the tegumental syncytium of F. gigantica. While in S. mansoni, tubulin appeared as vertical lines stretching across the whole thickness of the syncytium. Cytokeratin, representing one type of intermediate filaments, was detected in the tegumental cell bodies, their cytoplasmic processes, tegumental syncytium and spines of F. gigantica. In contrast, cytokeratin was evident only in the syncytium of S. mansoni, but not in the spines. Phalloidin, which could bind to actin, a subunit of microfilament, was detected in the tegumental cell bodies, their processes, and the microtrabecular network which form the scaffold of the tegumental syncytium of F. gigantica. In S. mansoni, actin was localized in similar tissues except the syncytium was not stained while spines exhibited intense staining. In F. gigantica, the presence of microtubules and actin filaments in the tegumental cells, their processes and in the syncytium could mediate the movement of secretory granules from the cell bodies towards the basal as well as the apical layer of the tegument. Cytokeratin filaments may serve to reinforce the integrity of the tegumental syncytium as well as the spines.

Confocal Laser Scanning Microscopy to Study Molecular Mechanism of Mast Cell Activation

In the immune system, mast cells are a key cell type in the pathogenesis of immunoglobulin E (IgE)-dependent hypersensitivity reactions. Engagement of the high-affinity IgE receptors by multivalent antigens initiates the downstream activation of signal-transducing enzymes and evokes degranulation and cytokine production via an increase in the intracellular Ca2+ concentration. In addition, mast cells also play a prominent role in non-IgE-mediated hypersensitivity reactions. Mast cells are closely apposed to nerves in vivo and are likely to be regulated functionally by nerves. However, the molecular mechanisms for mast cell activation in an IgE-dependent and -independent manner have not been fully clarified. Confocal laser scanning microscopy has played an essential role in cell biology by allowing visualization of specific intracellular signaling molecules with high spatiotemporal resolution in living cells. We have studied intracellular movements of Ca2+ using a specific fluorescent probe and several types of signaling molecules using derivatives of green fluorescent protein in a living single mast cell using a microscopic strategy. We here describe our imaging analysis of the calcium signals to the nucleus, the movement of secretory granules in the degranulation process, and the nucleocytoplasmic shuttling of mitogen-activated protein kinase in mast cells. Further, we demonstrate that direct communication between mast cells and nerves occurs. These findings provide useful information from a new perspective to understand the molecular mechanisms of allergic reaction and inflammation.

Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis.

Compound exocytosis of inflammatory mediators from mast cells requires SNARE and a series of accessory proteins. However, the molecular steps that regulate secretory granule movement and membrane fusion as well as the role of the cytoskeleton are still poorly understood. Here, we report on our investigation of the role of syntaxin-binding Munc18 isoforms and the microtubule network in this process. We found that mast cells express Munc18-2, which interacts with target SNAREs syntaxin 2 or 3, as well as Munc18-3, which interacts with syntaxin 4. Munc18-2 was localised to secretory granules, whereas Munc18-3 was found on the plasma membrane. Increased expression of Munc18-2 and derived peptides containing an interfering effector loop inhibited IgE-triggered exocytosis, while increased expression of Munc18-3 showed no effect. Munc18-2 localisation on granules is polarised; however, upon stimulation Munc18-2 redistributed into forming lamellipodia and persisted on granules that were aligned along microtubules, but was excluded from F-actin ruffles. Disruption of the microtubule network with nocodazole provoked Munc18-2 redistribution and affected mediator release. These findings suggest a role for Munc18-2 and the microtubule network in the regulation of secretory granule dynamics in mast cells.

Localization of myosin XVA in endocrine tumors of gut and pancreas.

The myosin superfamily includes conventional and unconventional myosin proteins. Among unconventional myosins, myosin XVA has recently been characterized, and it has been suggested that it may be involved in cytoplasmic organelle movement, including secretory granules in pituitary cells and pituitary adenomas. In this study, we investigated the expression of myosin XVA protein and mRNA in normal endocrine cells and in a series of 53 endocrine tumors of the gut and pancreas. Myosin XVA was expressed in rare normal endocrine cells of the gut and in almost all pancreatic islet cells. In addition, myosin XVA was detected in several cells of all endocrine tumors investigated, and its expression was not related to malignancy, type, site, or functional status of tumors. These results indicate that myosin XVA protein and mRNA are widely distributed in endocrine cells of the gut and pancreas. Although the role of this protein in endocrine cells is unknown, previous studies suggest that it may have a role in secretory granule movement and/or hormone secretion.

Myosin XVA Expression in the Pituitary and in Other Neuroendocrine Tissues and Tumors

The myosin superfamily of molecular motor proteins includes conventional myosins and several classes of unconventional myosins. Recent studies have characterized the human and mouse unconventional myosin XVA, which has a role in the formation and/or maintenance of the unique actin-rich structures of inner ear sensory hair cells. Myosin XVA is also highly expressed in human anterior pituitary cells. In this study we examined the distribution of myosin XVA protein and mRNA in normal and neoplastic human pituitaries and other neuroendocrine cells and tumors. Myosin XVA was expressed in all types of normal anterior pituitary cells and pituitary tumors and in other neuroendocrine cells and tumors including those of the adrenal medulla, parathyroid, and pancreatic islets. Most nonneuroendocrine tissues examined including liver cells were negative for myosin XVA protein and mRNA, although the distal and proximal tubules of normal kidneys showed moderate immunoreactivity for myosin XVA. Ultrastructural immunohistochemistry localized myosin XVA in association with secretory granules of human anterior pituitary cells and human pituitary tumors. These data suggest that in neuroendocrine cells myosin XVA may have a role in secretory granule movement and/or secretion.

Role of Actin in Regulated Exocytosis and Compensatory Membrane Retrieval: Insights from an Old Acquaintance

This review summarizes new insights into the role of the actin cytoskeleton in exocytosis and compensatory membrane retrieval from mammalian regulated secretory cells. Data from our lab and others now indicate that the actin cytoskeleton is involved in exocytosis both as a negative regulator of membrane fusion under resting conditions and as a facilitator of movement of secretory granules to their site of fusion with the apical plasmalemma. Coating of docked secretory granules with actin filaments correlates with the dissociation of secretory-granule-associated rab3D, pointing out a novel role for rab proteins in modulating the actin cytoskeleton during regulated exocytosis. Compensatory membrane retrieval following regulated exocytosis is also critically dependent on the actin cytoskeleton both in initiating the formation of clathrin-coated retrieval vesicles and subsequent trafficking back into the cell. We propose that insertion of secretory granule membrane into the plasmalemma initiates a trigger for membrane retrieval, possibly by exposing sites where proteins involved in compensatory membrane retrieval are assembled. The results summarized in this review were derived primarily from investigations on the pancreatic acinar cell, an old friend who is providing modern wisdom not attainable in other simpler systems.

Roles of intracellular Ca2+ receptors in the pancreatic beta-cell in insulin secretion.

Ca2+ is the central second messenger in the regulation of insulin release from the pancreatic beta-cell; and intracellular Ca2+ -binding proteins, classified into two groups, the EF hand proteins and the Ca2+/phospholipid binding proteins, are considered to mediate Ca2+ signaling. A number of Ca binding proteins have been suggested to participate in the secretory machinery in the beta-cell. Calmodulin, the ubiquitous EF hand protein, is the predominant intracellular Ca2+ receptor that modulates insulin release via the multiplicity of its binding to target proteins including protein kinases. Other Ca binding proteins such as calcyclin and the Ca2+/phospholipid binding proteins may also be suggested to be involved. Ca2+ influx from the extracellular space appears to be responsible for exocytosis of insulin via Ca2+ -dependent protein/protein interactions. On the other hand, intracellular Ca2+ mobilization resulting in secretory granule movement may be controlled by Ca2+/calmodulin-dependent protein phosphorylation. Thus, Ca2+ exerts versatile effects on the secretory cascade via binding to specific binding proteins in the pancreactic beta-cells.

Acetylcholine activates intracellular movement of insulin granules in pancreatic beta-cells via inositol trisphosphate-dependent [correction of triphosphate-dependent] mobilization of intracellular Ca2+.

Intracellular movement of secretory granules is a proximal stage in the secretory cascade that ends in the release product from cells. We investigated mechanisms underlying the control of this movement by acetylcholine using an insulinoma cell line, MIN6, in which acetylcholine increases both insulin secretion and granule movement. The peak activation of movement was observed 3 min after an acetylcholine challenge. The effects were nullified by the muscarinic inhibitor atropine, phospholipase C (PLC) inhibitors (D 609 and compound 48/80), and pretreatment with the Ca2+ pump inhibitor, thapsigargin. Inhibitors of Ca2+-dependent phospholipase A2 (arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphate) also partially inhibited the movement caused by acetylcholine, but downregulation of protein kinase C by overnight incubation with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate failed to exert any influence. Acetylcholine stimulation of granule movement was not reproduced by membrane depolarization with high K+. Phosphorylation of the endogenous myosin light chain in MIN6 cells was increased by addition of acetylcholine and decreased by the Ca2+ chelator BAPTA (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid). The calmodulin inhibitor W-7 and the myosin light-chain kinase inhibitor ML-9 decreased the motile events in the beta-cells under both nonstimulated and acetylcholine-stimulated conditions. These findings led us to conclude that inositol trisphosphate [corrected] causes Ca2+ mobilization by muscarinic activation of PLC, leading to intracellular translocation of insulin granules to the ready-releasable pool in pancreatic beta-cells via Ca2+/calmodulin-dependent phosphorylation of myosin light chains.

Trimeric G Proteins Control Exocytosis in Chromaffin Cells: Go REGULATES THE PERIPHERAL ACTIN NETWORK AND CATECHOLAMINE SECRETION BY A MECHANISM INVOLVING THE SMALL GTP-BINDING PROTEIN Rho

Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles and its activation by mastoparan inhibits the ATP-dependent priming of exocytosis. The effectors by which Go controls exocytosis are currently unknown. The subplasmalemmal actin network is one candidate, since it modulates secretion by controlling the movement of secretory granules to the plasma membrane. In streptolysin-O-permeabilized chromaffin cells, activation of exocytosis produces disassembly of cortical actin filaments. Mastoparan blocks the calcium-evoked disruption of cortical actin, and this effect is specifically inhibited by antibodies against Galphao and by a synthetic peptide corresponding to the COOH-terminal domain of Galphao. Disruption of actin filaments with cytochalasin E and Clostridium perfringens iota toxin partially reverses the mastoparan-induced inhibition of secretion. Furthermore, the effects of mastoparan on cortical actin and exocytosis are greatly reduced in cells treated with Clostridium botulinum C3 exoenzyme, which specifically inactivates the small G protein Rho. We propose that the control exerted by the granule-associated Go on exocytosis may be related to effects on the cortical actin network through a sequence of events which eventually involves the participation of Rho.