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A robust and efficient cellular response to lysosomal membrane damage prevents leakage from the lysosome lumen into the cytoplasm. This response is understood to happen through either lysosomal membrane repair or lysophagy. Here we report exocytosis as a third response mechanism to lysosomal damage, which is further potentiated when membrane repair or lysosomal degradation mechanisms are impaired. We show that Connexin43 (Cx43), a protein canonically associated with gap junctions, is recruited from the plasma membrane to damaged lysosomes, promoting their secretion and accelerating cell recovery. The effects of Cx43 on lysosome exocytosis are mediated by a reorganization of the actin cytoskeleton that increases plasma membrane fluidity and decreases cell stiffness. Furthermore, we demonstrate that Cx43 interacts with the actin nucleator Arp2, the activity of which was shown to be necessary for Cx43-mediated actin rearrangement and lysosomal exocytosis following damage. These results define a novel mechanism of lysosomal quality control whereby Cx43-mediated actin remodelling potentiates the secretion of damaged lysosomes. Synopsis: Damaged lysosomes are marked for either membrane repair or removal by lysophagy. This report describes a third pathway, Connexin43-mediated lysosome exocytosis, that is also able to restore cell homeostasis. Lysosomal exocytosis upon injury is enhanced by inhibiting either lysophagy or lysosomal membrane repair. Connexin43 (Cx43) at the plasma membrane is recruited to Galectin3-positive damaged lysosomes via AP2-dependent endocytosis. Exocytosis of damaged lysosomes is potentiated by Cx43 through an Arp2-mediated remodelling of the actin cytoskeleton. Cx43-mediated lysosome exocytosis is relevant in pathological conditions such as Salmonella infection or the lysosomal storage disorder Niemann-Pick disease. Damaged lysosomes can be expelled from the cell in a Connexin43-dependent manner. [ABSTRACT FROM AUTHOR]

Key points Somatostatin, a peptide hormone that activates G‐protein‐coupled receptors, inhibits the secretion of many hormones. This study investigated the mechanisms of this inhibition using amperometry recording of Ca2+‐triggered catecholamine secretion from mouse chromaffin cells. Two distinct stimulation protocols, high‐KCl depolarization and caffeine, were used to trigger exocytosis, and confocal fluorescence imaging was used to monitor the rise in intracellular free Ca2+. Analysis of single‐vesicle fusion events (spikes) resolved the action of somatostatin on fusion pores at different stages. Somatostatin reduced spike frequency, and this reduction was accompanied by prolongation of pre‐spike feet and slowing of spike rise times. This indicates that somatostatin stabilizes initial fusion pores and slows their expansion. This action on the initial fusion pore impacted the release mode to favour kiss‐and‐run over full‐fusion. During a spike the permeability of a fusion pore peaks, declines and then settles into a plateau. Somatostatin had no effect on the plateau, suggesting no influence on late‐stage fusion pores. These actions of somatostatin were indistinguishable between exocytosis triggered by high‐KCl and caffeine, and fluorescence imaging showed that somatostatin had no effect on stimulus‐induced rises in cytosolic Ca2+. Our findings thus demonstrate that the signalling cascades activated by somatostatin target the exocytotic machinery that controls the initial and expanding stages of fusion pores, while having no effect on late‐stage fusion pores. As a result of its stronger inhibition of full‐fusion compared to kiss‐and‐run, somatostatin will preferentially inhibit the secretion of large peptides over the secretion of small catecholamines. Somatostatin inhibits the secretion of various hormones by activating G‐protein‐coupled receptors. In this study, we used amperometry to investigate the mechanism by which somatostatin inhibits catecholamine release from mouse chromaffin cells. Somatostatin increased pre‐spike foot lifetime and slowed fusion pore expansion. Somatostatin inhibited full‐fusion more strongly than kiss‐and‐run. Our results suggest that the initial fusion pore is the target of somatostatin‐mediated regulation of hormone release. The stronger inhibition of full‐fusion by somatostatin will result in preferential inhibition of peptide release. [ABSTRACT FROM AUTHOR]

[Display omitted] Lanthanide-doped up-converting nanoparticles (UCNPs) have emerged as promising biomedical tools in recent years. Most research efforts were devoted to the synthesis of inorganic cores with the optimal physicochemical properties. However, the careful design of UCNPs with the adequate surface coating to optimize their biological performance still remains a significant challenge. Here, we propose the functionalization of UCNPs with four distinct types of surface coatings, which were compared in terms of the provided colloidal stability and resistance to degradation in different biological-relevant media, including commonly avoided analysis in acidic lysosomal-mimicking fluids. Moreover, the influence of the type of particle surface coating on cell cytotoxicity and endocytosis/exocytosis was also evaluated. The obtained results demonstrated that the functionalization of UCNPs with poly(isobutylene- alt -maleic anhydride) grafted with dodecylamine (PMA- g -dodecyl) constitutes an outstanding strategy for their subsequent biomedical application, whereas poly(ethylene glycol) (PEG) coating, although suitable for colloidal stability purposes, hinders extensive cell internalization. Conversely, surface coating with small ligand were found not to be suitable, leading to large degradation degrees of UCNPs. The analysis of particle' behavior in different biological media and in vitro conditions here performed pretends to help researchers to improve the design and implementation of UCNPs as theranostic nanotools. [ABSTRACT FROM AUTHOR]

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 Ca2+ measurements were carried out in Fluo‐4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane‐tethered Ca2+ indicator Lck‐GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ 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 Ca2+. 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 Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ 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 Ca2+ microdomains. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

Following exocytosis, the recapture of plasma membrane‐stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been proposed to act as a ‘pre‐assembly’ mechanism for endocytosis that ensures high‐fidelity retrieval of SV cargo. Here, we used single‐molecule imaging to examine the nanoclustering of synaptotagmin‐1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1K326A/K328A) and SV2A knockdown. SV2A co‐clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2AT84A). Surprisingly, impairing SV2A‐Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin‐1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5‐positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs. [ABSTRACT FROM AUTHOR]

Exocytosis involving the fusion of intracellular vesicles with cell membrane, is thought to be modulated by the mechanical cues in the microenvironment. Single‐cell electrochemistry can offer unique information about the quantification and kinetics of exocytotic events; however, the effects of mechanical force on vesicular release have been poorly explored. Herein, we developed a stretchable microelectrode with excellent electrochemical stability under mechanical deformation by microfabrication of functionalized poly(3,4‐ethylenedioxythiophene) conductive ink, which achieved real‐time quantitation of strain‐induced vesicular exocytosis from a single cell for the first time. We found that mechanical strain could cause calcium influx via the activation of Piezo1 channels in chromaffin cell, initiating the vesicular exocytosis process. Interestingly, mechanical strain increases the amount of catecholamines released by accelerating the opening and prolonging the closing of fusion pore during exocytosis. This work is expected to provide revealing insights into the regulatory effects of mechanical stimuli on vesicular exocytosis. [ABSTRACT FROM AUTHOR]

Fluid and enzyme secretion from exocrine glands is initiated by Ca2+ signalling in acinar cells and is activated by external neural or hormonal signals. A wealth of information has been derived from studies in acutely isolated exocrine cells but Ca2+ signalling has until recently not been studied in undisrupted intact tissue in live mice. Our in vivo observations using animals expressing genetically encoded Ca2+ indicators in specific cell types in exocrine glands revealed both similarities to and differences from the spatiotemporal characteristics previously reported in isolated cells. These in vivo studies facilitate further understanding of how both neuronal and hormonal input shapes Ca2+ signalling events in a physiological setting and how these signals are translated into the stimulation of fluid secretion and exocytosis. [ABSTRACT FROM AUTHOR]

Pathogenic variants in STXBP1 /MUNC18-1 cause severe encephalopathies that are among the most common in genetic neurodevelopmental disorders. Different molecular disease mechanisms have been proposed, and pathogenicity prediction is limited. In this study, we aimed to define a generalized disease concept for STXBP1-related disorders and improve prediction. A cohort of 11 disease-associated and 5 neutral variants (detected in healthy individuals) were tested in 3 cell-free assays and in heterologous cells and primary neurons. Protein aggregation was tested using gel filtration and Triton X-100 insolubility. PRESR (predicting STXBP1-related disorder), a machine learning algorithm that uses both sequence- and 3-dimensional structure–based features, was developed to improve pathogenicity prediction using 231 known disease-associated variants and comparison to our experimental data. Disease-associated variants, but none of the neutral variants, produced reduced protein levels. Cell-free assays demonstrated directly that disease-associated variants have reduced thermostability, with most variants denaturing around body temperature. In addition, most disease-associated variants impaired SNARE-mediated membrane fusion in a reconstituted assay. Aggregation/insolubility was observed for none of the variants in vitro or in neurons. PRESR outperformed existing tools substantially: Matthews correlation coefficient = 0.71 versus <0.55. These data establish intrinsic protein instability as the generalizable, primary cause for STXBP1-related disorders and show that protein-specific ortholog and 3-dimensional information improve disease prediction. PRESR is a publicly available diagnostic tool. [ABSTRACT FROM AUTHOR]

The presynapse performs an essential role in brain communication via the activity‐dependent release of neurotransmitters. However, the sequence of events through which a presynapse acquires functionality is relatively poorly understood, which is surprising, since mutations in genes essential for its operation are heavily implicated in neurodevelopmental disorders. We addressed this gap in knowledge by determining the developmental trajectory of synaptic vesicle (SV) recycling pathways in primary cultures of rat hippocampal neurons. Exploiting a series of optical and morphological assays, we revealed that the majority of nerve terminals displayed activity‐dependent calcium influx from 3 days in vitro (DIV), immediately followed by functional evoked exocytosis and endocytosis, although the number of responsive nerve terminals continued to increase until the second week in vitro. However, the most intriguing discovery was that activity‐dependent bulk endocytosis (ADBE) was only observed from DIV 14 onwards. Importantly, optimal ADBE recruitment was delayed until DIV 21 in Fmr1 knockout neurons, which model Fragile X Syndrome (FXS). This implicates the delayed recruitment of ADBE as a potential contributing factor in the development of circuit dysfunction in FXS, and potentially other neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory autophagy‐mediated exocytosis of IL‐1β, insulin, and TIMP1 (tissue inhibitor of 54 metalloproteinase 1). Other Rab family members participating in secretory autophagy are poorly understood. Herein, we identified 26 overlapped Rab proteins in purified autophagosomes of mouse pancreatic β‐cell "Min‐6" and human lung cancer cell "CL1‐5‐Q89L" with high secretory autophagy tendency by LC–MS/MS proteomics analysis. Six Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, Rab37, and Rab7a) were detected in autophagosomes of four cell lines, associating them with autophagy‐related vesicle trafficking. We used CL1‐5‐Q89L cell line model to evaluate the levels of Rab proteins colocalization with autophagy LC3 proteins and presence in purified autophagosomes. We found five Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, and Rab37) are highly expressed in the autophagosome compared to the normal control by immunoblotting under active secretion conditions. However, only Rab8a, Rab35, and Rab37 showing high colocalization with LC3 protein by cofocal microscopy. Despite the discrepancy between the image and immunoblotting analysis, our data sustains the speculation that Rab8a, Rab11b, Rab27a, Rab35, and Rab37 are possibly associated with the secretory autophagy machinery. In contrast, Rab7a shows low colocalization with LC3 puncta and low level in the autophagosome, suggesting it regulates different vesicle trafficking machineries. Our findings open a new direction toward exploring the role of Rab proteins in secretory autophagy‐related cargo exocytosis and identifying the cargoes and effectors regulated by specific Rab proteins. [ABSTRACT FROM AUTHOR]

Abstract Background Theclinical presentation of atopic dermatitis (AD) varies in different skin phototypes and has been evaluated previously using reflectance confocal microscopy (RCM). Line‐field confocal optical coherence tomography (LC‐OCT) is a new noninvasive imaging modality with additional capabilities. Objectives This prospective cohort study aimed to assess the capabilities of LC‐OCT and RCM in visualizing and quantifying microscopic features of AD lesions. Methods Ten patients with clinically diagnosed AD and visible active lesions were included in the study. LC‐OCT and RCM imaging were performed on lesional and perilesional skin during routine dermatology visits. Image analysis included quantitative measurements of living and total epidermal thickness and dermo‐epidermal junction (DEJ) undulation using LC‐OCT, as well as visual assessment of characteristic features such as spongiosis, exocytosis, perivascular inflammation, and atypical blood vessels using both LC‐OCT and RCM. Results LC‐OCT provided precise measurements of epidermal landmarks and shared similar microscopic features compared to RCM. AD lesions exhibited increased stratum corneum thickness, living epidermis thickness, total epidermis thickness, and DEJ undulation compared to clinically normal skin. Spongiosis, exocytosis, perivascular inflammation, blood vessel proliferation, and dilation were prominent features observed in AD lesions on both LC‐OCT and RCM images. Interestingly, some inflammatory features were also detected in the visually normal skin of AD patients. Conclusions LC‐OCT showed potential as a fast and noninvasive imaging tool for assessing inflammatory skin pathologies, including AD. Its ability to visualize microscopic features and quantify epidermal changes may aid in diagnosis and treatment monitoring. These findings suggest that LC‐OCT could provide valuable insights, particularly for patients with darker skin types where clinical severity assessment may be challenging. Further studies are warranted to explore the full potential of LC‐OCT and its integration into clinical practice.

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 template complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.

Abstract Background The use of stem cell-derived exosomes (Exos) as therapeutic vehicles is receiving increasing attention. Exosome administration has several advantages over cell transplantation, thus making exosomes promising candidates for large-scale clinical implementation and commercialization. However, exosome extraction and purification efficiencies are relatively low, and therapeutic heterogeneity is high due to differences in culture conditions and cell viability. Therefore, in this study, we investigated a priming procedure to enhance the production and therapeutic effects of exosomes from human umbilical cord mesenchymal stem cells (hucMSCs). After preconditioning hucMSCs with agonists/inhibitors that target the Wnt/β-catenin pathway, we assessed both the production of exosomes and the therapeutic efficacy of the optimized exosomes in the context of diabetic wound healing, hoping to provide a safer, more stable and more effective option for clinical application. Results The Wnt signalling pathway agonist CHIR99021 increased exosome production by 1.5-fold without causing obvious changes in the characteristics of the hucMSCs or the size of the exosome particles. Further studies showed that CHIR99021 promoted the production of exosomes by facilitating exocytosis. This process was partly mediated by SNAP25. To further explore whether CHIR99021 changed the cargo that was loaded into the exosomes and its therapeutic effects, we performed proteomic and transcriptomic analyses of exosomes from primed and control hucMSCs. The results showed that CHIR99021 significantly upregulated the expression of proteins that are associated with cell migration and wound healing. Animal experiments confirmed that, compared to control hucMSC-derived exosomes, CHIR99021-pretreated hucMSC-derived exosomes (CHIR-Exos) significantly accelerated wound healing in diabetic mice, enhanced local collagen deposition, promoted angiogenesis, and reduced chronic inflammation. Subsequent in vitro experiments confirmed that the CHIR-Exos promoted wound healing by facilitating cell migration, inhibiting oxidative stress-induced apoptosis, and preventing cell cycle arrest. Conclusions The Wnt agonist CHIR99021 significantly increased exosome secretion by hucMSCs, which was partly mediated by SNAP25. Notably, CHIR99021 treatment also significantly increased the exosomal levels of proteins that are associated with wound healing and cell migration, resulting in enhanced acceleration of wound healing. All of these results suggested that pretreatment of hucMSCs with CHIR99021 not only promoted exosome production but also improved the exosome therapeutic efficacy, thus providing a promising option for large-scale clinical implementation and commercialization. Graphical Abstract

Background: The use of stem cell-derived exosomes (Exos) as therapeutic vehicles is receiving increasing attention. Exosome administration has several advantages over cell transplantation, thus making exosomes promising candidates for large-scale clinical implementation and commercialization. However, exosome extraction and purification efficiencies are relatively low, and therapeutic heterogeneity is high due to differences in culture conditions and cell viability. Therefore, in this study, we investigated a priming procedure to enhance the production and therapeutic effects of exosomes from human umbilical cord mesenchymal stem cells (hucMSCs). After preconditioning hucMSCs with agonists/inhibitors that target the Wnt/β-catenin pathway, we assessed both the production of exosomes and the therapeutic efficacy of the optimized exosomes in the context of diabetic wound healing, hoping to provide a safer, more stable and more effective option for clinical application. Results: The Wnt signalling pathway agonist CHIR99021 increased exosome production by 1.5-fold without causing obvious changes in the characteristics of the hucMSCs or the size of the exosome particles. Further studies showed that CHIR99021 promoted the production of exosomes by facilitating exocytosis. This process was partly mediated by SNAP25. To further explore whether CHIR99021 changed the cargo that was loaded into the exosomes and its therapeutic effects, we performed proteomic and transcriptomic analyses of exosomes from primed and control hucMSCs. The results showed that CHIR99021 significantly upregulated the expression of proteins that are associated with cell migration and wound healing. Animal experiments confirmed that, compared to control hucMSC-derived exosomes, CHIR99021-pretreated hucMSC-derived exosomes (CHIR-Exos) significantly accelerated wound healing in diabetic mice, enhanced local collagen deposition, promoted angiogenesis, and reduced chronic inflammation. Subsequent in vitro experiments confirmed that the CHIR-Exos promoted wound healing by facilitating cell migration, inhibiting oxidative stress-induced apoptosis, and preventing cell cycle arrest. Conclusions: The Wnt agonist CHIR99021 significantly increased exosome secretion by hucMSCs, which was partly mediated by SNAP25. Notably, CHIR99021 treatment also significantly increased the exosomal levels of proteins that are associated with wound healing and cell migration, resulting in enhanced acceleration of wound healing. All of these results suggested that pretreatment of hucMSCs with CHIR99021 not only promoted exosome production but also improved the exosome therapeutic efficacy, thus providing a promising option for large-scale clinical implementation and commercialization. [ABSTRACT FROM AUTHOR]

Formins are a large, evolutionarily old family of cytoskeletal regulators whose roles include actin capping and nucleation, as well as modulation of microtubule dynamics. The plant class I formin clade is characterized by a unique domain organization, as most of its members are transmembrane proteins with possible cell wall-binding motifs exposed to the extracytoplasmic space—a structure that appears to be a synapomorphy of the plant kingdom. While such transmembrane formins are traditionally considered mainly as plasmalemma-localized proteins contributing to the organization of the cell cortex, we review, from a cell biology perspective, the growing evidence that they can also, at least temporarily, reside (and in some cases also function) in endomembranes including secretory and endocytotic pathway compartments, the endoplasmic reticulum, the nuclear envelope, and the tonoplast. Based on this evidence, we propose that class I formins may thus serve as 'active cargoes' of membrane trafficking—membrane-embedded proteins that modulate the fate of endo- or exocytotic compartments while being transported by them. [ABSTRACT FROM AUTHOR]

Small proteins represent a significant portion of the cargo transported through plant secretory pathways, playing crucial roles in developmental processes, fertilization, and responses to environmental stresses. Despite the importance of small secreted proteins, substantial knowledge gaps persist regarding the regulatory mechanisms governing their trafficking along the secretory pathway, and their ultimate localization or destination. To address these gaps, we conducted a comprehensive literature review, focusing particularly on trafficking and localization of Arabidopsis small secreted proteins with potential biochemical and/or signaling roles in the extracellular space, typically those within the size range of 101–200 amino acids. Our investigation reveals that while at least six members of the 21 mentioned families have a confirmed extracellular localization, eight exhibit intracellular localization, including cytoplasmic, nuclear, and chloroplastic locations, despite the presence of N-terminal signal peptides. Further investigation into the trafficking and secretion mechanisms of small protein cargo could not only deepen our understanding of plant cell biology and physiology but also provide a foundation for genetic manipulation strategies leading to more efficient plant cultivation. [ABSTRACT FROM AUTHOR]

Front cover: A puffed‐up fugu is wrestling with a toad. The body of the fugu has numerous spots that look like insulin secretory granules and a nucleus‐like pattern, just like the pancreatic β‐cells. Perhaps triggered by the wrestling, the fugu's body releases insulin. Inspired by a review article in this journal (Izumi (2023). Multiple pathways and independent functional pools in insulin granule exocytosis. Genes Cells 28: 471‐481, DOI: 10.1111/gtc.13029). Designed by TRAIS Co., Ltd. (Kobe, Japan). [ABSTRACT FROM AUTHOR]

SUMMARY: Plant cell walls are essential for defining plant growth and development, providing structural support to the main body and responding to abiotic and biotic cues. Cellulose, the main structural polymer of plant cell walls, is synthesized at the plasma membrane by cellulose synthase complexes (CSCs). The construction and transport of CSCs to and from the plasma membrane is poorly understood but is known to rely on the coordinated activity of cellulose synthase‐interactive protein 1 (CSI1), a key regulator of CSC trafficking. In this study, we found that Trs85, a TRAPPIII complex subunit, interacted with CSI1 in vitro. Using functional genetics and live‐cell imaging, we have shown that trs85‐1 mutants have reduced cellulose content, stimulated CSC delivery, an increased population of static CSCs and deficient clathrin‐mediated endocytosis in the primary cell wall. Overall, our findings suggest that Trs85 has a dual role in the trafficking of CSCs, by negatively regulating the exocytosis and clathrin‐mediated endocytosis of CSCs. Significance Statement: The synthesis of cellulose requires the transportation of cellulose synthase complexes (CSCs) to and from the plasma membrane. Our study has identified that Trs85, a TRAPPIII subunit, is important in both the delivery and endocytosis of CSCs at the plasma membrane in Arabidopsis. [ABSTRACT FROM AUTHOR]

This obituary pays tribute to Odile Grynszpan-Winograd, a French ultrastructural morphologist who made significant contributions to the study of adrenal gland anatomy. Her research in the 1970s provided the first convincing evidence of exocytosis in chromaffin cells, supporting biochemical experiments conducted by other researchers. Despite facing skepticism, her work on the vesicular secretion of catecholamines had a significant impact on the understanding of hormone release mechanisms. Grynszpan-Winograd also contributed to understanding intercellular junctions in chromaffin cells. The article acknowledges the importance of electron microscopy in biology and recognizes Grynszpan-Winograd's achievements in the field. [Extracted from the article]

The intracellular coral–dinoflagellate symbiosis is the engine that underpins the success of coral reefs, one of the most diverse ecosystems on the planet. However, the breakdown of the symbiosis and the loss of the microalgal symbiont (i.e. coral bleaching) due to environmental changes are resulting in the rapid degradation of coral reefs globally. There is an urgent need to understand the cellular physiology of coral bleaching at the mechanistic level to help develop solutions to mitigate the coral reef crisis. Here, at an unprecedented scope, we present novel models that integrate putative mechanisms of coral bleaching within a common framework according to the triggers (initiators of bleaching, e.g. heat, cold, light stress, hypoxia, hyposalinity), cascades (cellular pathways, e.g. photoinhibition, unfolded protein response, nitric oxide), and endpoints (mechanisms of symbiont loss, e.g. apoptosis, necrosis, exocytosis/vomocytosis). The models are supported by direct evidence from cnidarian systems, and indirectly through comparative evolutionary analyses from non‐cnidarian systems. With this approach, new putative mechanisms have been established within and between cascades initiated by different bleaching triggers. In particular, the models provide new insights into the poorly understood connections between bleaching cascades and endpoints and highlight the role of a new mechanism of symbiont loss, i.e. 'symbiolysosomal digestion', which is different from symbiophagy. This review also increases the approachability of bleaching physiology for specialists and non‐specialists by mapping the vast landscape of bleaching mechanisms in an atlas of comprehensible and detailed mechanistic models. We then discuss major knowledge gaps and how future research may improve the understanding of the connections between the diverse cascade of cellular pathways and the mechanisms of symbiont loss (endpoints). [ABSTRACT FROM AUTHOR]

Red light (670 nm) energy controls vasodilation via the formation of a transferable endothelium-derived nitric oxide (NO)-precursor-containing substance, its intracellular traffic, and exocytosis. Here we investigated the underlying mechanistic effect of oxidative stress on light-mediated vasodilation by using pressure myography on dissected murine arteries and immunofluorescence on endothelial cells. Treatment with antioxidants Trolox and catalase decreased vessel dilation. In the presence of catalase, a lower number of exosomes were detected in the vessel bath. Light exposure resulted in increased cellular free radical levels. Mitochondrial reactive oxygen species were also more abundant but did not alter cellular ATP production. Red light enhanced the co-localization of late exosome marker CD63 and cellular S-nitrosoprotein to a greater extent than high glucose, suggesting that a mild oxidative stress favors the localization of NO precursor in late exosomes. Exocytosis regulating protein Rab11 was more abundant after irradiation. Our findings conclude that red-light-induced gentle oxidative stress facilitates the dilation of blood vessels, most likely through empowering the traffic of vasodilatory substances. Application of antioxidants disfavors this mechanism. [ABSTRACT FROM AUTHOR]

Exocytosis plays an essential role in delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces. Accurate secretion through exocytosis is key to normal plant development as well as responses to biotic and abiotic stresses. During exocytosis, an octameric protein complex named the exocyst facilitates the tethering of secretory vesicles to the plasma membrane. Despite some understanding of molecular and cellular aspects of exocyst function obtained through reverse genetics and direct interaction assays, knowledge about upstream modulators and genetic interactors remains limited. Traditional genetic screens encounter practical issues in exocyst subunit mutant backgrounds, such as lethality of certain knockout mutants and/or potential redundancy of EXO70 homologs. To address these challenges, this study leverages the tunable and reversible nature of chemical genetics, employing Endosidin2 (ES2)—a synthetic inhibitor of EXO70—for a large‐scale chemical genetic mutant screen in Arabidopsis. This approach led to the identification of 70 ES2‐hypersensitive mutants, named es2s. Through a whole‐genome sequencing‐based mapping strategy, 14 nonallelic es2s mutants were mapped and the candidate mutations reported here. In addition, T‐DNA insertion lines were tested as alternative alleles to identify causal mutations. We found that T‐DNA insertion alleles for DCP5, VAS1/ISS1, ArgJ, and MEF11 were hypersensitive to ES2 for root growth inhibition. This research not only offers new genetic resources for systematically identifying molecular players interacting with the exocyst in Arabidopsis but also enhances understanding of the regulation of exocytosis. [ABSTRACT FROM AUTHOR]

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction. [ABSTRACT FROM AUTHOR]

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation--a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges--unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla. [ABSTRACT FROM AUTHOR]

This article explores the presence of secretory vesicles in astrocytes and their role in synaptic transmission. It discusses the debate surrounding whether astrocytes release gliotransmitters through vesicular exocytosis or through non-vesicular pathways. The use of electron microscopy to study astrocytic ultrastructure is also discussed, as well as the identification of astrocytic processes and organelles in unstained tissue. The article concludes by reviewing the current state of knowledge regarding exocytotic synaptic-like microvesicles in astrocytes. The document also contains a list of references related to the study of astrocytes, providing a starting point for library patrons conducting research on astrocytes and their role in brain function. [Extracted from the article]

Lysosomes and lysosome related organelles (LROs) are dynamic organelles at the intersection of various pathways involved in maintaining cellular hemostasis and regulating cellular functions. Vesicle trafficking of lysosomes and LROs are critical to maintain their functions. The lysosomal trafficking regulator (LYST) is an elusive protein important for the regulation of membrane dynamics and intracellular trafficking of lysosomes and LROs. Mutations to the LYST gene result in Chédiak-Higashi syndrome, an autosomal recessive immunodeficiency characterized by defective granule exocytosis, cytotoxicity, etc. Despite eight decades passing since its initial discovery, a comprehensive understanding of LYST's function in cellular biology remains unresolved. Accumulating evidence suggests that dysregulation of LYST function also manifests in other disease states. Here, we review the available literature to consolidate available scientific endeavors in relation to LYST and discuss its relevance for immunomodulatory therapies, regenerative medicine and cancer applications. [ABSTRACT FROM AUTHOR]

Vesicles carry out many essential functions within cells through the processes of endocytosis, exocytosis, and passive and active transport. This includes transporting and delivering molecules between different parts of the cell, and storing and releasing neurotransmitters in neurons. To date, computational simulation of these key biological players has been rather limited and has not advanced at the same pace as other aspects of cell modeling, restricting the realism of computational models. We describe a general vesicle modeling tool that has been designed for wide application to a variety of cell models, implemented within our software STochastic Engine for Pathway Simulation (STEPS), a stochastic reaction-diffusion simulator that supports realistic reconstructions of cell tissue in tetrahedral meshes. The implementation is validated in an extensive test suite, parallel performance is demonstrated in a realistic synaptic bouton model, and example models are visualized in a Blender extension module. A computational study describing a parallel extension to software "STochastic Engine for Pathway Simulation" that simulates vesicles and their interactions with the cellular environment, including endocytosis, docking, fusion and active transport. [ABSTRACT FROM AUTHOR]

In all cell types, small EVs, very abundant extracellular vesicles, are generated and accumulated within MVB endocytic cisternae. Upon MVB fusion and exocytosis with the plasma membrane, the EVs are released to the extracellular space. In the central nervous system, the release of neuronal EVs was believed to occur only from the surface of the body and dendrites. About 15 years ago, MVB cisternae and EVs were shown to exist and function at synaptic boutons, the terminals' pre- and post-synaptic structures essential for canonical neurotransmitter release. Recent studies have revealed that synaptic EVs are peculiar in many respects and heterogeneous with respect to other neuronal EVs. The distribution of synaptic EVs and the effect of their specific molecules are found at critical sites of their distribution. The role of synaptic EVs could consist of the modulation of canonical neurotransmitter release or a distinct, non-canonical form of neurotransmission. Additional roles of synaptic EVs are still not completely known. In the future, additional investigations will clarify the role of synaptic EVs in pathology, concerning, for example, circuits, trans-synaptic transmission, diagnosis and the therapy of diseases. [ABSTRACT FROM AUTHOR]

Mast cells play important role in acquired and natural immunity making these favorable therapeutic targets in various inflammatory diseases. Here we observed that, pentacyclic tri terpenoid betulinic acid (BA) treatment resulted in a significantly high number (9%) of cells positive for Hoechst and negative for annexin-V indicating that BA could interfere with plasma membrane integrity. The degranulation of both activated and non-activated mast cells was enhanced upon treatment with BA. The pre-treatment of BA had remarkable effect on calcium response in activated mast cells which showed increased calcium influx relative compared to untreated cells. The results also showed potentially less migration of BA treated mast cells signifying the possible effect of BA on cell membrane. BA treatment resulted in a significant increase in mRNA levels of IL-13 while as mRNA levels of other target cytokines, IL-6 and TNF-α seem to be not affected. Moreover, there was global Increase in phosphorylation of signaling proteins and no significant change in phosphorylation of FcεRI receptors indicating that the effect of BA was independent of signaling cascade or FcεRI receptor mediated mast cell aggregation. Overall, these results portray BA potentiates mast cell effector functions by compromising the membrane integrity and independent of FcεRI involvement. [ABSTRACT FROM AUTHOR]

In this study, the constant temperature control system of a heating plate was established, ethanol–diesel fuel with different proportions was prepared, and a series of experiments were carried out. The experimental system was used to observe, summarize, and analyze four evaporation and crushing modes of mixed droplets, which were explosion, liquid filament stretching, exocytosis, and ejection mode. The evaporation process of four kinds of mixed droplets in their life cycle was analyzed by normalizing the diameter square. It was proposed that the evaporation process of droplets could be divided into the following three stages: a heating stage, a fluctuating evaporation stage, and an equilibrium evaporation stage. It was also pointed out that the expansion, ejection, and micro-explosion of droplets were the causes of fluctuating evaporation. The concept of expansion and crushing intensity was put forward and the expansion and crushing intensity of ethanol/diesel mixed droplets with different proportions were calculated. The reasons why expansion and crushing intensity first increased and decreased with the increase in ethanol blending ratio were analyzed. Finally, the time proportion of ethanol–diesel mixed droplets in each evaporation stage was calculated, which explained that the time proportion of the instantaneous heating stage showed a parabolic law with the increase in ethanol content. [ABSTRACT FROM AUTHOR]

Background/Aims: Adrenaline quickly inhibits the release of histamine from mast cells. Besides α2-adrenergic receptors, several in vitro studies also indicate the involvement of a-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 α2-adrenergic receptor antagonist. Among &#946-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 α2-adrenergic receptors. [ABSTRACT FROM AUTHOR]

Weibel Palade bodies (WPB) are lysosome‐related secretory organelles of endothelial cells. Commonly known for their main cargo, the platelet and leukocyte receptors von‐Willebrand factor (VWF) and P‐selectin, WPB play a crucial role in hemostasis and inflammation. Here, the authors identify the glycerophosphodiester phosphodiesterase domain‐containing protein 5 (GDPD5) as a WPB cargo protein and show that GDPD5 is transported to WPB following uptake from the plasma membrane via an unique endocytic transport route. GDPD5 cleaves GPI‐anchored, plasma membrane‐resident proteins within their GPI‐motif, thereby regulating their local activity. The authors identify a novel target of GDPD5 , the complement regulator CD59, and show that it is released from the endothelial surface by GDPD5 following WPB exocytosis. This results in increased deposition of complement components and can enhance local inflammatory and thrombogenic responses. Thus, stimulus‐induced WPB exocytosis can modify the endothelial cell surface by GDPD5‐mediated selective release of a subset of GPI‐anchored proteins. [ABSTRACT FROM AUTHOR]

This work presents a novel multielectrode array (MEA) to quantitatively assess the dose enhancement factor (DEF) produced in a medium by embedded nanoparticles. The MEA has 16 nanocrystalline diamond electrodes (in a cell-culture well), and a single-crystal diamond divided into four quadrants for X-ray dosimetry. DEF was assessed in water solutions with up to a 1000 µg/mL concentration of silver, platinum, and gold nanoparticles. The X-ray detectors showed a linear response to radiation dose (r2 ≥ 0.9999). Overall, platinum and gold nanoparticles produced a dose enhancement in the medium (maximum of 1.9 and 3.1, respectively), while silver nanoparticles produced a shielding effect (maximum of 37%), lowering the dose in the medium. This work shows that the novel MEA can be a useful tool in the quantitative assessment of radiation dose enhancement due to nanoparticles. Together with its suitability for cells' exocytosis studies, it proves to be a highly versatile device for several applications. [ABSTRACT FROM AUTHOR]

Aim: In neuroendocrine cells, large dense‐core vesicles (LDCVs) undergo highly regulated pre‐fusion processes before releasing hormones via membrane fusion. Significant heterogeneity has been found for LDCV population based on the dynamics of membrane fusion. However, how the pre‐fusion status impacts the heterogeneity of LDCVs still remains unclear. Hence, we explored pre‐fusion determinants of heterogeneous membrane fusion procedure of LDCV subpopulations. Methods: We assessed the pre‐fusion motion of two LDCV subpopulations with distinct membrane fusion dynamics individually, using total internal reflection fluorescence microscopy. These two subpopulations were isolated by blocking Rho GTPase‐dependent actin reorganization using Clostridium difficile toxin B (ToxB), which selectively targets the fast fusion vesicle pool. Results: We found that the fast fusion subpopulation was in an active motion mode prior to release, termed "active" LDCV pool, while vesicles from the slow fusion subpopulation were also moving but in a significantly more confined status, forming an "inert" pool. The depletion of the active pool by ToxB also eliminated fast fusion vesicles and was not rescued by pre‐treatment with phorbol ester. A mild actin reorganization blocker, latrunculin A, that partially disrupted the active pool, only slightly attenuated the fast fusion subpopulation. Conclusion: The pre‐fusion motion state of LDCVs also exhibits heterogeneity and dictates the heterogeneous fusion pore dynamics. Rearrangement of F‐actin network mediates vesicle pre‐fusion motion and subsequently determines the membrane fusion kinetics. [ABSTRACT FROM AUTHOR]

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage. [ABSTRACT FROM AUTHOR]

Insulin secretion from pancreatic β cells is essential to the maintenance of glucose homeostasis. Defects in this process result in diabetes. Identifying genetic regulators that impair insulin secretion is crucial for the identification of novel therapeutic targets. Here, we show that reduction of ZNF148 in human islets, and its deletion in stem cell-derived β cells (SC-β cells), enhances insulin secretion. Transcriptomics of ZNF148-deficient SC-β cells identifies increased expression of annexin and S100 genes whose proteins form tetrameric complexes involved in regulation of insulin vesicle trafficking and exocytosis. ZNF148 in SC-β cells prevents translocation of annexin A2 from the nucleus to its functional place at the cell membrane via direct repression of S100A16 expression. These findings point to ZNF148 as a regulator of annexin-S100 complexes in human β cells and suggest that suppression of ZNF148 may provide a novel therapeutic strategy to enhance insulin secretion.

The Ca2+-dependent activator protein for secretion (CAPS/CADPS) family protein facilitates catecholamine release through the dense-core vesicle exocytosis in model neuroendocrine cell lines. However, it remains unclear if it induces dopamine release in the central neurons. This study aimed to examine the expression and function of CADPS2, one of the two CADPS paralogs, in dopamine neurons of the mouse midbrain. This study shows that CADPS2 was expressed in tyrosine hydroxylase and the vesicular monoamine transporter 2 (VMAT2)-positive dopaminergic neurons of the midbrain samples and primary mesencephalic cell cultures. Subcellular fractions rich in dopamine were collected using immunoaffinity for CADPS2 from midbrain protein extracts. Cell imaging using fluorescent false neurotransmitter FFN511 as a substrate for VMAT2 showed decreased activity-dependent dopamine release in Cadps2-deficient cultures, compared to that in wild-type cultures. These results suggest that CADPS2 is involved in dopamine release from the central neurons, indicating its involvement in the central dopamine pathway.

Chromaffin cells have been used as a physiological model to understand neurosecretion in mammals for many years. Nicotinic receptors located in the cells’ membrane are stimulated by acetylcholine, and they participate in the exocytosis of chromaffin granules, releasing catecholamines in response to stress. In this work, we discuss how the participation of nicotinic receptors and the localization of active zones in the borders of the cytoskeleton can generate local calcium signals leading to secretion. We use a computational model of a cytoskeleton cage to simulate Ca2+ levels in response to voltage and acetylcholine pulses. We find that nicotinic receptors are able to enhance the differences between local and average calcium values, as well as the heterogeneous distributions around the active zones, producing a non-linear, highly localized Ca2+ entry that, although consisting of a few ions, is able to improve secretion responses in chromaffin cells. Our findings emphasize the intricate interplay among nicotinic receptors, the cytoskeleton, and active zones within chromaffin cells as an example of Ca2+-dependent neurosecretion in mammals.

Many types of plastic products, including polystyrene, have long been used in commercial and industrial applications. Microplastics and nanoplastics, plastic particles derived from these plastic products, are emerging as environmental pollutants that can pose health risks to a wide variety of living organisms, including humans. However, it is not well understood how microplastics and nanoplastics affect cellular functions and induce stress responses. Humans can be exposed to polystyrene-microplastics and polystyrene-nanoplastics through ingestion, inhalation, or skin contact. Most ingested plastics are excreted from the body, but inhaled plastics may accumulate in the lungs and can even reach the brain via the nose-to-brain route. Small-sized polystyrene-nanoplastics can enter cells by endocytosis, accumulate in the cytoplasm, and cause various cellular stresses, such as inflammation with increased pro-inflammatory cytokine production, oxidative stress with generation of reactive oxygen species, and mitochondrial dysfunction. They induce autophagy activation and autophagosome formation, but autophagic flux may be impaired due to lysosomal dysfunction. Unless permanently exposed to polystyrene-nanoplastics, they can be removed from cells by exocytosis and subsequently restore cellular function. However, neurons are very susceptible to this type of stress, thus even acute exposure can lead to neurodegeneration without recovery. This review focuses specifically on recent advances in research on polystyrene-nanoplastic-induced cytotoxicity and neurotoxicity. Furthermore, in this review, based on mechanistic studies of polystyrene-nanoplastics at the cellular level other than neurons, future directions for overcoming the negative effects of polystyrene-nanoplastics on neurons were suggested.

Introduction. The Tay-Sachs disease is a progressive neurodegenerative disorder that is caused by a genetic mutation in the HEXA gene coding the lysosomal α-subunit of β-hexosaminidase A. Currently, there is no effective treatment for Tay-Sachs. Induction of exocytosis as a potential treatment approach is suggested to restore lysosomal enlargement in several lysosomal storage diseases. Here, we aimed to test the therapeutic potential of two small molecules, δ-tocopherol and hydroxypropyl-β-cyclodextrin, in fibroblast and neuroglia cells derived from Hexa-/-Neu3-/- mice and Tay-Sachs patients. Method. The effect of two small molecules on lysosomal enlargement and GM2 accumulation in lysosomes was examined by LysoTracker staining and immunocytochemical colocalization analysis for GM2 and LAMP1. qRT-PCR and fluorometric enzyme assay were also used to investigate the effect of combined treatment on the level of neuraminidase 1, a negative regulator of exocytosis. Results. Single treatment with δ-tocopherol (5-40 μM) and hydroxypropyl-β-cyclodextrin (10-50 μM) for 48 hours led to significant induction of lysosomal exocytosis. We demonstrated that the combined treatment with δ-tocopherol (10 μM) and hydroxypropyl-β-cyclodextrin (25 μM) resulted in a significant reduction of lysosomal GM2 and downregulation of lysosomal Neu1 expression. Conclusion. In this study, we demonstrated that inducing exocytosis by δ-tocopherol and hydroxypropyl-β-cyclodextrin might have therapeutic potential to reduce GM2 storage and pathology in Tay-Sachs cells. [ABSTRACT FROM AUTHOR]

HIV-associated neurocognitive disorders (HAND) affect 15–55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER)-plasma membrane (PM) tethering is crucial for the non-vesicular lipid transport between the ER membrane and the PM. However, the PM-associated ER can impede the PM binding of cytoskeletons and other organelles. It is poorly understood how the competition between the ER and cytoskeletons/organelles on the PM is resolved. Here, we show that, upon septin collar assembly, ER-PM tethering proteins are excluded from the yeast bud sites, and the PM-associated ER is locally detached from the PM. Our results suggest that PM flows by polarized exocytosis extrude PM proteins, including ER-PM tethering proteins, from the bud sites. When the reorganization of the ER-PM tethering was inhibited by exocytosis repression, septin localization was restricted to the PM sites poor in ER-PM tethering proteins. This study proposes machinery reconciling ER-septin competition on the PM, providing mechanistic insights into the spatial organization of PM-associated organelles and cytoskeletons. [ABSTRACT FROM AUTHOR]

Host immune systems serving as crucial defense lines are vital resisting mechanisms against biofilm‐associated implant infections. Nevertheless, biofilms hinder the penetration of anti‐bacterial species, inhibit phagocytosis of immune cells, and frustrate host inflammatory responses, ultimately resulting in the weakness of the host immune system for biofilm elimination. Herein, a cell‐like construct is developed through encapsulation of erythrocyte membrane fragments on the surface of Fe3O4 nanoparticle‐fabricated microbubbles and then loaded with hydroxyurea (EMB‐Hu). Under ultrasound (US) stimulation, EMB‐Hu undergoes a stable oscillation manner to act in an "exocytosis" mechanism for disrupting biofilm, releasing agents, and enhancing penetration of catalytically generated anti‐bacterial species within biofilms. Additionally, the US‐stimulated "exocytosis" by EMB‐Hu can activate pro‐inflammatory macrophage polarization and enhance macrophage phagocytosis for clearance of disrupted biofilms. Collectively, this work has exhibited cell‐like microbubbles with US‐stimulated "exocytosis" mechanisms to overcome the biofilm barrier and signal macrophages for inflammatory activation, finally achieving favorable therapeutic effects against implant infections caused by methicillin‐resistant Staphylococcus aureus (MRSA) biofilms. [ABSTRACT FROM AUTHOR]

Introduction: Cystic Fibrosis (CF) is the commonest genetically inherited disease (1 in 4,500 newborns) and 70% of people with CF (pwCF) harbour the F508Del mutation, resulting in misfolding and incorrect addressing of the channel CFTR to the epithelial membrane and subsequent dysregulation of fluid homeostasis. Although studies have underscored the importance and over-activation of myeloid cells, and in particular neutrophils in the lungs of people with CF (pwCF), relatively less emphasis has been put on the potential immunological bias in CF blood cells, at homeostasis or following stimulation/infection. Methods: Here, we revisited, in an exhaustive fashion, in pwCF with mild disease (median age of 15, median % FEV1 predicted = 87), whether their PBMCs, unprimed or primed with a 'non specific' stimulus (PMA+ionomycin mix) and a 'specific' one (live P.a =PAO1 strain), were differentially activated, compared to healthy controls (HC) PBMCs. Results: 1) we analysed the lymphocytic and myeloid populations present in CF and Control PBMCs (T cells, NKT, Tgd, ILCs) and their production of the signature cytokines IFN-g, IL-13, IL-17, IL-22. 2) By q-PCR, ELISA and Luminex analysis we showed that CF PBMCs have increased background cytokines and mediators production and a partial functional tolerance phenotype, when restimulated. 3) we showed that CF PBMCs low-density neutrophils release higher levels of granule components (S100A8/A9, lactoferrin, MMP-3, MMP-7, MMP-8, MMP-9, NE), demonstrating enhanced exocytosis of potentially harmful mediators. Discussion: In conclusion, we demonstrated that functional lymphoid tolerance and enhanced myeloid protease activity are key features of cystic fibrosis PBMCs. [ABSTRACT FROM AUTHOR]

SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors) facilitate docking and fusion of vesicles with their target membranes, playing a crucial role in vesicle trafficking and exocytosis. However, the spatial assembly and roles of plasma membrane (PM)‐associated SNAREs in phytopathogen development and pathogenicity are not clearly understood. In this study, we analysed the roles and molecular mechanisms of PM‐associated SNARE complexes in the banana Fusarium wilt fungus Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4). Our findings demonstrate that FocSso1 is important for the fungal growth, conidiation, host penetration and colonization. Mechanistically, FocSso1 regulates protein secretion by mediating vesicle docking and fusion with the PM and hyphal apex. Interestingly, a FocSso1–FocSec9–FocSnc1 complex was observed to assemble not only at the fungal PM but also on the growing hyphal apex, facilitating exocytosis. FocSso2, a paralogue of FocSso1, was also found to form a ternary SNARE complex with FocSec9 and FocSnc1, but it mainly localizes to the PM in old hyphae. The functional analysis of this protein demonstrated that it is dispensable for the fungal growth but necessary for host penetration and colonization. The other subunits, FocSec9 and FocSnc1, are involved in the fungal development and facilitate host penetration. Furthermore, FocSso1 and FocSnc1 are functionally interdependent, as loss of FocSso1 leads to mis‐sorting and degradation of FocSnc1 in the vacuole and vice versa. Overall, this study provides insight into the formation of two spatially and functionally distinct PM SNARE complexes and their involvement in vesicle exocytosis to regulate development and pathogenicity of FocTR4. [ABSTRACT FROM AUTHOR]

Although it has been known for decades that lysosomes are central for degradation and recycling in the cell, their pivotal role as nutrient sensing signaling hubs has recently become of central interest. Since lysosomes are highly dynamic and in constant change regarding content and intracellular position, fusion/fission events allow communication between organelles in the cell, as well as cell-to-cell communication via exocytosis of lysosomal content and release of extracellular vesicles. Lysosomes also mediate different forms of regulated cell death by permeabilization of the lysosomal membrane and release of their content to the cytosol. In cancer cells, lysosomal biogenesis and autophagy are increased to support the increased metabolism and allow growth even under nutrient- and oxygen-poor conditions. Tumor cells also induce exocytosis of lysosomal content to the extracellular space to promote invasion and metastasis. However, due to the enhanced lysosomal function, cancer cells are often more susceptible to lysosomal membrane permeabilization, providing an alternative strategy to induce cell death. This review summarizes the current knowledge of cancer-associated alterations in lysosomal structure and function and illustrates how lysosomal exocytosis and release of extracellular vesicles affect disease progression. We focus on functional differences depending on lysosomal localization and the regulation of intracellular transport, and lastly provide insight how new therapeutic strategies can exploit the power of the lysosome and improve cancer treatment. [ABSTRACT FROM AUTHOR]

Angiogenesis is a key player in the pathogenesis of rheumatoid arthritis. Exocytosis from Weibel–Palade bodies is a prerequisite for angiopoietin‐2 (Ang‐2) to activate endothelial cells and initiate angiogenesis. Geniposide (GE) was previously reported to exert anti‐angiogenic effects. The aim of this study was to shed light on whether and how GE regulates Ang‐2 exocytosis. A rat model of adjuvant arthritis (AA) was established to evaluate the therapeutic effect of GE (60 and 120 mg/kg) especially in synovial angiogenesis. In addition, the Matrigel plug assay was used to detect the effect of GE (120 and 240 mg/kg) on angiogenesis in AA mice. In vitro, sphingosine‐1‐phosphate (S1P)‐stimulated human umbilical vein endothelial cells (HUVECs) were used to investigate the effect and mechanism of GE on Ang‐2 exocytosis. It was found that GE improved the symptoms of AA rats and inhibited angiogenesis in AA, which may be related to the down‐regulation of S1P receptors 1, 3 (S1PR1, S1PR3), phospholipase Cβ3 (PLCβ3), inositol 1,4,5‐trisphosphate receptor (IP3R) and Ang‐2 expression. The results of in vitro experiments showed that S1P induced rapid release of Ang‐2 from HUVECs with multigranular exocytosis. Suppression of the S1P/S1PR1/3/PLCβ3/Ca2+ signal axis by the S1PR1/3 inhibitor VPC23019 and the IP3R inhibitor 2‐APB blocked Ang‐2 exocytosis, accompanied by diminished angiogenesis in vitro. GE dose‐dependently weakened S1P/S1PR1/3/PLCβ3/Ca2+ signal axis activation, Ang‐2 exocytosis and angiogenesis in HUVECs (p < 0.05, p < 0.01). Overall, these findings revealed that angiogenesis inhibition of GE was partly attributed to the intervention of Ang‐2 exocytosis through negatively modulating the S1P/S1PR1/3/PLCβ3/Ca2+ signal axis, providing a novel strategy for rheumatoid arthritis anti‐angiogenic therapy. [ABSTRACT FROM AUTHOR]

SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced readily releasable pool (RRP) size, due to increased spontaneous (miniature Excitatory Postsynaptic Current, mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y > V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion. [ABSTRACT FROM AUTHOR]