Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

Mesenchymal Stromal Cell Secretome for Therapeutic Application in Skin Wound Healing: A Systematic Review of Preclinical Studies.

Non-healing skin wounds remain a challenge in the healthcare system. In this sense, it is suggested that the secretome of mesenchymal stromal cells (MSCs) can be effective as a therapeutic strategy for regenerative medicine. Therefore, this systematic review aimed to determine the effects of treatment with a secretome derived from MSCs on the healing of skin wounds in a preclinical model of rodents (mice and rats). Studies were systematically retrieved from 6 databases and gray literature that provided 1,172 records, of which 25 met the inclusion criteria for qualitative analysis. Results revealed substantial heterogeneity among studies concerning experimental designs and methodologies, resulting in a high risk of bias. Together, the selected studies reported that treatment improved wound healing by (1) accelerating wound closure and improving skin repair quality; (2) reducing inflammation by decreasing the number of cells and inflammatory cytokines, accompanied by polarization of the M2 macrophage; (3) complete re-epithelialization and epidermal reorganization; (4) neovascularization promoted by proliferation of endothelial cells (CD34+) and increased levels of pro-angiogenic mediators; (5) better scar quality promoted by increased expression of collagen types I and III, as well as improved deposition and remodeling of collagen fibers. In conclusion, despite the need for alignment of methodological protocols and transparent reports in future studies, results show that the secretome of MSCs from different tissue sources corresponds to a promising tool of regenerative medicine for the treatment of skin wounds.

Organelle movement and apical accumulation of secretory vesicles in pollen tubes of Arabidopsis thaliana depend on class XI myosins.

F-actin and myosin XI play important roles in plant organelle movement. A few myosin XI genes in the genome of Arabidopsis are mainly expressed in mature pollen, which suggests that they may play a crucial role in pollen germination and pollen tube tip growth. In this study, a genetic complementation assay was conducted in a myosin xi-c (myo11c1) myosin xi-e (myo11c2) double mutant, and fluorescence labeling combined with microscopic observation was applied. We found that myosin XI-E (Myo11C2)-green fluorescent protein (GFP) restored the slow pollen tube growth and seed deficiency phenotypes of the myo11c1 myo11c2 double mutant and Myo11C2-GFP partially colocalized with mitochondria, peroxisomes and Golgi stacks. Furthermore, decreased mitochondrial movement and subapical accumulation were detected in myo11c1 myo11c2 double mutant pollen tubes. Fluorescence recovery after photobleaching experiments showed that the fluorescence recoveries of GFP-RabA4d and AtPRK1-GFP at the pollen tube tip of the myo11c1 myo11c2 double mutant were lower than those of the wild type were after photobleaching. These results suggest that Myo11C2 may be associated with mitochondria, peroxisomes and Golgi stacks, and play a crucial role in organelle movement and apical accumulation of secretory vesicles in pollen tubes of Arabidopsis thaliana.

TGFß1 and TGFß2 proteins in corneas with and without stromal fibrosis: Delayed regeneration of apical epithelial growth factor barrier and the epithelial basement membrane in corneas with stromal fibrosis.

The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) ß1 and TGFß2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, -4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFß1 or TGFß2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to -4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFß1 and tear TGFß2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFß1 and/or TGFß2 in the stroma in some corneas. TGFß1 and TGFß2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four -4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in -4.5D PRK corneas without fibrosis, compared to -4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFß1 and TGFß2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.

Geoffrey Burnstock, our friend and magister: the diadenosine polyphosphate connection.

Development of science needs the cooperation of many creative brains. Sometimes, ideas on a specific area get suddenly exhausted and then it is the time for a privileged mind to think in a different way and reach the turning point to introduce a new paradigm. This happened to Geoffrey Burnstock, a heterodox thinker and nonconformist scientist that has been the paladin of purinergic signalling since 1972, opening neuroscience to the understanding of organs and tissues functioning and development of a new pharmacology. This review summarizes the contribution of our group to the understanding of the role of the diadenosine polyphosphates, ApnA, as signalling molecules, describing their tissue and organ distribution, their transport and storage in secretory vesicles and their release and interaction with purinergic receptors. We also have to acknowledge the friendly and kindly support of Professor Burnstock that showed a great interest in the field from our initial findings and actively stimulated our efforts to establish the extracellular roles and biological significance of these dinucleotides.

Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion.

Astrocytes are housekeepers of the central nervous system (CNS) and are important for CNS development, homeostasis and defence. They communicate with neurones and other glial cells through the release of signalling molecules. Astrocytes secrete a wide array of classic neurotransmitters, neuromodulators and hormones, as well as metabolic, trophic and plastic factors, all of which contribute to the gliocrine system. The release of neuroactive substances from astrocytes occurs through several distinct pathways that include diffusion through plasmalemmal channels, translocation by multiple transporters and regulated exocytosis. As in other eukaryotic cells, exocytotic secretion from astrocytes involves divergent secretory organelles (synaptic-like microvesicles, dense-core vesicles, lysosomes, exosomes and ectosomes), which differ in size, origin, cargo, membrane composition, dynamics and functions. In this review, we summarize the features and functions of secretory organelles in astrocytes. We focus on the biogenesis and trafficking of secretory organelles and on the regulation of the exocytotic secretory system in the context of healthy and diseased astrocytes.

The Emerging Role of Spinal Dynorphin in Chronic Pain: A Therapeutic Perspective.

Notable findings point to the significance of the dynorphin peptide neurotransmitter in chronic pain. Spinal dynorphin neuropeptide levels are elevated during development of chronic pain and sustained during persistent chronic pain. Importantly, knockout of the dynorphin gene prevents development of chronic pain in mice, but acute nociception is unaffected. Intrathecal (IT) administration of opioid and nonopioid dynorphin peptides initiates allodynia through a nonopioid receptor mechanism; furthermore, antidynorphin antibodies administered by the IT route attenuate chronic pain. Thus, this review presents the compelling evidence in the field that supports the role of dynorphin in facilitating the development of a persistent pain state. These observations illustrate the importance of elucidating the control mechanisms responsible for the upregulation of spinal dynorphin in chronic pain. Also, spinal dynorphin regulation of downstream signaling molecules may be implicated in hyperpathic states. Therapeutic strategies to block the upregulation of spinal dynorphin may provide a nonaddictive approach to improve the devastating condition of chronic pain that occurs in numerous human diseases.

Gliocrine System: Astroglia as Secretory Cells of the CNS.

Astrocytes are secretory cells, actively participating in cell-to-cell communication in the central nervous system (CNS). They sense signaling molecules in the extracellular space, around the nearby synapses and also those released at much farther locations in the CNS, by their cell surface receptors, get excited to then release their own signaling molecules. This contributes to the brain information processing, based on diffusion within the extracellular space around the synapses and on convection when locales relatively far away from the release sites are involved. These functions resemble secretion from endocrine cells, therefore astrocytes were termed to be a part of the gliocrine system in 2015. An important mechanism, by which astrocytes release signaling molecules is the merger of the vesicle membrane with the plasmalemma, i.e., exocytosis. Signaling molecules stored in astroglial secretory vesicles can be discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This leads to a fusion pore formation, a channel that must widen to allow the exit of the Vesiclal cargo. Upon complete vesicle membrane fusion, this process also integrates other proteins, such as receptors, transporters and channels into the plasma membrane, determining astroglial surface signaling landscape. Vesiclal cargo, together with the whole vesicle can also exit astrocytes by the fusion of multivesicular bodies with the plasma membrane (exosomes) or by budding of vesicles (ectosomes) from the plasma membrane into the extracellular space. These astroglia-derived extracellular vesicles can later interact with various target cells. Here, the characteristics of four types of astroglial secretory vesicles: synaptic-like microvesicles, dense-core vesicles, secretory lysosomes, and extracellular vesicles, are discussed. Then machinery for vesicle-based exocytosis, second messenger regulation and the kinetics of exocytotic vesicle content discharge or release of extracellular vesicles are considered. In comparison to rapidly responsive, electrically excitable neurons, the receptor-mediated cytosolic excitability-mediated astroglial exocytotic vesicle-based transmitter release is a relatively slow process.

ATP: The crucial component of secretory vesicles.

The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

Protein secretion in plants: conventional and unconventional pathways and new techniques.

Protein secretion is an essential process in all eukaryotic cells and its mechanisms have been extensively studied. Proteins with an N-terminal leading sequence or transmembrane domain are delivered through the conventional protein secretion (CPS) pathway from the endoplasmic reticulum (ER) to the Golgi apparatus. This feature is conserved in yeast, animals, and plants. In contrast, the transport of leaderless secretory proteins (LSPs) from the cytosol to the cell exterior is accomplished via the unconventional protein secretion (UPS) pathway. So far, the CPS pathway has been well characterized in plants, with several recent studies providing new information about the regulatory mechanisms involved. On the other hand, studies on UPS pathways in plants remain descriptive, although a connection between UPS and the plant defense response is becoming more and more apparent. In this review, we present an update on CPS and UPS. With the emergence of new techniques, a more comprehensive understanding of protein secretion in plants can be expected in the future.

Characterization of Acyl-CoA synthetase isoforms in pancreatic beta cells: Gene silencing shows participation of ACSL3 and ACSL4 in insulin secretion.

Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ∼50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion.

Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases.

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.

Abnormally Increased Secretion in Olfactory Neuronal Precursors from a Case of Schizophrenia Is Modulated by Melatonin: A Pilot Study.

The alterations that underlie the pathophysiology of schizophrenia (SCZ) include the dysregulation of structural and functional properties of neurons. Among these, the secretion of neurotransmitters and hormones, which plays a key role for neuronal communication and development, is altered. Neuronal precursors from the human olfactory epithelium have been recently characterized as a reliable model for studying the etiopathogenesis of neuropsychiatric diseases. Our previous work has shown that melatonin enhances the development of morphological and functional features of cloned olfactory neuronal precursors (ONPs) from a healthy subject. In this work we found that primary cultures of ONPs obtained from a schizophrenic patient display an increased potassium-evoked secretion, when compared with ONPs from an age- and gender-matched healthy control subject (HCS). Secretion was evaluated by FM1-43 fluorescence cumulative changes in response to depolarization. Interestingly, a 12 h-melatonin treatment modulated the abnormally increased secretion in SCZ ONPs and brought it to levels similar to those found in the HCS ONPs. Our results suggest that the actin cytoskeleton might be a target for melatonin effects, since it induces the thickening of actin microfilament bundles. Further research will address the mechanisms by which melatonin modulates neurochemical secretion from ONPs.

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION.

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075-11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.

Gelsolin role in microapocrine secretion.

A role of gelsolin in opening the way along the microvilli for secretory vesicles during microapocrine secretion is proposed here. Data obtained with different techniques showed that many digestive enzymes are released by microapocrine secretion in insects. Proteins that might be involved in the machinery of microapocrine secretion were selected from our transcriptomes and literature searches. The proteins were annexin, Complex actin-related proteins 2 and 3 (ARP 2/3) cofilin, fimbrin, gelsolin 1, gelsolin 2, moesin, myosin 1, myosin 6, protein disulphide isomerase 1 (PDI 1), PDI 2 and profilin. The cDNAs coding for annexin, fimbrin, gelsolin 1, myosin 1, PDI 1 and PDI 2 were cloned and their sequences deposited in GenBank. Only gelsolin 1 and myosin 1 are expressed exclusively in the midgut (semiquantitative reverse transcriptase PCR). As myosin 1 may have a structural role in microvilli, gelsolin 1 is the best guess to be involved in the secretory machinery. A truncated recombinant gelsolin 1 was used to generate antibodies with which it was shown labelling inside and around midgut cell microvilli shown in an electron microscope, reinforcing a microvillar role for gelsolin 1. Suppression of gelsolin 1 synthesis by RNA interference prevents secretory vesicles from advancing inside the microvilli, in agreement with its putative role in severing the actin filaments to free the way for the vesicles.

Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis.

Munc18-1 is believed to prime or stimulate SNARE-mediated membrane fusion/exocytosis through binding to the SNARE complex, in addition to chaperoning its cognate syntaxins. Nevertheless, a Munc18-1 mutant that selectively loses the priming function while retaining the syntaxin chaperoning activity has not been identified. As a consequence, the mechanism that mediates Munc18-1-dependent priming remains unclear. In the course of analyzing the functional outcomes of a variety of point mutations in domain 3a of Munc18-1, we discovered insertion mutants (K332E/K333E with insertions of 5 or 39 residues). These mutants completely lose their ability to rescue secretion whereas they effectively restore syntaxin-1 expression at the plasma membrane as well as dense-core vesicle docking in Munc18-1 and Munc18-2 double-knockdown PC12 cells. The mutants can bind syntaxin-1A in a stoichiometric manner. However, binding to the SNARE complex is impaired compared with the wild type or the hydrophobic pocket mutant (F115E). Our results suggest that the domain 3a of Munc18-1 plays a crucial role in priming of exocytosis, which is independent of its syntaxin-1 chaperoning activity and is downstream of dense-core vesicle docking. We also suggest that the priming mechanism of Munc18-1 involves its domain-3a-dependent interaction with the SNARE complex.

Ketotifen is a microglial stabilizer by inhibiting secretory vesicle acidification.

AIMS: Microglia survey the brain environment by sensing alarm signals to provide the first line of defense against injury or infection after which they acquire an activated phenotype, but they also respond to chemical signals sent from brain mast cells, sentinels of the immune system, when these are degranulated in response to noxious agents. Nevertheless, excessive microglia activation damages the surrounding healthy neural tissue causing progressive loss of neurons and inducing chronic inflammation. Thus, it would be of intense interest the development and application of agents which prevent mast cell mediator release and inhibit the actions of such mediators once released on microglia. MAIN METHODS: Fluorescence measurements of fura-2 and quinacrine were used to measure intracellular Ca2+ signaling and exocytotic vesicle fusion in resting and activated microglia. KEY FINDINGS: We show that treatment of microglia with a cocktail of mast cell mediators induces microglia activation, phagocytosis, and exocytosis, and reveal by the first-time microglia undergo a phase of vesicular acidification just before the exocytotic fusion occurs. This acidification is an important process for vesicular maturation and contributes with ∼25 % to the content that the vesicle can store and later release by exocytosis. Pre-incubation with ketotifen, a mast cell stabilizer and H1R antagonist completely abolished histamine-mediated calcium signaling and acidification of microglial organelles, and concomitantly reduced the discharge of vesicle contents. SIGNIFICANCE: These results highlight a key role for vesicle acidification in microglial physiology and provide a potential therapeutic target for diseases related to mast cell and microglia-mediated neuroinflammation.

Quantitative super-resolution imaging of platelet degranulation reveals differential release of von Willebrand factor and von Willebrand factor propeptide from alpha-granules.

BACKGROUND: Von Willebrand factor (VWF) and VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body exocytosis in endothelial cells; however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3-dimensional super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo, we studied alpha-granule cargo release in response to different physiological stimuli. OBJECTIVES: To investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli. METHODS: Platelets were activated with protease-activated receptor 1 (PAR-1) activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, secreted protein acidic and cysteine rich (SPARC), and fibrinogen were imaged using 3-dimensional structured illumination microscopy, followed by semiautomated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. RESULTS: VWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap with VWF than SPARC or fibrinogen. Activation of PAR-1 (0.6-20 µM PAR-1 ap) or glycoprotein VI (GPVI) (0.25-1 µg/mL CRP-XL) signaling pathways caused a dose-dependent increase in alpha-granule exocytosis. More than 80% of alpha-granules remained positive for VWF, even at the highest agonist concentrations. In contrast, the residual fraction of alpha-granules containing VWFpp decreased in a dose-dependent manner to 23%, whereas SPARC and fibrinogen were detected in 60% to 70% of alpha-granules when stimulated with 20 µM PAR-1 ap. Similar results were obtained using CRP-XL. Using an extracellular anti-VWF nanobody, we identified VWF in postexocytotic alpha-granules. CONCLUSION: We provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules.

Measurements of Calcium in Chromaffin Cell Organelles Using Targeted Aequorins.

The molecular mechanisms that mediate and regulate calcium (Ca2+) fluxes through the membranes of intracellular organelles play a key role in the generation and shaping of the local and global cytosolic Ca2+ signals triggering the process of regulated exocytosis in chromaffin cells. Beyond that role, intraorganellar Ca2+ homeostasis also regulates organelle-specific processes such as oxidative phosphorylation in mitochondria, maturation of secretory granules, or stress in the endoplasmic reticulum. In this chapter, we describe current methods to study mitochondrial, endoplasmic reticulum, and secretory vesicle calcium homeostasis in living chromaffin cells using engineered targeted aequorins.

Quantification of Secretory Granule Exocytosis by TIRF Imaging and Capacitance Measurements.

Hormones and neurotransmitters are released from (neuro)endocrine cells by regulated exocytosis of secretory granules. During exocytosis, the granule membrane fuses with the plasma membrane, which allows release of the stored content into the bloodstream or the surrounding tissue. Here, we give a detailed description of two complementary methods to observe and quantify exocytosis in single cells: high-resolution TIRF microscopy and patch-clamp capacitance recordings. Precise stimulation of exocytosis is achieved by local pressure application or voltage-clamp depolarizations. While the chapter is focused on insulin-secreting cells as an accessible and disease-relevant model system, the methodology is applicable to a wide variety of secretory cells including chromaffin and PC12 cells.