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Acrosomal dysfunction has been considered as a cause of subfertility in males of different species, including stallions. A subset of subfertile stallions with acrosomal dysfunction is unique because they have normal sperm quality (motility, morphology, viability, and DNA quality). The current work aims to describe the clinical characteristics of subfertile stallions that were diagnosed with Impaired Acrosomal Exocytosis (IAE) by using two high throughput methods: flow cytometry and molecular genetic analysis, and to identify the prevalence of subfertility due to IAE in stallions evaluated at Texas AM University. Clinical data from 1,128 stallions evaluated during 17 years at a Veterinary Teaching Hospital was retrospectively analyzed. Only stallions with a history of subfertility not explained following a breeding soundness examination and/or conventional semen analysis, were included. For those stallions, the acrosomal exocytosis test (AE test), in which sperm is incubated at 37 °C for up to 2 h in the presence of the calcium ionophore A23187, was used to determine IAE. The difference in AE-Rate (AE-Diff) between each pair of fertile control stallion and subfertile stallion was categorized as: Normal: AE-Diff14%; Questionable: AE-Diff 15-29%; Abnormal: AE-Diff30%. In selected cases, blood or hair was procured for identification of the susceptibility genotype for IAE, A/A-A/A, in the FKBP6 gene, exon 5. Twenty-one (21) stallions (1.86% total population analyzed) had reduced fertility despite having acceptable sperm quality. Sperm from these stallions were subjected to the AE Test. Of these, 8 stallions had reduced sperm AE-rate, based on the AE Test (8/21; 38.1%). Subsequently, blood or hair samples from these 8 stallions which had either questionable (AE-Diff 15 - 29%; n = 5) or abnormal (AE-Diff30%; n = 3) responses to the AE Test were analyzed for the susceptibility genotype for IAE, A/A-A/A (FKBP6 gene, exon 5). Seven out of the eight (7/8) stallions carried this susceptibility genotype. All of these were Thoroughbreds. After 2 h of incubation, the viability in fertile stallion sperm was lower than in A/A-A/A stallions (4% vs. 25%, respectively; P 0.05), while the AE-rate was higher for fertile than for A/A-A/A stallions (85% vs. 56%, respectively; P 0.05). The use of two high throughput tests (i.e., flow cytometry and molecular genetic analysis) may complement each other in the diagnosis of IAE in breeding stallions. In this study, 5/7 subfertile stallions diagnosed with the IAE susceptibility genotype would have been diagnosed as normal with the AE Test. This study introduces a subset of stallions with the IAE genotype with fertility higher than has been previously reported (i.e.,15% per-cycle pregnancy rate), suggesting that IAE manifests as a broader range of subfertility.

The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that suppresses T cell receptor (TCR) signaling. We developed a phenotypic assay to screen for small molecules that interfere with this pathway using TALL-104 human leukemic cytotoxic T lymphocytes pretreated with prostaglandin E2 to elevate cAMP. Beads coated with anti-CD3 antibodies stimulate lytic granule exocytosis, which is detected via binding of an antibody against lysosome associated membrane protein 1 (LAMP-1) measured with flow cytometry. Confirming that the assay can find compounds with desired activity, treating cells with a phorbol ester restores exocytosis. The assay behaves well in 96-well format and we screened a collection of compounds expected to have effects on epigenetic regulatory proteins. Compounds in this collection affected lytic granule exocytosis after 24-hour treatment, but none prevented cAMP from suppressing lytic granule exocytosis. We used a fully automated 384-well version of the assay to screen the Prestwick Compound Library but obtained no confirmed hits. Analyzing this assay's performance reveals two points of interest. First, cytometry offers multiple ways to quantify signals. Z' was higher using percent positive cells than mean fluorescence because the relationship between the two measures saturates, but using percent positive could make it harder to find hits in some assays. Second, variance was higher in positive controls than in negative controls in this assay, which degrades assay performance less than if variance was higher in negative controls.

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.

Transporters are traditionally considered to transport small molecules rather than large-sized nanoparticles due to their small pores. In this study, we demonstrate that the upregulated intestinal transporter (PCFT), which reaches a maximum of 12.3-fold expression in the intestinal epithelial cells of diabetic rats, mediates the uptake of the folic acid-grafted nanoparticles (FNP). Specifically, the upregulated PCFT could exert its function to mediate the endocytosis of FNP and efficiently stimulate the traverse of FNP across enterocytes by the lysosome-evading pathway, Golgi-targeting pathway and basolateral exocytosis, featuring a high oral insulin bioavailability of 14.4% in the diabetic rats. Conversely, in cells with relatively low PCFT expression, the positive surface charge contributes to the cellular uptake of FNP, and FNP are mainly degraded in the lysosomes. Overall, we emphasize that the upregulated intestinal transporters could direct the uptake of ligand-modified nanoparticles by mediating the endocytosis and intracellular trafficking of ligand-modified nanoparticles via the transporter-mediated pathway. This study may also theoretically provide insightful guidelines for the rational design of transporter-targeted nanoparticles to achieve efficient drug delivery in diverse diseases.

Human mesenchymal stem cells (hMSCs) were labeled with Dotarem or (Gd-DOTA)2- EM7 (EM7Gd2) via electroporation (EP). Cellular transmission electron microscopy (TEM) reveals free distribution of Gd agents and formation of EM7Gd2 clusters in the cytosol. Cellular magnetic resonance imaging (MRI) reveals that the free Gd agents induce MRI signal enhancement effect due to its fast exocytosis and subsequent interaction with intercellular water molecules. The EM7Gd2 clusters exhibits a longer intracellular retention time and induce a persistent MRI signal reduction effect. The cellular MRI results are interpreted by taking into account both T1 and T2 relaxation rates and their correlation with cellular binding structures of Dotarem and EM7Gd2.

Visual Abstract, COVID-19 is characterized by vascular inflammation and thrombosis, including elevations in P-selectin, a mediator of inflammation released by endothelial cells. We tested the effect of P-selectin inhibition on biomarkers of thrombosis and inflammation in patients with COVID-19. Hospitalized patients with moderate COVID-19 were randomly assigned to receive either placebo or crizanlizumab, a P-selectin inhibitor, in a double-blind fashion. Crizanlizumab reduced P-selectin levels by -89%. Crizanlizumab increased D-dimer levels by 77% and decreased prothrombin fragment. There were no significant differences between crizanlizumab and placebo for clinical endpoints. Crizanlizumab was well tolerated. Crizanlizumab may induce thrombolysis in the setting of COVID-19. (Crizanlizumab for Treating COVID-19 Vasculopathy [CRITICAL]; NCT04435184)

Background The cellular and molecular mechanisms by which general anaesthesia occurs is poorly understood. Hippocampal interneurone subpopulations, which are critical regulators of cognitive function, have diverse neurophysiological and synaptic properties, but their responses to anaesthetics are unclear. Methods We used live-cell imaging of fluorescent biosensors expressed in mouse hippocampal neurones to delineate interneurone subtype-specific effects of isoflurane on synaptic vesicle exocytosis. The role of voltage-gated sodium channel (Nav) subtype expression in determining isoflurane sensitivity was probed by overexpression or knockdown of specific Nav subtypes in identified interneurones. Results Clinically relevant concentrations of isoflurane differentially inhibited synaptic vesicle exocytosis: to 83.1% (11.7%) of control in parvalbumin-expressing interneurones, and to 58.6% (13.3%) and 64.5% (8.5%) of control in somatostatin-expressing interneurones and glutamatergic neurones, respectively. The relative expression of Nav1.1 (associated with lower sensitivity) and Nav1.6 (associated with higher sensitivity) determined the sensitivity of exocytosis to isoflurane. Conclusions Isoflurane inhibits synaptic vesicle exocytosis from hippocampal glutamatergic neurones and GABAergic interneurones in a cell-type-specific manner depending on their expression of voltage-gated sodium channel subtypes.

Peptide nucleic acids (PNAs), a synthetic DNA mimic, have been extensively utilized for antisense- and antigene-based biomedical applications. Significant efforts have been made to increase the cellular uptake of PNAs, but here we examined relatively unexplored aspects of intracellular trafficking and endocytic recycling of PNAs. For proof-of-concept, we used anti-microRNA (miR) PNA targeting miR-155. The sub-cellular localization of PNA was studied via confocal and flow-cytometry-based assays in HeLa cells. A comprehensive characterization of PNA-containing extracellular vesicles revealed spherical morphology, negative surface charge density, and the presence of tetraspanin markers. Most importantly, we investigated rab11a and rab27b GTPases’ role in regulating the exocytosis of PNAs. Organelle staining, followed by confocal imaging, showed higher localization of PNA in lysosomes. Gene-expression analysis established the enhanced functional activity of PNA after inhibition of endocytic recycling. Multiple studies report the exocytosis of single-stranded oligonucleotides, short interfering RNAs (siRNAs), and nanocarriers. To our knowledge, this is the first mechanistic study to establish that PNA undergoes endocytic recycling and exocytosis out of tumor cells. The results presented here can serve as a platform to develop and optimize strategies for improving the therapeutic efficacy of PNAs by avoiding the recycling pathways., Graphical abstract, Authors investigated that peptide nucleic acid undergoes exocytosis in the cancer cells in a series of cell-culture-based functional assays. It was noted that membrane trafficking proteins play a significant role in the exocytosis and intracellular trafficking of peptide nucleic acid.

International audience; Apicomplexan parasites are unicellular eukaryotes that invade the cells in which they proliferate. The development of genetic tools in Toxoplasma, and then in Plasmodium, in the 1990s allowed the first description of the molecular machinery used for motility and invasion, revealing a crucial role for two different secretory organelles, micronemes and rhoptries. Rhoptry proteins are injected directly into the host cytoplasm not only to promote invasion but also to manipulate host functions. Nonetheless, the injection machinery has remained mysterious, a major conundrum in the field. Here we review recent progress in uncovering structural components and proteins implicated in rhoptry exocytosis and explain how revisiting early findings and considering the evolutionary origins of Apicomplexa contributed to some of these discoveries.

RIN4 is an important immunomodulator in Arabidopsis, which is targeted by multiple pathogenic effectors, and consequently guarded by different immune receptors. Although RIN4 plays a significant role in plant immunity, its molecular function is not fully understood. We found that RIN4 interacts with the exocyst subunit EXO70E2. Transiently expressed RIN4 can recruits EXO70E2 vesicles to the plasma membrane, and promote the transport of the vesicles to the extracellular matrix. RIN4 also can decrease the protein level of EXO70E2. Base on the fact that EXO70 proteins positively mediates plant immunity, the function of RIN4 is to promote the extracellular export of defense related vesicles. Pathogens will secret effectors to modify or cleavage it to interfere this exocytosis.

SNARE-mediated membrane fusion proceeds via the formation of a fusion pore. This intermediate structure is highly dynamic and can flicker between open and closed states. In cells, cholesterol has been reported to affect SNARE-mediated exocytosis and fusion pore dynamics. Here, we address the question of whether cholesterol directly affects the flickering rate of reconstituted fusion pores in vitro. These experiments were enabled by the recent development of a nanodisc⋅black lipid membrane recording system that monitors dynamic transitions between the open and closed states of nascent recombinant pores with submillisecond time resolution. The fusion pores formed between nanodiscs that bore the vesicular SNARE synaptobrevin 2 and black lipid membranes that harbored the target membrane SNAREs syntaxin 1A and SNAP-25B were markedly affected by cholesterol. These effects include strong reductions in flickering out of the open state, resulting in a significant increase in the open dwell-time. We attributed these effects to the known role of cholesterol in altering the elastic properties of lipid bilayers because manipulation of phospholipids to increase membrane stiffness mirrored the effects of cholesterol. In contrast to the observed effects on pore kinetics, cholesterol had no effect on the current that passed through individual pores and, hence, did not affect pore size. In conclusion, our results show that cholesterol dramatically stabilizes fusion pores in the open state by increasing membrane bending rigidity.

In plants, defense-associated signal transduction involves key membrane-related processes, such as phospholipid-based signaling and membrane trafficking. Coordination of these processes occurs in the lipid bilayer of plasma membrane (PM) and luminal/extracellular membranes. Deciphering the spatiotemporal organization of phospholipids and lipid-protein interactions provides crucial information on the mechanisms that link phospholipid-based signaling and membrane trafficking in plant immunity. In this review, we summarize recent advances in our understanding of these connections, including deployment of key enzymes and molecules in phospholipid pathways, and roles of lipid diversity in membrane trafficking. We highlight the mechanisms that mediate feedback between phospholipid-based signaling and membrane trafficking to regulate plant immunity, including their novel roles in balancing endocytosis and exocytosis.

The dominant role of CaV2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of CaV2 and CaV1 channels, and less so CaV3 channels, it is unclear why there have not been major shifts toward dependence on other CaV channels for synaptic transmission. Here, we provide a structural and functional profile of the CaV2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for CaV1–CaV3 channels. Remarkably, the highly divergent channel possesses similar features as human CaV2.1 and other CaV2 channels, including high voltage–activated currents that are larger in external Ba2+ than in Ca2+; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax CaV2 suggests that the core features of presynaptic CaV2 channels were established early during animal evolution, after CaV1 and CaV2 channels emerged via proposed gene duplication from an ancestral CaV1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian CaV2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd2+ and Ni2+. Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gβγ subunits, which nevertheless inhibited the human CaV2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.

Most proteins in the secretory pathway are glycosylated. However, the role of glycans in membrane trafficking is still unclear. Here, we discovered that transmembrane secretory cargos, such as interleukin 2 receptor α subunit or Tac, transferrin receptor and cluster of differentiation 8a, unexpectedly displayed substantial Golgi localization when their O-glycosylation was compromised. By quantitatively measuring their Golgi residence times, we found that the observed Golgi localization of O-glycan deficient cargos is due to their slow Golgi export. Using a super-resolution microscopy method that we previously developed, we revealed that O-glycan deficient Tac chimeras localize at the interior of the trans-Golgi cisternae. O-glycans were observed to be both necessary and sufficient for the efficient Golgi export of Tac chimeras. By sequentially introducing O-glycosylation sites to ST6GAL1, we demonstrated that O-glycan’s effect on Golgi export is probably additive. Finally, the finding that N-glycosylated GFP substantially reduces the Golgi residence time of a Tac chimera suggests that N-glycans might have a similar effect. Therefore, both O- and N-glycans might function as a generic Golgi export signal at the trans-Golgi to promote the constitutive exocytic trafficking. Ministry of Education (MOE) This work was supported by the following grants to L.L.: MOE AcRF Tier1 RG35/17, Tier2 MOE2015-T2-2-073 and Tier2 MOE2018-T2-2- 026.

A rise in cytosolic Ca2+ concentration is a universal signalling mechanism that regulates myriad physiological responses including exocytosis, contraction, cell growth and cell death. Cytosolic Ca2+ signals often occur in the form of repetitive Ca2+ transients, or oscillations, particularly following activation of G-protein-coupled receptors that engage the phospholipase C pathway. Ca2+ oscillations have also been seen within organelles including mitochondria and the nucleus. It is well established that signalling information is contained within the amplitude and frequency of the Ca2+ oscillations but recent studies reveal that the spatial profile of the Ca2+ transients is also used to selectively activate downstream responses.

The cell wall is essential for cell viability and pathogenesis of fungi. It was previously shown that the exocytosis landmark Sec3 is an effector of the cell wall integrity (CWI) master regulator Rho1 GTPase. However, disruption of the interaction between Sec3 and Rho1 did not inhibit exocytic secretion and cell growth. The physiological role of Sec3 in fungi is unclear. We have examined the growth, cell wall sensitivity, exocyst localization, and exocytic secretion of Sec3-binding deficient rho1 mutants and Rho1-binding deficient sec3 mutants. We found that the Sec3 N-terminal deletion mutant was defective in cell wall integrity. The cells harboring binding mutation between Rho1 and Sec3 N-terminus were sensitive to cell wall antagonists. We also found that the polarized localization of exocyst subunits was disrupted in these mutants. Our study demonstrates that the N-terminus of Sec3 mediates cell wall integrity in yeast. Pathogenic fungi may use similar regulatory mechanisms because components of the exocytic signaling pathways are conserved.

Like other cells, fungal hyphae show exquisite sensitivity to their environment. This reactiveness is demonstrated at many levels, from changes in the form of the hypha resulting from alterations in patterns of exocytosis, to membrane excitation, and mechanisms of wound repair. Growing hyphae detect ridges on surfaces and respond to restrictions in their physical space. These are expressions of cellular consciousness. Fungal mycelia show decision-making and alter their developmental patterns in response to interactions with other organisms. Mycelia may even be capable of spatial recognition and learning coupled with a facility for short-term memory. Now is a fruitful time to recognize the study of fungal ethology as a distinctive discipline within mycology.

In a presynaptic nerve terminal, the active zone is composed of sophisticated protein machinery that enables secretion on a sub-millisecond time scale and precisely targets it towards postsynaptic receptors. The past two decades have provided deep insight into the roles of active zone proteins in exocytosis, but we are only beginning to understand how a neuron assembles active zone protein complexes into effective molecular machines. In this review, we outline the fundamental processes that are necessary for active zone assembly and discuss recent advances in understanding assembly mechanisms that arise from genetic, morphological and biochemical studies. We further outline the challenges ahead for understanding this important problem.

Phosphoinositides are important signaling molecules involved in the regulation of vesicular trafficking. It has been implicated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in insulin-regulated GLUT4 translocation in adipocytes. However, it remains unclear where and how PI(4,5)P2 regulates discrete steps of GLUT4 vesicle translocation in adipocytes, especially on the exocytic arm of regulation. Here, we employed optogenetic tools to acutely control the PI(4,5)P2 metabolism in living cells. By combination of TIRFM imaging, we were able to monitor the temporal-spatial-dependent PI(4,5)P2 regulation on discrete steps of GLUT4 translocation in adipocytes. We found that the plasma membrane localized PI(4,5)P2 is crucial for proper insulin signaling propagation and for insulin-stimulated GLUT4 vesicle translocation in 3T3-L1 adipocytes. Global depletion of PI(4,5)P2 on the cell surface blunted insulin-stimulated Akt phosphorylation and abolished insulin effects in promotion of the docking and fusion of GLUT4 vesicle with the plasma membrane. Furthermore, by development of a novel optogenetic module to selectively modulate PI(4,5)P2 levels on the GLUT4 vesicle docking site, we identified an important regulatory role of PI(4,5)P2 in controlling of vesicle docking process. Local depletion of PI(4,5)P2 at the vesicle docking site promoted GLUT4 vesicle undocking, diminished insulin-stimulated GLUT4 vesicle docking and fusion, but without perturbation of insulin signaling propagation in adipocytes. Our results provide strong evidence that cell surface PI(4,5)P2 plays two distinct functions on regulation of the exocytic trafficking of GLUT4 in adipocytes. PI(4,5)P2 not only regulates the proper activation of insulin signaling in general but also controls GLUT4 vesicle docking process at the vesicle-membrane contact sites.

Extracellular adenosine triphosphate (ATP) participates in maintaining the vascular tone in the CNS, particularly in the retina, via the tonic activity of ligand gated activated P2X1 receptors. P2X1 receptors are characterized by their high affinity for ATP and their strong desensitization to concentrations of ATP that are 200-fold lower than their EC50. The mechanism behind P2X1 tonic activity remains unclear. In this study, we expressed human P2X1 (hP2X1) homomeric receptors in Xenopus oocytes to explore the relationship between ATP release from oocytes at rest, hP2X1, and Ca2+-activated Cl− channels. Our results indicate that Xenopus oocytes release ATP at rest via vesicular exocytosis, and this process is a constitutive phenomenon independent of extracellular Ca2+. Our results also indicate that hP2X1 receptors are able to sustain a tonic activity of Ca2+-activated Cl− channels. In the presence of extracellular Ca2+ the activity of hP2X1 receptors is greatly amplified by its coupling with Ca2+-activated Cl− channels. Future studies addressing the relationship between hP2X1 receptors and Ca2+-activated Cl− channels in vascular smooth muscle cells should provide information about additional mechanisms that regulate the vascular tone and their potential as pharmaceutical targets. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Insight into the exocytosis process of diagnostical nanomaterials is critical for their clinical transformation. However, it is unclear whether nanomaterials will be eliminated during cell migration. Herein, gold nanoparticles (AuNPs) were utilized as plasmonic imaging probes and dark-field microscopy was applied to examine the movement as well as exocytosis of AuNPs during cell migration. The results showed that internalized AuNPs could localize into the retraction fibers. The mobility of nanoparticles reduced with the increased distance from the cell body. When the retraction fibers disconnected from the cell body, AuNPs were left outside the cell. This study demonstrated the migracytosis of nanomaterials, which could benefit the future design of safer and more efficient nanomaterials for diagnostics.

Background We reported that the pancreas of the interferon-regulatory factor (IRF) 2 knock-out (KO) mouse represents an early phase of acute pancreatitis, including defective regulatory exocytosis, intracellular activation of trypsin, and disturbance of autophagy. The significantly upregulated and downregulated genes in the IRF2 KO pancreas have been reported. The catalogue of gene transcripts included two types of calcium-binding proteins (S100 calcium binding protein G [S100g] and Annexin A10 [Anxa10]), which were highly upregulated in the IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and its disturbance is related to pancreatitis, we then evaluated the role of S100g and Anxa10 in acute pancreatitis. Method We induced cerulein-pancreatitis in wild-type mice and examined the changes in the expression of these genes by qPCR and immunohistochemistry. We constructed S100g-overexpressing or Anxa10-overexpressing AR42J cells (AR42J-S100g, AR42J-Anxa10). We examined the changes in amylase secretion, intracellular calcium ([Ca2+]i), and cell viability in these cells, when incubated with cholecystokinin (CCK). Results The expression of S100g and Anxa10 was increased in cerulean-induced pancreatitis. The acini were patchily stained for S100g and the cytosol of acini was evenly but weakly stained for Anxa10. Stimulation with 100pM CCK-8, decreased amylase secretion and inhibited the [Ca2+]i increase in AR42J-S100g cells. These effects were weak in AR42J-Anxa10 cells. Cell viability was not changed by incubation with cerulein. Conclusion In cerulean pancreatitis, the expression of S100g and Anxa10 was induced in the acini. S100g may work as a Ca2+ buffer in acute pancreatitis.

We previously found that VAMP721/722 SNARE proteins guide secretory vesicles to pathogen-attacking sites during immune responses in Arabidopsis, which suggests that these vesicles should deliver immune molecules. However, the lethality of vamp721 vamp722 double null mutant makes it difficult to understand the nature of cargo transported via VAMP721/722 vesicles. Since VAMP721/722-depleted (VAMP721+/-VAMP722-/- and VAMP721-/-VAMP722+/-) plants show compromised resistance to extracellular pathogens, we assume that an immune protein secreted through the VAMP721/722-engaged exocytosis would be remained more in VAMP721/722-depleted plants than WT. By comparing intracellular proteins between WT and VAMP721/722-depleted plants, we found caffeoyl-CoA O-methyltransferase 1 (CCOAOMT1) involved in the lignin biosynthesis was more abundantly detected in both VAMP721/722-depleted lines than WT. Plants are well-known to deposit secondary cell walls as physical barriers at pathogen-attempting sites. Therefore, extracellular detection of CCOAOMT1 and impaired resistance to Pseudomonas syringae DC3000 in ccoaomt1 plants suggest that plants secrete cell wall-modifying enzymes at least including CCOAOMT1 to reinforce the secondary cell walls for immunity.

Cochlear ribbon synapses formed between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are immature at birth and they require dramatic morphological and functional developments to achieve auditory maturation in postnatal mice. However, the mechanism underlying this remodeling process of cochlear ribbon synapse remains elusive. Here, we report that autophagy is necessary for the development and maturation of cochlear ribbon synapses in mice. In this study, significantly high levels of LC3B (a widespread marker of autophagy) were found in the cochlea from postnatal day 1 (P1) to P15, which then decreased at P28 to P30. Treatment of mice at P7 with rapamycin or 3-methyladenine (activator and inhibitor of autophagy, respectively) for 7 days led to the significant elevations of hearing threshold across frequencies from P15 to P30. Moreover, abnormal morphology of cochlear ribbon synapses and reduced IHC exocytosis function were detected from P15 to P30, which were likely associated to hearing impairment. Thus, our study demonstrated that autophagy was required for remodeling of cochlear ribbon synapses and provided a new insight into autophagy-related hearing disorder during auditory development. Furthermore, we implicated a novel therapeutic target for sensorineural hearing loss.

Hyperhidrosis is a dermatological condition defined by excessive sweating beyond thermoregulatory needs with significant effects on patients' quality of life. Hyperhidrosis is categorized as primary or secondary: primary hyperhidrosis is mostly focal and idiopathic, whereas secondary hyperhidrosis is commonly generalized and caused by an underlying medical condition or use of medications. Various surgical and nonsurgical therapies exist for primary hyperhidrosis. Although botulinum toxin is one of the deadliest toxins known, when used in small doses, it is one of the most effective therapies for primary hyperhidrosis. Botulinum toxin injections are widely used as a second-line primary hyperhidrosis treatment option once topical treatment strategies have failed. This article provides an overview of the commercially available botulinum toxin formulations and their applications in the treatment of primary hyperhidrosis.

17α-ethynylestradiol (EE2), a synthetic hormone that derives from the natural hormone estradiol, has been reported to alter the sex determination, sexual maturity and secondary sexual characteristics of exposed organisms. However, the adverse effects of EE2 on the oocyte quality have not fully determined. Here, we found that EE2 exposure compromised the fertilization capacity of mouse oocytes, while treatment of melatonin remarkably elevated the fertilization rate. Specifically, we observed that EE2 exposure led to the abnormal distribution and premature exocytosis of ovastacin, leading to the reduced number of sperm binding to the EE2-exposed oocytes. In addition, we found that the abundance of Juno, the sperm receptor on the oocyte membrane, was also diminished, which might be another potential cause leading to the fertilization failure of EE2-exposed oocytes. Finally, we demonstrated that melatonin improved the fertilization ability of EE2-exposed oocytes through eliminating the excessive ROS and inhibiting apoptosis.

For metastasis to occur, cancer cells must exocytose proteases, like matrix metalloproteinases (MMPs), that are key in extracellular matrix (ECM) degradation. Growing evidence suggests that cancer cells use distinct spatial and temporal clustering patterns or organizing 'elements' that facilitate secretory vesicle fusion and the subsequent exocytosis of proteins that contribute to metastasis.

As one of the leading causes of morbidity and mortality worldwide, diabetes affects an estimated 422 million adults, and it is expected to continue expanding such that by 2050, 30% of the U.S. population will become diabetic within their lifetime. Out of the estimated 422 million people currently afflicted with diabetes worldwide, about 5% have type 1 diabetes (T1D), while the remaining ~95% of diabetics have type 2 diabetes (T2D). Type 1 diabetes results from the autoimmune-mediated destruction of functional β-cell mass, whereas T2D results from combinatorial defects in functional β-cell mass plus peripheral glucose uptake. Both types of diabetes are now believed to be preceded by β-cell dysfunction. T2D is increasingly associated with numerous reports of deficiencies in the exocytosis proteins that regulate insulin release from β-cells, specifically the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE protein's functionality is further regulated by a variety of accessory factors such as Sec1/Munc18 (SM), double C2-domain proteins (DOC2), and additional interacting proteins at the cell surface that influence the fidelity of insulin release. As new evidence emerges about the detailed mechanisms of exocytosis, new questions and controversies have come to light. This emerging information is also contributing to dialogue in the islet biology field focused on how to correct the defects in insulin exocytosis. Herein we present a balanced review of the role of exocytosis proteins in T2D, with thoughts on novel strategies to protect functional β-cell mass.

Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses.

Insulin promotes glucose uptake by triggering the translocation of glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane through exocytosis. GLUT4 exocytosis is a vesicle fusion event involving fusion of GLUT4-containing vesicles with the plasma membrane. For GLUT4 vesicle fusion to occur, GLUT4 vesicles must first be tethered to the plasma membrane. A key tethering factor in exocytosis is a heterooctameric protein complex called the exocyst. The role of the exocyst in GLUT4 exocytosis, however, remains incompletely understood. Here we first systematically analyzed data from a genome-scale CRISPR screen in HeLa cells that targeted virtually all known genes in the human genome, including 12 exocyst genes. The screen recovered only a subset of the exocyst genes, including exocyst complex component 7 (Exoc7/Exo70). Other exocyst genes, however, were not isolated in the screen, likely because of functional redundancy. Our findings suggest that selection of an appropriate exocyst gene is critical for genetic studies of exocyst functions. Next we developed an inducible adipocyte genome editing system that enabled Exoc7 gene deletion in adipocytes without interfering with adipocyte differentiation. We observed that insulin-stimulated GLUT4 exocytosis was markedly inhibited in Exoc7 KO adipocytes. Insulin signaling, however, remained intact in these KO cells. These results indicate that the exocyst plays a critical role in insulin-stimulated GLUT4 exocytosis in adipocytes. We propose that the strategy outlined in this work could be instrumental in genetically dissecting other membrane-trafficking pathways in adipocytes.

In central synapses, synaptobrevin-2 (also called VAMP-2) is the predominant synaptic vesicle SNARE protein that interacts with the plasma membrane SNAREs, SNAP-25 and syntaxin-1 to execute exocytosis. Mice deficient in synaptobrevin-2 or SNAP-25 show embryonic lethality, which precludes investigation of the complete loss-of-function of these proteins in the adult nervous system. However, mice that carry heterozygous null mutations survive into adulthood and are fertile. In order to elucidate how loss-of-function mutations in these proteins may result in human disease phenotypes it is important to develop bona fide animal models. Therefore, given the importance of these two critical SNAREs in central synaptic transmission and their association with several neurological or neuropsychiatric disorders, we performed a comprehensive behavioral analysis of SNAP-25 heterozygous null (SNAP25(+/−)) mice as well as the synaptobrevin-2 heterozygous null (+/−) mice. This analysis revealed only mild phenotypes, SNAP-25 (+/−) mice exhibited marked hypoactivity, whereas synaptobrevin-2 (+/−) mice showed enhanced performance on the rotarod. The two mouse lines did not manifest significant deficits in anxiety-related behaviors, learning and memory measures, or prepulse inhibition. The rather mild behavioral deficits indicate that these key proteins, SNAP25 and synaptobrevin-2, are expressed in excess to circumvent the impact of potential fluctuations in expression levels on nervous system function.

The twin goals of determining how neural secretion is unique and how proteins in the presynaptic terminal can be targeted in the treatment of neurological disorders are both met in the study of SV2. Unlike the machinery of vesicle fusion, SV2 is unique to regulated secretion and has no functional homolog in the vesicles that mediate membrane trafficking at other cellular locations. SV2 is, to date, the only synaptic vesicle protein that is a viable therapeutic target in the treatment of nervous system diseases. This may be linked to the fact that its function(s) are limited to quiescent synapses. By playing an essential role in regulating initial release probability, SV2 action has special relevance to neural network synchrony. Given the role of aberrant excitability in numerous neurological disorders, the relevance of SV2 to drug development will likely only grow.

Neurons have the remarkable ability to release a batch of neurotransmitters into the synapse immediately after an action potential. This signature event is made possible by the simultaneous fusion of a number of synaptic vesicles to the plasma membrane upon Ca2+ entry into the active zone. The outcomes of both cellular and in vitro studies suggest that soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) and synaptotagmin 1 (Syt1) constitute the minimal fast exocytosis machinery in the neuron. Syt1 is the major Ca2+-sensor and orchestrates the synchronous start of individual vesicle fusion events while SNAREs are the membrane fusion machinery that dictates the kinetics of each single fusion event. The data also suggest that Ca2+-bound Syt1 is involved in the upstream docking step which leads to an increase in the number of fusion events or the size of the release, leaving the SNARE complex alone to carry out membrane fusion by themselves.

Summary The invasion of a suitable host hepatocyte by Plasmodium sporozoites is an essential step in malaria infection. We demonstrate that in infected hepatocytes, lysosomes are redistributed away from the nucleus, and surface exposure of lysosome-associated membrane protein 1 (LAMP1) is increased. Lysosome exocytosis in infected cells occurs independently of sporozoite traversal. Instead, a sporozoite-secreted factor is sufficient for the process. Knockdown of SNARE proteins involved in lysosome-plasma membrane fusion reduces lysosome exocytosis and Plasmodium infection. In contrast, promoting fusion between the lysosome and plasma membrane dramatically increases infection. Our work demonstrates parallels between Plasmodium sporozoite entry of hepatocytes and infection by the excavate pathogen Trypanosoma cruzi and raises the question of whether convergent evolution has shaped host cell invasion by divergent pathogens., Graphical Abstract, Highlights • Plasmodium sporozoites induce host lysosome exocytosis during invasion • Hepatocyte lysosome exocytosis occurs in a SPECT2-independent manner • Inhibition of lysosome-plasma membrane fusion inhibits sporozoite invasion • Secreted parasite factors are sufficient to induce lysosome exocytosis, Biological Sciences; Molecular Biology; Parasitology; Microbiology Parasite; Molecular Microbiology; Cell Biology

Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.

Multilamellar bodies (MLBs) are produced and secreted by many cell types. In this study, we report the existence and ultrastructure of MLBs that are produced by Leydig cells and identification of telocytes in the testicular interstitium of naked mole rat. This study was performed on both breeder and non-breeder male naked mole rats using light microscopy, transmission electron microscopy, and morphometric approaches. In the testicular interstitium, the most prominent cells were Leydig cells, which contained numerous lipid droplets (LDs) in the cytoplasm. We found that MLBs were associated with the LDs of Leydig cells and were secreted into the extracellular or interstitial environment via exocytosis. After their release from Leydig cells, MLBs localized to the space between Leydig cells near blood vessels and attached to telocytes. We also identified telocytes in the testicular interstitium, and their cellular extensions were distributed throughout the interstitium. MLBs were aligned along the cellular extensions of telocytes, and membrane-to-membrane contact was observed between the cellular extensions of telocytes and MLBs, suggesting that telocytes may play a role in the transport of MLBs within the interstitial space. No ultrastructural differences were found in Leydig cells, telocytes, or MLBs between breeder and non-breeder testes. However, morphometric analysis revealed a significant difference in the number of MLBs between the breeder and non-breeder animals. Furthermore, both selective autophagy of LDs and non-selective autophagy were observed in Leydig cells. Typical features of macrolipophagy were also observed, as a few LDs were entirely enclosed by a limiting membrane. Remarkably, autophagy may be a key factor in the biogenesis of MLBs and steroid hormone production. The appearance of MLBs in the testicular interstitium of naked mole rats could thus be related to lipid storage and trafficking.

With the purpose of investigating the bio-interaction between CNC and cells, the end-fluorescence CNC (FCNC) and surface-adsorbed glucosamine [(g)FCNC] were prepared by the region-selective modification and electrostatic adsorption strategy in this study. The cell growth was arrested in a time dependent manner when incubated with CNC in the low glucose environment. The small-size FCNC was preferred by the cells with the observation of higher affinity. Specifically, the affinity of (g)FCNC to the phagocytic cell RAW264.7 cell and kidney derived cell 293 t was generally increased in response to the "Warburg effect", which on the contrary was observed with the weak effect in the tumor cells. We confirmed the exocytosis of internalized rod-like nanocrystal was achieved by the packaging effect of exosomes. Our results revealed the underappreciated bio-interaction between CNC and different cells, which contributed the evidence of an inspiration for this natural nanomaterial in the cell-level application.

The objective of this study is to optimize the cryopreservation of dissociated islet cells and obtain functional cells that can be used in single-cell transcriptome studies on the pathology and treatment of diabetes. Using an iterative graded freezing approach we obtained viable cells after cooling in 10% dimethyl sulfoxide and 6% hydroxyethyl starch at 1°C/min to –40°C, storage in liquid nitrogen, rapid thaw, and removal of cryoprotectants by serial dilution. The expression of epithelial cell adhesion molecule declined immediately after thaw, but recovered after overnight incubation, while that of an endocrine cell marker (HPi2) remained high after cryopreservation. Patch-clamp electrophysiology revealed differences in channel activities and exocytosis of various islet cell types; however, exocytotic responses, and the biophysical properties of voltage-gated Na+and Ca2+channels, are sustained after cryopreservation. Single-cell RNA sequencing indicates that overall transcriptome and crucial exocytosis genes are comparable between fresh and cryopreserved dispersed human islet cells. Thus, we report an optimized procedure for cryopreserving dispersed islet cells that maintained their membrane integrity, along with their molecular and functional phenotypes. Our findings will not only provide a ready source of cells for investigating cellular mechanisms in diabetes but also for bio-engineering pseudo-islets and islet sheets for modeling studies and potential transplant applications.

Measurements of the time elapsed during synaptic transmission has shown that synaptic vesicle (SV) fusion lags behind Ca

Pancreatic beta cells secrete insulin postprandially and during fasting to maintain glucose homeostasis. Although glucose-stimulated insulin secretion (GSIS) has been extensively studied, much less is known about basal insulin secretion. Here, we performed a genome-wide CRISPR/Cas9 knockout screen to identify novel regulators of insulin secretion.To identify genes that cell autonomously regulate insulin secretion, we engineered a Cas9-expressing MIN6 subclone that permits irreversible fluorescence labeling of exocytic insulin granules. Using a fluorescence-activated cell sorting assay of exocytosis in low glucose and high glucose conditions in individual cells, we performed a genome-wide CRISPR/Cas9 knockout screen.We identified several members of the COMMD family, a conserved family of proteins with central roles in intracellular membrane trafficking, as positive regulators of basal insulin secretion, but not GSIS. Mechanistically, we show that the Commander complex promotes insulin granules docking in basal state. This is mediated, at least in part, by its function in ITGB1 recycling. Defective ITGB1 recycling reduces its membrane distribution, the number of focal adhesions and cortical ELKS-containing complexes.We demonstrated a previously unknown function of the Commander complex in basal insulin secretion. We showed that by ITGB1 recycling, Commander complex increases cortical adhesions, which enhances the assembly of the ELKS-containing complexes. The resulting increase in the number of insulin granules near the plasma membrane strengthens basal insulin secretion.

Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P

Trypanosoma brucei brucei (T.b.brucei) is an extra-cellular parasite that causes Animal African Trypanosomiasis (AAT) disease in animals. Till day, this disease is more difficult to treat and control due to lack of efficient vaccines and early diagnosis of the parasite infection. T.b.brucei Excretory/Secretory (ES) proteins were involved in pathogenesis and key for understanding the host-parasite interactions. Functions of T.b.brucei's ES proteins were poorly investigated and experimental identification is expensive and time-consuming. Bioinformatics approaches are cost-effective by facilitating the experimental analysis of potential drug targets for parasitic diseases. Here we applied several bioinformatics tools to predict and functionalize the annotation of 1104 ES proteins and immunoinformatics approaches carried out to predict and evaluate the epitopes in T.b.brucei. Secretory information, functional annotations and potential epitopes of each ES proteins were available at http://tbb.insilico.in . This study provides functional information of T.b.brucei for experimental studies to identify potential targets for diagnosis and therapeutics development.

This is the accepted manuscript of the following article: Folgueira I, Lamas J, De Felipe AP, Sueiro RA, Leiro JM. (2019). Evidence for the role of extrusomes in evading attack by the host immune system in a scuticociliate parasite. Fish Shellfish Immunol. 2019 Jul 5;92:802-812. doi: 10.1016/j.fsi.2019.07.008 Like other ciliates, Philasterides dicentrarchi, the scuticociliate parasite of turbot, produces a feeding-only or growing stage called a trophont during its life cycle. Exposure of the trophonts to heat-inactivated serum extracted from the turbot host and containing specific antibodies that induce agglutination/immobilization leads to the production of a mucoid capsule from which the trophonts later emerge. We investigated how these capsules are generated, observing that the mechanism was associated with the process of exocytosis involved in the release of a matrix material from the extrusomes. The extruded material contains mucin-like glycoproteins that were deposited on the surface of the cell and whose expression increased with time of exposure to the heat-inactivated immune serum, at both protein expression and gene expression levels. Stimulation of the trophonts with the immune serum also caused an increase in discharge of the intracellular storage compartments of calcium necessary for the exocytosis processes in the extrusomes. The results obtained suggest that P. dicentrarchi uses the extrusion mechanism to generate a physical barrier protecting the ciliate from attack by soluble factors of the host immune system. Data on the proteins involved and the potential development of molecules that interfere with this exocytic process could contribute to improving the prevention and control of scuticociliatosis in turbot This study was financially supported by grants from the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional -FEDER- (European Union) (AGL2017-83577-R) and from the Xunta de Galicia (ED431C2017/31) and also by the PARAFISHCONTROL project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634429 SI

Unique functions of specialised cells such as those of the immune and haemostasis systems, skin, blood vessels, lung, and bone require specialised compartments, collectively referred to as lysosome-related organelles (LROs), that share features of endosomes and lysosomes. LROs harbour unique morphological features and cell type-specific contents, and most if not all undergo regulated secretion for diverse functions. Ongoing research, largely driven by analyses of inherited diseases and their model systems, is unravelling the mechanisms involved in LRO generation, maturation, transport and secretion. A molecular understanding of these features will provide targets and markers that can be exploited for diagnosis and therapy of a myriad of diseases.

Glutamic acid modified Pluronic P123 (P123-G) was synthesized to coat hydrophobic mesoporous silica nanoparticles (PGMSN) aiming to target large amino acid transporter 1 (LAT1) overexpressed cancer cells. We discovered that, once internalized, PGMSN could be transported out of the cells with their cargo, and the exported nanoparticles were then taken up by neighboring cells. This intercellular delivery of particles led to a deeper tumor penetration, presumably through tandem cycles of LAT1 mediated endocytosis and exocytosis. Taking advantage of high drug loading capability of MSN, a kind of cyanine dye Cypate acting as a photothermal converting agent was loaded into MSN along with the chemotherapeutic drug doxorubicin hydrochloride (DOX) to improve the anti-cancer effect by the combination of chemotherapy and photothermal therapy (PTT). Both DOX and Cypate stably dispersed in the pores of MSN, and the coating of P123-G serving as diffusion barrier blocked drug preleakage. The system possessed pH and near-infrared (NIR) light dual-responsive DOX release property. Additionally, PGMSN exhibited targeted PTT effect which strengthened cytotoxic activity of the system due to the enhanced endocytosis. So the deep tumor penetration and targeted PTT effect made PGMSN especially suitable for cancers overexpressing LAT1 receptors.

Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca(2+) and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca(2+) sensor that penetrates membranes upon binding Ca(2+) to trigger synchronous vesicle fusion. The closely related Doc2β is a slow-acting, high-affinity Ca(2+) sensor that triggers spontaneous and asynchronous vesicle fusion, but whether it also penetrates membranes is unknown. Both syt1 and Doc2β bind the dynamically regulated plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)), but it is unclear whether PIP(2) serves only as a membrane contact or enables specialized membrane-binding modes by these Ca(2+) sensors. Furthermore, it has been shown that PIP(2) uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca(2+) influx, suggesting that current models for the action of these Ca(2+) sensors are incomplete. Here, using a series of steady-state and time-resolved fluorescence measurements, we show that Doc2β, like syt1, penetrates membranes in a Ca(2+)-dependent manner. Unexpectedly, we observed that PIP(2) can drive membrane penetration by both syt1 and Doc2β in the absence of Ca(2+), providing a plausible mechanism for Ca(2+)-independent, PIP(2)-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca(2+), PIP(2) drives Doc2β, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca(2+) sensor. Our results provide evidence of a novel role for PIP(2) in regulating, and under some circumstances triggering, exocytosis.

Weibel-Palade bodies (WPBs) are specialized secretory organelles of endothelial cells that serve important functions in the response to inflammation and vascular injury. WPBs actively respond to different stimuli by regulated exocytosis leading to full or selective release of their contents. Cellular conditions and mechanisms that distinguish between these possibilities are only beginning to emerge. To address this we analyzed dynamic rearrangements of the actin cytoskeleton during histamine-stimulated, Ca2+-dependent WPB exocytosis. We show that most WPB fusion events are followed by a rapid release of von-Willebrand factor (VWF), the large WPB cargo, and that this occurs concomitant with a softening of the actin cortex by the recently described Ca2+-dependent actin reset (CaAR). However, a considerable fraction of WPB fusion events is characterized by a delayed release of VWF and observed after the CaAR reaction peak. These delayed VWF secretions are accompanied by an assembly of actin rings or coats around the WPB post-fusion structures and are also seen following direct elevation of intracellular Ca2+ by plasma membrane wounding. Actin ring/coat assembly at WPB post-fusion structures requires Rho GTPase activity and is significantly reduced upon expression of a dominant-active mutant of the formin INF2 that triggers a permanent CaAR peak-like sequestration of actin to the endoplasmic reticulum. These findings suggest that a rigid actin cortex correlates with a higher proportion of fused WPB which assemble actin rings/coats most likely required for efficient VWF expulsion and/or stabilization of a WPB post-fusion structure. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

The canonical Phospholipase A2 (PLA2) metabolites lysophosphatidylcholine (LPC) and arachidonic acid (ARA) affect regulated exocytosis in a wide variety of cells and are proposed to directly influence membrane merger owing to their respective spontaneous curvatures. According to the Stalk-pore hypothesis, negative curvature ARA inhibits and promotes bilayer merger upon introduction into the distal or proximal monolayers, respectively; in contrast, with positive curvature, LPC has the opposite effects. Using fully primed, release-ready native cortical secretory vesicles (CV), well-established fusion assays and standardized lipid analyses, we show that exogenous ARA and LPC, as well as their non-metabolizable analogous, ETYA and ET-18-OCH3, inhibit the docking/priming and membrane merger steps, respectively, of regulated exocytosis.

Background and purpose Studies demonstrated that oxidative damage decreased intracellular ATP level in astrocytes. However, the pathway mediated ATP level decrease is obscure. Our previous study found intracellular ATP could be released via lysosome exocytosis in astrocytes. Here, we explored whether lysosome exocytosis was involved in ATP release during oxidative stress induced by H2O2 in astrocytes. Methods Astrocytes were isolated from the cortex of neonatal rats. Intracellular lysosomes and calcium signals were stained in astrocytes before and after H2O2 stimulation. ATP molecules location and ATP level were detected by immunostaining and bioluminescence method, respectively. Extracellular β-Hexosaminidase and LDH were examined by colorimetric method. Results We found that ATP located in lysosome of astrocytes. H2O2 stimulation resulted in the decrease of lysosomes staining and the increase of extracellular ATP, compared to the control (p Conclusion Our results indicated that H2O2 induced ATP release from intracellular to extracellular via lysosome exocytosis. The increase of intracellular Ca2+ was necessary for lysosome release under oxidative stress induced by H2O2.