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Resistant starch (RS) is present in various natural and processed foods as well as medications. It has garnered significant attention from both scientists and consumers due to its notable health benefits. However, there is a limited understanding of how RS particles are absorbed at the cellular level and their metabolic behavior, resulting in a lack of clarity regarding the intestinal safety implications of prolonged RS exposure. Here, we demonstrate that rice-derived RS nanoparticles (RSNs) can lead to colitis in mice by triggering lysosomal exocytosis. The research shows that RSNs enter the cells through macropinocytosis and clathrin- and caveolin-mediated endocytosis and activate TRPML1 thereafter, causing the release of lysosomal calcium ions. This, in turn, triggered the TFEB signaling pathway and thus upregulated the lysosomal exocytosis level, leading to lysosomal enzymes to be released to the intestinal lumen. As a result, a decreased number of intestinal goblet cells, diminished tight junction protein expression, and imbalanced intestinal flora in mice were observed. These damages to the intestinal barrier ultimately led to the occurrence of colitis. Our study offers important insights into the cellular bioeffects and detrimental effects on intestinal health caused by RS particles and emphasizes the need to re-evaluate the safety of long-term RS consumption.

As the most prescribed psychotropic drugs in current medical practice, antidepressant drugs (ADs) of the selective serotonin reuptake inhibitor (SSRI) class represent prime candidates for drug repurposing. The mechanisms underlying their mode of action, however, remain unclear. Here, we show that common SSRIs and selected representatives of other AD classes bidirectionally regulate fluid-phase uptake at therapeutic concentrations and below. We further characterize membrane trafficking induced by a canonical SSRI fluvoxamine to show that it involves enhancement of clathrin-mediated endocytosis, endosomal system, and exocytosis. RNA sequencing analysis showed few fluvoxamine-associated differences, consistent with the effect being independent of gene expression. Fluvoxamine-induced increase in membrane trafficking boosted transcytosis in cell-based blood-brain barrier models, while a single injection of fluvoxamine was sufficient to enable brain accumulation of a fluid-phase fluorescent tracer in vivo. These findings reveal modulation of membrane trafficking by ADs as a possible cellular mechanism of action and indicate their clinical repositioning potential for regulating drug delivery to the brain., (© 2024. The Author(s).)

In assisted fertility protocols, in vitro culture conditions mimic physiological conditions to preserve gametes in the best conditions. After collection, oocytes are maintained in a culture medium inside the incubator until in vitro fertilization (IVF) is performed. This time outside natural and physiological conditions exposes oocytes to an oxidative stress that renders in vitro aging. It has been described that in vitro aging produces a spontaneous cortical granule (CG) release decreasing the fertilization rate of oocytes. Nevertheless, this undesirable phenomenon has not been investigated, let alone prevented. In this work, we characterized the spontaneous CG secretion in in vitro aged oocytes. Using immunofluorescence indirect, quantification, and functional assays, we showed that the expression of regulatory proteins of CG exocytosis was affected. Our results demonstrated that in vitro oocyte aging by 4 and 8 h altered the expression and localization of alpha-SNAP and reduced the expression of NSF and Complexin. These alterations were prevented by supplementing culture medium with dithiothreitol (DTT), which in addition to having a protective effect on those proteins, also had an unexpected effect on the actin cytoskeleton. Indeed, DTT addition thickened the cortical layer of fibrillar actin. Both DTT effects, together, prevented the spontaneous secretion of CG and recovered the IVF rate in in vitro aged oocytes. We propose the use of DTT in culture media to avoid the spontaneous CG secretion and to improve the success rate of IVF protocols in in vitro aged oocytes., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Serotonin, a monoamine neurotransmitter, is important in both the central nervous system (CNS) and the peripheral nervous system. Malfunction of serotonin signaling leads to various disorders. We studied serotonin signaling from serotonergic neurons inside the ventral nerve cord of Drosophila melanogaster. Serotonergic neurons and stimulated release were visualized and achieved with mCherry and channelrhodopsin-2 (an optogenetically transfected ion channel), respectively, and two electrochemical techniques quantified serotonin release and vesicular content. Mean vesicular serotonin content released during exocytosis from these neurons was 84 %, considerably higher than reported in previous studies regarding octopamine (4.5 %) and glutamate release (31 %). Serotonin content within all vesicles is uniformly changed when serotonin concentration is inhibited or enhanced. However, serotonin release exhibits two Gaussian distributions: higher frequency of small release events, and similar or slightly higher frequency of large events, resulting in differential release fractions ranging from partial (13-18 %) to full (100 %) release after treatment with agents to either enhance or diminish release. This is the first example of consistent full exocytotic release events we have observed in any system. We suggest one pool of vesicles can release significantly diverse fractions of transmitter load during exocytosis, a potentially novel pathway to regulate exocytosis and neuronal signaling., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Afferent synapses between inner hair cells (IHCs) and the type I spiral ganglion neurons (SGNs) in the cochlea provide over 95% of sensory signals for auditory perception in the brain. However, these afferent synapses are particularly vulnerable to damage, for example from excitotoxicity, and exposure to noise in the environment which often leads to noise-induced cochlear synaptopathy (NICS). In this study, we simulated excitotoxic trauma by incubating kainic acid, a non-desensitizing agonist for AMPA type glutamate receptors on cultured cochleae. The possible protective effects of amitriptyline against NICS were examined. We found that, in IHCs, amitriptyline reversed the decrease of Ca 2+ current and exocytosis caused by excitotoxic trauma. In SGNs, amitriptyline promoted the recovery of neurite loss caused by excitotoxic trauma. Furthermore, we found that the protective effects of amitriptyline are likely mediated by suppressing apoptosis factors that were upregulated during excitotoxic trauma. In conclusion, our results suggest that amitriptyline could protect afferent synapses in the cochlea from NICS, making it a potential drug candidate for hearing protection., (© 2024 Federation of European Biochemical Societies.)

β2-Adrenoceptors (β2-ARs) are the most abundant subtype of adrenergic receptors in skeletal muscles. Their activation via a stabilization of postsynaptic architecture has beneficial effects in certain models of neuromuscular disorders. However, the ability of β2-ARs to regulate neuromuscular transmission at the presynaptic level is poorly understood. Using electrophysiological recordings and fluorescent FM dyes, we found that β2-AR activation with fenoterol enhanced an involvement of synaptic vesicles in exocytosis and neurotransmitter release during intense activity at the neuromuscular junctions of mouse diaphragm. This was accompanied by an improvement of contractile responses to phrenic nerve stimulation (but not direct stimulation of the muscle fibers) at moderate-to-high frequencies. β2-ARs mainly reside in lipid microdomains enriched with cholesterol and sphingomyelin. The latter is hydrolyzed by sphingomyelinases, whose upregulation occurs in many conditions characterized by muscle atrophy and sympathetic nerve hyperactivity. Sphingomyelinase treatment reversed the effects of β2-AR agonist on the neurotransmitter release and synaptic vesicle recruitment to the exocytosis during intense activity. Inhibition of G i protein with pertussis toxin completely prevented the sphingomyelinase-mediated inversion in the β2-AR agonist action. Note that lipid raft disrupting enzyme cholesterol oxidase had the same effect on β2-AR agonist-mediated changes in neurotransmission as sphingomyelinase. Thus, β2-AR agonist fenoterol augmented recruitment and release of synaptic vesicles during intense activity in the diaphragm neuromuscular junctions. Sphingomyelin hydrolysis inversed the effects of β2-AR agonist on neurotransmission probably via switching to G i protein-dependent signaling. This phenomenon may reflect a dependence of the β2-AR signaling on lipid raft integrity in the neuromuscular junctions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background/aims: Lemons ( Citrus limon ) contain various nutrients and are among the most popular citrus fruit. Besides their antioxidant, anticancer, antibacterial, and anti-inflammatory properties, clinical studies have indicated their anti-allergic properties., Methods: Using the differential-interference contrast (DIC) microscopy, we examined the effects of lemon juice and peel constituents, such as citric acid, ascorbic acid, hesperetin and eriodictyol, on the degranulation from rat peritoneal mast cells. Using fluorescence imaging with a water-soluble dye, Lucifer Yellow, we also examined their effects on the deformation of the plasma membrane., Results: Lemon juice dose-dependently decreased the number of degranulated mast cells. At concentrations equal to or higher than 0.25 mM, citric acid, hesperetin, and eriodictyol significantly reduced the number of degranulating mast cells in a dose-dependent manner, while ascorbic acid required much higher doses to exert significant effects. At 1 mM, citric acid, hesperetin, and eriodictyol almost completely inhibited exocytosis and washed out the Lucifer Yellow trapped on the mast cell surface, while ascorbic acid did not., Conclusion: This study provides in vitro evidence for the first time that lemon constituents, such as citric acid, hesperetin, and eriodictyol, potently exert mast cell-stabilizing properties. These properties are attributable to their inhibitory effects on plasma membrane deformation in degranulating mast cells., Competing Interests: The authors have no competing interests to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic G i protein-coupled P 2 Y 13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P 2 Y 13 receptors (MRS 2211), Ca 2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca 2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca 2+ with subsequent elevation of ROS levels. The elevation of [Ca 2+ ] in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of G i protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P 2 Y 13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P 2 Y 13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P 2 Y 13 receptor/G i protein/Ca 2+ /protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Purpose: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated., Experimental Approach: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca 2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca 2+ was examined in PC12 cells transfected with a membrane-tethered Ca 2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca 2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct., Key Results: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca 2+ . A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca 2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca 2+ microdomains inhibited by A740003., Conclusion and Implications: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca 2+ microdomains., (© 2024 British Pharmacological Society.)

Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental in vivo and in vitro studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the in vitro validation of our findings to improve therapeutic efficacy and safety of p5.

Unveiling molecular mechanisms that dominate protein phase dynamics has been a pressing need for deciphering the intricate intracellular modulation machinery. While ions and biomacromolecules have been widely recognized for modulating protein phase separations, effects of small molecules that essentially constitute the cytosolic chemical atmosphere on the protein phase behaviors are rarely understood. Herein, we report that vitamin C (V C ), a key small molecule for maintaining a reductive intracellular atmosphere, drives reentrant phase transitions of myosin II/F-actin (actomyosin) cytoskeletons. The actomyosin bundle condensates dissemble in the low-V C regime and assemble in the high-V C regime in vitro or inside neuronal cells, through a concurrent myosin II protein aggregation-dissociation process with monotonic V C concentration increase. Based on this finding, we employ in situ single-cell and single-vesicle electrochemistry to demonstrate the quantitative modulation of catecholamine transmitter vesicle exocytosis by intracellular V C atmosphere, i.e., exocytotic release amount increases in the low-V C regime and decreases in the high-V C regime. Furthermore, we show how V C regulates cytomembrane-vesicle fusion pore dynamics through counteractive or synergistic effects of actomyosin phase transitions and the intracellular free calcium level on membrane tensions. Our work uncovers the small molecule-based reversive protein phase regulatory mechanism, paving a new way to chemical neuromodulation and therapeutic repertoire expansion.

During the process of decidualization, the stromal cells of the endometrium change dynamically to create a favorable environment for embryo implantation. Lysosome activity has often been associated with physiological changes in the endometrium during the preimplantation period and early pregnancy. In this study, the effect of para-nonylphenol (p-NP), an endocrine disruptor, on human immortalized endometrial stromal cells (tHESCs) was investigated. After exposure to p-NP (1 nM and 1 pM), the cells were examined for the decidualization markers connexin-43, insulin like growth factor binding protein 1 (IGFBP1), and prolactin. In addition, the effect of p-NP on lysosome biogenesis and exocytosis was investigated by examining the expression and localization of the transcription factor EB (TFEB) and that of the lysosomal-associated membrane protein 1 (LAMP-1). Finally, we evaluated the effect of p-NP on extracellular matrix (ECM) remodeling using a fibronectin assay. Our results showed that p-NP reduced the expression of prolactin protein, increased the nuclear localization of TFEB, and induced the increase and translocation of the lysosomal protein LAMP-1 to the membrane of tHESCs. The data indicate an impairment of decidualization and suggest an increase in lysosomal biogenesis and exocytosis, which is supported by the higher release of active cathepsin D by tHESCs. Given the importance of cathepsins in the processing and degradation of the ECM during trophoblast invasiveness and migration into the decidua, our results appear to be clear evidence of the negative effects of p-NP on endometrial processes that are fundamental to reproductive success and the establishment of pregnancy. NEW & NOTEWORTHY Endocrine disruptors, such as para-nonylphenol, affect the decidualization of human endometrial stromal cells with an impact on decidualization itself, lysosome biogenesis and exocytosis, and extracellular matrix remodeling. All these alterations may negatively impact embryo implantation with the success of reproduction and the establishment of pregnancy.

Exocytosis is a critical factor for designing efficient nanocarriers and determining cytotoxicity. However, the research on the exocytosis mechanism of nanoparticles, especially the role of long non-coding RNAs (lncRNAs), has not been reported. In this study, the exocytosis of AuNPs in the KYSE70 cells and the involved molecular pathways of exocytosis are analyzed. It demonstrates that nanoparticles underwent time-dependent release from the cells by exocytosis, and the release of β-hexosaminidase confirms that AuNPs are excreted through lysosomes. Mechanistic studies reveal that lncRNA ESCCAL-1 plays a vital role in controlling the exocytosis of AuNPs through activation of the MAPK pathway, including the phosphorylation of ERK and JNK. The study implies that the ESCCAL-1-mediated pathway plays an important role in the exocytosis of AuNPs in KYSE70 cells. This finding has implications for the role of ESCCAL-1 on the drug resistance of esophagus cancer by controlling lysosome-mediated exocytosis., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the author(s)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca 2+ -dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca 2+ ] i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca 2+ ] i microdomains around single or clustered Ca 2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca 2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca 2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca 2+ spikes or incidentally overlap with subplasmalemmal Ca 2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca 2+ microdomains around voltage-gated Ca 2+ channels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background/aims: Adrenaline quickly inhibits the release of histamine from mast cells. Besides β2-adrenergic receptors, several in vitro studies also indicate the involvement of α-adrenergic receptors in the process of exocytosis. Since exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of drugs would determine their mast cell-stabilizing properties., Methods: Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of adrenaline on the degranulation of mast cells and the increase in the Cm during exocytosis. We also examined the degranulation of mast cells in the presence or absence of α-adrenergic receptor agonists or antagonists., Results: Adrenaline dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells, which was almost completely erased in the presence of butoxamine, a β2-adrenergic receptor antagonist. Among α-adrenergic receptor agonists or antagonists, high dose prazosin, a selective α1-adrenergic receptor antagonist, significantly reduced the ratio of degranulating mast cells and suppressed the increase in the Cm. Additionally, prazosin augmented the inhibitory effects of adrenaline on the degranulation of mast cells., Conclusion: This study provided electrophysiological evidence for the first time that adrenaline dose-dependently inhibited the process of exocytosis, confirming its usefulness as a potent mast cell-stabilizer. The pharmacological blockade of α1-adrenergic receptor by prazosin synergistically potentiated such mast cell-stabilizing property of adrenaline, which is primarily mediated by β2-adrenergic receptors., Competing Interests: The authors declared no competing interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5β1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5β1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lithium is a mood stabilizer broadly used to prevent and treat symptoms of mania and depression in people with bipolar disorder (BD). Little is known, however, about its mode of action. Here, we analyzed the impact of lithium on synaptic vesicle (SV) cycling at presynaptic terminals releasing glutamate, a neurotransmitter previously implicated in BD and other neuropsychiatric conditions. We used the pHluorin-based synaptic tracer vGpH and a fully automated image processing pipeline to quantify the effect of lithium on both SV exocytosis and endocytosis in hippocampal neurons. We found that lithium selectively reduces SV exocytic rates during electrical stimulation, and markedly slows down SV recycling post-stimulation. Analysis of single-bouton responses revealed the existence of functionally distinct excitatory synapses with varying sensitivity to lithium-some terminals show responses similar to untreated cells, while others are markedly impaired in their ability to recycle SVs. While the cause of this heterogeneity is unclear, these data indicate that lithium interacts with the SV machinery and influences glutamate release in a large fraction of excitatory synapses. Together, our findings show that lithium down modulates SV cycling, an effect consistent with clinical reports indicating hyperactivation of glutamate neurotransmission in BD., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: Neurotransmitter release from the synaptic vesicles can occur through two modes of exocytosis: "full-collapse" or "kiss-and-run". Here we investigated how increasing the nerve activity and pharmacological stimulation of adrenoceptors can influence the mode of exocytosis in the motor nerve terminal., Methods: Recording of endplate potentials with intracellular microelectrodes was used to estimate acetylcholine release. A fluorescent dye FM1-43 and its quenching with sulforhodamine 101 were utilized to visualize synaptic vesicle recycling., Key Findings: An increase in the frequency of stimulation led to a decrease in the rate of FM1-43 unloading despite the higher number of quanta released. High frequency activity promoted neurotransmitter release via the kiss-and-run mechanism. This was confirmed by experiments utilizing (I) FM1-43 dye quencher, that is able to pass into the synaptic vesicle via fusion pore, and (II) loading of FM1-43 by compensatory endocytosis. Noradrenaline and specific α2-adrenoreceptors agonist, dexmedetomidine, controlled the mode of synaptic vesicle recycling at high frequency activity. Their applications favored neurotransmitter release via full-collapse exocytosis rather than the kiss-and-run pathway., Significance: At the diaphragm neuromuscular junctions, neuronal commands are translated into contractions necessary for respiration. During stress, an increase in discharge rate of the phrenic nerve shifts the exocytosis from the full-collapse to the kiss-and-run mode. The stress-related molecule, noradrenaline, restricts neurotransmitter release in response to a high frequency activity, and prevents the shift in the mode of exocytosis through α2-adrenoceptor activation. This may be a component of the mechanism that limits overstimulation of the respiratory system during stress., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The fusion of membranes is a central part of the physiological processes involving the intracellular transport and maturation of vesicles and the final release of their contents, such as neurotransmitters and hormones, by exocytosis. Traditionally, in this process, proteins, such SNAREs have been considered the essential components of the fusion molecular machinery, while lipids have been seen as merely structural elements. Nevertheless, sphingosine, an intracellular signalling lipid, greatly increases the release of neurotransmitters in neuronal and neuroendocrine cells, affecting the exocytotic fusion mode through the direct interaction with SNAREs. Moreover, recent studies suggest that FTY-720 (Fingolimod), a sphingosine structural analogue used in the treatment of multiple sclerosis, simulates sphingosine in the promotion of exocytosis. Furthermore, this drug also induces the intracellular fusion of organelles such as dense vesicles and mitochondria causing cell death in neuroendocrine cells. Therefore, the effect of sphingosine and synthetic derivatives on the heterologous and homologous fusion of organelles can be considered as a new mechanism of action of sphingolipids influencing important physiological processes, which could underlie therapeutic uses of sphingosine derived lipids in the treatment of neurodegenerative disorders and cancers of neuronal origin such neuroblastoma.

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia-a locus for craniofacial sensory nerves-were used to examine how NGF affects the Ca 2+ -dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca 2+ -dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca 2+ -independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181-197 and 198-206) to support CAP-evoked Ca 2+ -dependent exocytosis at different intensities of stimulation.

In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC 50 ) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.

Alpha-synuclein (α-Syn) localizes at presynaptic terminal and modulates synaptic functions. Increasing evidence demonstrate that α-Syn oligomers, forming at the early of aggregation, are cytotoxic and is thus related to brain neurodegenerative diseases. Herein, we find that vitamin D (VD) can reduce neurocytotoxicity. The reduced neurocytotoxicity might be attributed to the less amount of large-sized α-Syn oligomers inhibited by VD, measured by electrochemical collision at single particle level, which are not observable with traditionally ensembled method. Single-cell amperometry (SCA) results show that VD can recover the amount of neurotransmitter release during exocytosis induced by α-Syn oligomers, further verifying the neuroprotection of VD. Our study reveals the neuroprotective role of VD through inhibiting α-Syn aggregation, which is envisioned to be of great importance in treatment and prevention of the neurodegenerative diseases., (© 2021 Wiley-VCH GmbH.)

Vitamin D 3 is associated with improvements in insulin resistance and glycemia. In this study, we investigated the short-term effect of 1α,25(OH) 2 Vitamin D 3 (1,25-D 3 ) and cholecalciferol (vitamin D 3 ) on the glycemia and insulin sensitivity of control and dexamethasone-induced insulin-resistance rats. 45 Ca 2+ influx responses to 1,25-D 3 and its role in insulin secretion were investigated in isolated pancreatic islets from control rats. In vivo, 5 d treatment with 1,25-D 3 (i.p.) prevented insulin resistance in dexamethasone-treated rats. Treatment with 1,25-D 3 improved the activities of hepatic enzymes, serum lipids and calcium concentrations in insulin-resistant rats. 25-D 3 (o.g.) does not affect insulin resistance. In pancreatic islets, 1,25-D 3 increased insulin secretion and stimulated rapid response 45 Ca 2+ influx. The stimulatory effect of 1,25-D 3 on 45 Ca 2+ influx was decreased by diazoxide, apamine, thapsigargin, dantrolene, 2-APB, nifedipine, TEA, PKA, PKC, and cytoskeleton inhibitor, while it was increased by glibenclamide and N-ethylmaleimide. The stimulatory effect of 1,25-D 3 on 45 Ca 2+ influx involves the activation of L-type VDCC, K + -ATP, K + -Ca 2+, and Kv channels, which augment cytosolic calcium. These ionic changes mobilize calcium from stores and downstream activation of PKC, PKA tethering vesicle traffic and fusion at the plasma membrane for insulin secretion. This is the first study highlighting the unprecedented role of 1,25-D 3 (short-term effect) in the regulation of glucose homeostasis and on prevention of insulin resistance. Furthermore, this study shows the intracellular β-cell signal transduction of 1,25-D 3 through the modulation of pivotal ionic channels and proteins exhibiting a coordinated exocytosis of vesicles for insulin secretion., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca 2+ microdomain (HCMD) generated by Ca 2+ entry through nearby voltage-activated calcium channels. HCMD is efficiently cleared by local mitochondria that avidly take up Ca 2+ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na + /Ca 2+ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K + -depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca 2+ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca 2+ concentrations ([Ca 2+ ] c ) K + -challenged BCCs, and (v) non-inactivated [Ca 2+ ] c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca 2+ handling during K + depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.

The pancreatic β-cells control insulin secretion in the body to regulate glucose homeostasis, and β-cell stress and dysfunction is characteristic of Type 2 Diabetes. Pharmacological targeting of the β-cell to increase insulin secretion is typically utilised, however, extended use of common drugs such as sulfonylureas are known to result in secondary failure. Moreover, there is evidence they may induce β-cell failure in the long term. Within β-cells, insulin secretory granules (ISG) serve as compartments to store, process and traffic insulin for exocytosis. There is now growing evidence that ISG exist in multiple populations, distinct in their protein composition, motility, age, and capacity for secretion. In this review, we discuss the implications of a heterogenous ISG population in β-cells and highlight the need for more understanding into how unique ISG populations may be targeted in anti-diabetic therapies., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) pathology progresses gradually via anatomically connected brain regions. Direct transfer of amyloid-β 1 - 42 oligomers (oAβ) between connected neurons has been shown, however, the mechanism is not fully revealed. We observed formation of oAβ induced tunneling nanotubes (TNTs)-like nanoscaled f-actin containing membrane conduits, in differentially differentiated SH-SY5Y neuronal models. Time-lapse images showed that oAβ propagate from one cell to another via TNT-like structures. Preceding the formation of TNT-like conduits, we detected oAβ - induced plasma membrane (PM) damage and calcium-dependent repair through lysosomal-exocytosis, followed by massive endocytosis to re-establish the PM. Massive endocytosis was monitored by an influx of the membrane-staining dye TMA-DPH and PM damage was quantified by propidium iodide influx in the absence of Ca 2+ . The massive endocytosis eventually caused accumulation of internalized oAβ in Lamp1 positive multivesicular bodies/lysosomes via the actin cytoskeleton remodulating p21-activated kinase1 (PAK1) dependent endocytic pathway. Three-dimensional quantitative confocal imaging, structured illumination superresolution microscopy, and flowcytometry quantifications revealed that oAβ induces activation of phospho-PAK1, which modulates the formation of long stretched f-actin extensions between cells. Moreover, the formation of TNT-like conduits was inhibited by preventing PAK1-dependent internalization of oAβ using the small-molecule inhibitor IPA-3, a highly selective cell-permeable auto-regulatory inhibitor of PAK1. The present study reveals that the TNT-like conduits are probably instigated as a consequence of oAβ induced PM damage and repair process, followed by PAK1 dependent endocytosis and actin remodeling, probably to maintain cell surface expansion and/or membrane tension in equilibrium., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The dense variant surface glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host's antibody response. To explore the epitope space of VSGs, we generate anti-VSG nanobodies and combine high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody causes rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endocytosis and exocytosis are mechanistically linked to Trypanosoma brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrate that this loss of motility is accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)