Exocytosis In Cells Research Articles

Membrane lipids couple synaptotagmin to SNARE-mediated granule fusion in insulin-secreting cells.

Insulin secretion depends on the Ca2+-regulated fusion of granules with the plasma membrane. A recent model of Ca2+-triggered exocytosis in secretory cells proposes that lipids in the plasma membrane couple the calcium sensor Syt1 to the membrane fusion machinery (Kiessling etal., 2018). Specifically, Ca2+-mediated binding of Syt1's C2 domains to the cell membrane shifts the membrane-anchored SNARE syntaxin-1a to a more fusogenic conformation, straightening its juxtamembrane linker. To test this model in live cells and extend it to insulin secretion, we enriched INS1 cells with a panel of lipids with different acyl chain compositions. Fluorescence lifetime measurements demonstrate that cells with more disordered membranes show an increase in fusion efficiency, and vice versa. Experiments with granules purified from INS1 cells and recombinant SNARE proteins reconstituted in supported membranes confirmed that lipid acyl chain composition determines SNARE conformation and that lipid disordering correlates with increased fusion. Addition of Syt1's C2AB domains significantly decreased lipid order in target membranes and increased SNARE-mediated fusion probability. Strikingly, Syt's action on both fusion and lipid order could be partially bypassed by artificially increasing unsaturated phosphatidylserines in the target membrane. Thus, plasma membrane lipids actively participate in coupling Ca2+/synaptotagmin-sensing to the SNARE fusion machinery in cells.

High-Throughput Screening of Surface Engineered Cyanine Nanodots for Active Transport of Therapeutic Antibodies into Solid Tumor.

The successful delivery of therapeutic biomacromolecules into solid tumorholds great challenge due to their high resistance to penetrate through the complex tumor microenvironments. Here, active-transporting nanoparticles are harnessed to efficiently deliver biomacromolecular drugs into solid tumors through cell transcytosis. A series of molecularly precise cyanine 5-cored polylysine G5 dendrimers (Cy5 nanodots) with different peripheral amino acids (G5-AA) is prepared. The capability of these positively charged nanodots to induce cell endocytosis, exocytosis, and transcytosis is evaluated via fluorescence-based high-throughput screen. The optimized nanodots (G5-R) are conjugated with αPD-L1 (a therapeutic monoclonal antibody binding to programmed-death ligand 1) (αPD-L1-G5-R) to demonstrate the nanoparticle-mediated tumor active transport. The αPD-L1-G5-R can greatly enhance the tumor-penetration capability through adsorption-mediated transcytosis (AMT). The effectiveness of αPD-L1-G5-R is tested in treating mice bearing partially resected CT26 tumors, mimicking the local immunotherapy of residual tumors post-surgery in clinic. The αPD-L1-G5-R embedded in fibrin gel can efficiently mediate tumor cell transcytosis, and deliver αPD-L1 throughout the tumor, thereby enhancing immune checkpoint blockade, reducing tumor recurrence, and significantly prolonging the survival time. The active-transporting nanodots are promising platforms for efficient tumor delivery of therapeutic biomacromolecules.

Rab7 localized on zymogen granules is involved in maturation but not in autophagy or regulated exocytosis in pancreatic acinar cells

Rab7 is known to function in the autophagy and endocytosis pathways in eukaryocytes and is related to various diseases. We recently reported that Rab7 plays a protective role against acute pancreatitis. However, its physiological function in exocytic cells remains unclear. Therefore, we investigated the role of Rab7 in pancreas-specific Rab7 knockout mice (Rab7Δpan). Immunofluorescence microscopy revealed that Rab7 colocalized with amylase in pancreatic acinar cells of wild-type mice, but not in Rab7Δpan mice. Western blotting confirmed Rab7 localization in the zymogen granule (ZG) membranes of wild-type mice. Cholecystokinin (CCK)-stimulated amylase secretion examined using isolated pancreatic acini was similar in Rab7Δpan and wild-type mice. In contrast, electron microscopy revealed that the diameters of ZGs were shorter and the number of ZGs was larger in the pancreatic acinar cells of Rab7Δpan mice than in those of wild-type mice. However, the number of ZGs decreased in both Rab7Δpan and wild-type mice after 24 h of starvation. In addition, the amount of amylase in the pancreas was decreased in both Rab7Δpan and wild-type mice. These data indicate that Rab7 localized on ZGs plays a crucial role in the maturation of ZGs but not in their autophagy or regulated exocytosis in pancreatic acinar cells.

RFX6 Maintains Gene Expression and Function of Adult Human Islet α-Cells.

Mutations in the gene encoding the transcription factor regulatory factor X-box binding 6 (RFX6) are associated with human diabetes. Within pancreatic islets, RFX6 expression is most abundant in islet α-cells, and α-cell RFX6 expression is altered in diabetes. However, the roles of RFX6 in regulating gene expression, glucagon output, and other crucial human adult α-cell functions are not yet understood. We developed a method for selective genetic targeting of human α-cells and assessed RFX6-dependent α-cell function. RFX6 suppression with RNA interference led to impaired α-cell exocytosis and dysregulated glucagon secretion in vitro and in vivo. By contrast, these phenotypes were not observed with RFX6 suppression across all islet cells. Transcriptomics in α-cells revealed RFX6-dependent expression of genes governing nutrient sensing, hormone processing, and secretion, with some of these exclusively expressed in human α-cells. Mapping of RFX6 DNA-binding sites in primary human islet cells identified a subset of direct RFX6 target genes. Together, these data unveil RFX6-dependent genetic targets and mechanisms crucial for regulating adult human α-cell function. Article Highlights

Ca2+ binding to the C2E domain of otoferlin is required for hair cell exocytosis and hearing.

Ca2+ binding to the C2E domain of otoferlin is required for hair cell exocytosis and hearing.

A missense mutant of ocrl1 promotes apoptosis of tubular epithelial cells and disrupts endocytosis and the cell cycle of podocytes in Dent-2 Disease

BackgroundThis study aimed to identify an orcl1 mutation in a patient with Dent-2 Disease and investigate the underlying mechanisms.MethodsThe ocrl1 mutation was identified through exome sequencing. Knockdown of orcl1 and overexpression of the orcl1 mutant were performed in HK-2 and MPC5 cells to study its function, while flow cytometry measured reactive oxygen species (ROS), phosphatidylserine levels, and cell apoptosis. Scanning electron microscopy observed crystal adhesion, while transmission electron microscopy examined kidney tissue pathology. Laser scanning confocal microscopy was used to examine endocytosis, and immunohistochemical and immunofluorescence assays detected protein expression. Additionally, podocyte-specific orcl1 knockout mice were generated to investigate the role of orcl1 in vivo.ResultsWe identified a mutation resulting in the replacement of Histidine with Arginine at position 318 (R318H) in ocrl1 in the proband. orcl1 was widely expressed in the kidney. In vitro experiments showed that knockdown of orcl1 and overexpression of ocrl1 mutant increased ROS, phosphatidylserine exocytosis, crystal adhesion, and cell apoptosis in HK-2 cells. Knockdown of orcl1 in podocytes reduced endocytosis and disrupted the cell cycle while increasing cell migration. In vivo studies in mice showed that conditional deletion of orcl1 in podocytes caused glomerular dysfunction, including proteinuria and fibrosis.ConclusionThis study identified an R318H mutation in orcl1 in a patient with Dent-2 Disease. This mutation may contribute to renal injury by promoting ROS production and inducing cell apoptosis in tubular cells, while disrupting endocytosis and the cell cycle, and promoting cell migration of podocytes.CNNDAeUPC_g2tT4JVHCgTwVideo

Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration

Synaptotagmin-1 and synaptotagmin-7 are two prominent calcium sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding calcium, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examine the biophysical properties of these two synaptotagmin isoforms and compare their interactions with phospholipid membranes. We discover that synaptotagmin-1—membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a super-penetrator. We exploit these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to calcium.

Dehydroleucodine and xanthatin, two natural anti-inflammatory lactones, inhibit mast cell degranulation by affecting the actin cytoskeleton.

Actin remodeling is a critical regulator of mast cell secretion. In previous work, we have shown that dehydroleucodine and xanthatin, two natural α,β-unsaturated lactones, exhibit anti-inflammatory and mast cell stabilizing properties. Based on this background, this study aimed to determine whether the mast cell stabilizing action of these lactones is associated with changes in the actin cytoskeleton. Rat peritoneal mast cells were preincubated in the presence of dehydroleucodine or xanthatin before incubation with compound 48/80. Comparative studies with sodium cromoglycate and latrunculin B were also made. After treatments, different assays were performed on mast cell samples: β-hexosaminidase release, cell viability studies, quantification of mast cells and their state of degranulation by light microscopy, transmission electron microscopy, and actin staining for microscopy observation. Results showed that dehydroleucodine and xanthatin inhibited mast cell degranulation, evidenced by the inhibition of β-hexosaminidase release and decreased degranulated mast cell percentage. At the same time, both lactones altered the F-actin cytoskeleton in mast cells resulting, similarly to Latrunculin B, in a higher concentration of nuclear F-actin when activated by compound 48/80. For the first time, this study describes the biological properties of dehydroleucodine and xanthatin concerning to the rearrangement of actin filaments during stimulated exocytosis in mast cells. These data have important implications for developing new anti-inflammatory and mast cell stabilizing drugs and for designing new small molecules that may interact with the actin cytoskeleton.

SYT7 is a key player in increasing exosome secretion and promoting angiogenesis in non-small-cell lung cancer

Lung cancer is the leading cause of cancer-related mortality, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. Our previous study confirmed that synaptotagmin 7 (SYT7) promoted NSCLC metastasis in vivo and in vitro. Studies have shown that SYT7 is an important regulatory molecule of exocytosis in various cells. However, the characteristics of SYT7 across cancers and the function of SYT7 in tumor exosome secretion remain unclear. In this study, we conducted systematic pancancer analyses of SYT7, namely, analyses of expression patterns, diagnostic and prognostic values, genetic alterations, methylation, immune infiltration, and potential biological pathways. Furthermore, we demonstrated that SYT7 increased the secretion of exosomes from A549 and H1299 cells, promoting the migration, proliferation, and tube formation of human umbilical vein endothelial cells (HUVECs). Notably, SYT7 promoted angiogenesis by transferring exosomes containing the molecule centrosomal protein of 55 kDa (CEP55) protein to HUVECs. The CEP55 protein levels was downregulated in STAT1 inhibitor-treating SYT7-overexpresion NSCLC cells. We further found that SYT7 activated the mTOR signaling pathway through the downstream molecule CEP55, thereby promoting the invasion and metastasis of NSCLC cells. SYT7 promoted exosome secretion by NSCLC cells through upregulating syntaxin-1a and syntaxin-3. In vivo, SYT7 promoted the tumorigenesis, angiogenesis and metastasis of A549 cells through the exosome pathway. Our study is of great importance for understanding the mechanism of tumor exosome secretion and the role of exosomes in tumor progression.

Knockdown of Secernin 1 inhibit cell invasion and migration by activating the TGF-β/Smad3 pathway in oral squamous cell carcinomas

Secernin-1 (SCRN1) is a regulator of exocytosis in mast cells. Recently, SCRN1 was reported to be correlated with the prognosis of colorectal cancer and gastric cancer, but its functional effects on oral squamous cell carcinoma (OSCC) remain unclear. Our aim was to explore the expression pattern and the migration and invasion effects of the newly identified SCRN1 in OSCC. Western blotting (WB) was performed to measure SCRN1 expression in human OSCC tissue samples and OSCC cell lines. The effects of SCRN1 on OSCC cell proliferation, invasion and migration were analyzed by cell counting kit-8 and Transwell assays. The expression levels of TGF-β, Smad3 and phosphorylated Smad3 (p-Smad3) were measured by WB. The secretion of matrix metalloproteinase (MMP)-2 and MMP-9 was determined by the enzyme-linked immunosorbent assay. The expression of SCRN1 was significantly elevated in OSCC tissues and cell lines. SCRN1 knockdown reduced the expression of TGF-β and p-Smad3 in OSCC cells. TGF-β stimulation promoted proliferation, invasion and migration and enhanced the expression of p-Smad3 and the secretion of MMP9 in SCRN1-knockdown OSCC cell lines. Our study demonstrated that SCRN1 is upregulated in OSCC. Further analyses demonstrated that SCRN1 promotes the proliferation, invasion and migration of OSCC cells via TGF-β/Smad3 signaling.

Interactions and conformational changes of annexin A2/p11 heterotetramer models on a membrane: a molecular dynamics study

Ca2+-dependent membrane-binding by the Annexin A2/p11 heterotetramer (A2t) plays an important role in various biological processes including fibrinogen activation and exocytosis in neuroendocrine cells. Two models where A2t associates with a single membrane surface were generated and used to perform molecular dynamics simulations. The first model mimics initial A2t-membrane binding through both Annexin A2 (A2) subunits of A2t (TS model) while the second model mimics A2t-binding through a single A2 subunit (OS model). Conformational changes were summarized using principal component analysis (PCA), simulation snapshots, and distance plots from the simulations. The full TS model, including the p11 dimer, fully associates with the membrane adopting a stable structure with little conformational variation as evidence by PCA. The unassociated subunits of the OS model moved toward the membrane. The molecular mechanics/Generalized-Born surface area (MMGBSA) method was applied to investigate the energetics of the models. The MMGBSA results demonstrated that R63 of p11 was the primary contributor to the p11-membrane interaction. The TS model results were both consistent with those found in the literature and provide novel insights about the specific residues driving the A2t-membrane interaction. Additionally, it represents the most complete model of A2t on the membrane surface available. Communicated by Ramaswamy H. Sarma

Clinicopathological Survey of 204 Rosacea Patients Regarding Rosacea Subgroups and Severity.

Few studies have evaluated the histopathological characteristics of clinical rosacea subtypes in detail. To assess rosacea histopathological features in correspondence to clinical subgroups. The histopathological findings of 204 rosacea patients were analyzed retrospectively and were compared among clinical subtypes. Thirt-Two Percent of patients were male and 68% were female. Seventy-three patients had erythematotelangiectatic rosacea (ETR) and 110 had papulopustular rosacea (PPR), 12 were ETR + PPR, 4 ocular, 2 phymatous, and 3 had Morbihan's edema. Perivascular and perifollicular lymphohistiocytic infiltration, perifollicular exocytosis, follicular spongiosis, and ectatic vessels were almost found in all subtypes. Solar elastosis was higher in ETR. Spongiosis, exocytosis of inflammatory cells into epidermis, acanthosis, and granulomatous reaction were higher in PPR. Inflammatory cells exocytosis was more in PPR and phymatous. Demodex folliculorum was identified in 27% of ETR, 33.6% of PPR, 50% of phymatous, one ocular patient, and none of Morbihan edema. Demodex brevis were found in 5% of ETR, 3% of PPR, and 50% of phymatous. Demodex brevis not folliculorum was more in phymatous. Spongiosis was the most common finding in ocular rosacea. Spongiosis, exocytosis of inflammatory cells, and granulomatous reactions were more in PPR. Solar elastosis was more in ETR. Histopathological findings were compatible with clinical subgroups.

Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion.

Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.

Activation of ryanodine-sensitive calcium store drives pseudo-allergic dermatitis via Mas-related G protein-coupled receptor X2 in mast cells.

Mast cell (MC) activation is implicated in the pathogenesis of multiple immunodysregulatory skin disorders. Activation of an IgE-independent pseudo-allergic route has been recently found to be mainly mediated via Mas-Related G protein-coupled receptor X2 (MRGPRX2). Ryanodine receptor (RYR) regulates intracellular calcium liberation. Calcium mobilization is critical in the regulation of MC functional programs. However, the role of RYR in MRGPRX2-mediated pseudo-allergic skin reaction has not been fully addressed. To study the role of RYR in vivo, we established a murine skin pseudo-allergic reaction model. RYR inhibitor attenuated MRGPRX2 ligand substance P (SP)-induced vascular permeability and neutrophil recruitment. Then, we confirmed the role of RYR in an MC line (LAD2 cells) and primary human skin-derived MCs. In LAD2 cells, RYR inhibitor pretreatment dampened MC degranulation (detected by β-hexosaminidase retlease), calcium mobilization, IL-13, TNF-α, CCL-1, CCL-2 mRNA, and protein expression activated by MRGPRX2 ligands, namely, compound 48/80 (c48/80) and SP. Moreover, the inhibition effect of c48/80 by RYR inhibitor was verified in skin MCs. After the confirmation of RYR2 and RYR3 expression, the isoforms were silenced by siRNA-mediated knockdown. MRGPRX2-induced LAD2 cell exocytosis and cytokine generation were substantially inhibited by RYR3 knockdown, while RYR2 had less contribution. Collectively, our finding suggests that RYR activation contributes to MRGPRX2-triggered pseudo-allergic dermatitis, and provides a potential approach for MRGPRX2-mediated disorders.

T cell exocytosis testing provides superior sensitivity for the diagnosis of familial HLH

The rapid diagnosis of primary immunodeficiencies (PID) affecting cytotoxic lymphocyte granule exocytosis is commonly performed by evaluating NK cell surface CD107a expression after K562 challenge. NK cell exocytosis can also be induced by stimulating NK cells with P815 and anti-CD16 antibody. We have previously described how these NK tests can be adapted to study cytotoxic T cell CD107a exocytosis by gating CD3+CD8 +CD57+ cells after TCR engagement with P815 and anti-CD3 antibody. Here we present data comparing 92 patients with variants in genes affecting exocytosis function against 58 other PID patients diagnosed over 10 years from one center. NK and cytotoxic T cell exocytosis was tested on all patient samples. The anti-CD3T cell test returns superior sensitivity and specificity of 97.8% and 94.7% while the K562 NK test a sensitivity of 86.7% and specificity of 96.1%. The anti-CD 16 NK test was found comparable to the T cell test with values of 97.8% and 93.4%. The data was additionally juxtaposed against 63 patients with secondary HLH, 198 healthy adult donors, and 84 transport controls, all giving similar results of high accuracy. We further stratified the data by time taken between venipuncture and arrival at the testing laboratory and found that control samples up to 36 hours old showed comparable function to < 24 hours old samples. This is important as transporting samples from remote primary care centers often cannot be accomplished within 24 hours.While all three exocytosis tests return superior diagnostic sensitivity, we find the anti-CD3 antibody induced exocytosis of T cells and the anti-CD16 antibody induced exocytosis of NK cells more accurate than the current standard K562 cell stimulation for the diagnosis of PIDs affecting cytotoxic lymphocyte exocytosis. Evaluating T cells alongside NK cells acts as a confirmatory test and buffers against technical failures and the lack of NK cells which can be seen in lymphopenic patients. These assays with improved sensitivity may help us better understand PIDs that partially impair exocytosis.

Requirement of Cholesterol for Calcium-Dependent Vesicle Fusion by Strengthening Synaptotagmin-1-Induced Membrane Bending.

Cholesterol is essential for neuronal activity and function. Cholesterol depletion in the plasma membrane impairs synaptic transmission. However, the molecular mechanisms by which cholesterol deficiency leads to defects in vesicle fusion remain poorly understood. Here, it is shown that cholesterol is required for Ca2+ -dependent native vesicle fusion using the in vitro reconstitution of fusion and amperometry to monitor exocytosis in chromaffin cells. Purified native vesicles are crucial for the reconstitution of physiological Ca2+ -dependent fusion, because vesicle-mimicking liposomes fail to reproduce the cholesterol effect. Intriguingly, cholesterol has no effect on the membrane binding of synaptotagmin-1, a Ca2+ sensor for ultrafast fusion. Cholesterol strengthens local membrane deformation and bending induced by synaptotagmin-1, thereby lowering the energy barrier for Ca2+ -dependent fusion to occur. The data provide evidence that cholesterol depletion abolishes Ca2+ -dependent vesicle fusion by disrupting synaptotagmin-1-induced membrane bending, and suggests that cholesterol is an essential lipid regulator for Ca2+ -dependent fusion.

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

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

V-ATPase modulates exocytosis in neuroendocrine cells through the activation of the ARNO-Arf6-PLD pathway and the synthesis of phosphatidic acid.

Although there is mounting evidence indicating that lipids serve crucial functions in cells and are implicated in a growing number of human diseases, their precise roles remain largely unknown. This is particularly true in the case of neurosecretion, where fusion with the plasma membrane of specific membrane organelles is essential. Yet, little attention has been given to the role of lipids. Recent groundbreaking research has emphasized the critical role of lipid localization at exocytotic sites and validated the essentiality of fusogenic lipids, such as phospholipase D (PLD)-generated phosphatidic acid (PA), during membrane fusion. Nevertheless, the regulatory mechanisms synchronizing the synthesis of these key lipids and neurosecretion remain poorly understood. The vacuolar ATPase (V-ATPase) has been involved both in vesicle neurotransmitter loading and in vesicle fusion. Thus, it represents an ideal candidate to regulate the fusogenic status of secretory vesicles according to their replenishment state. Indeed, the cytosolic V1 and vesicular membrane-associated V0 subdomains of V-ATPase were shown to dissociate during the stimulation of neurosecretory cells. This allows the subunits of the vesicular V0 to interact with different proteins of the secretory machinery. Here, we show that V0a1 interacts with the Arf nucleotide-binding site opener (ARNO) and promotes the activation of the Arf6 GTPase during the exocytosis in neuroendocrine cells. When the interaction between V0a1 and ARNO was disrupted, it resulted in the inhibition of PLD activation, synthesis of phosphatidic acid during exocytosis, and changes in the timing of fusion events. These findings indicate that the separation of V1 from V0 could function as a signal to initiate the ARNO-Arf6-PLD1 pathway and facilitate the production of phosphatidic acid, which is essential for effective exocytosis in neuroendocrine cells.

The C2 and PH domains of CAPS constitute an effective PI(4,5)P2-binding unit essential for Ca2+-regulated exocytosis

Ca2+-dependent activator proteins for secretion (CAPSs) are required for Ca2+-regulated exocytosis in neurons and neuroendocrine cells. CAPSs contain a pleckstrin homology (PH) domain that binds PI(4,5)P2-membrane. There is also a C2 domain residing adjacent to the PH domain, but its function remains unclear. In this study, we solved the crystal structure of the CAPS-1 C2PH module. The structure showed that the C2 and PH tandem packs against one another mainly via hydrophobic residues. With this interaction, the C2PH module exhibited enhanced binding to PI(4,5)P2-membrane compared with the isolated PH domain. In addition, we identified a new PI(4,5)P2-binding site on the C2 domain. Disruption of either the tight interaction between the C2 and PH domains or the PI(4,5)P2-binding sites on both domains significantly impairs CAPS-1 function in Ca2+-regulated exocytosis at the Caenorhabditis elegans neuromuscular junction (NMJ). These results suggest that the C2 and PH domains constitute an effective unit to promote Ca2+-regulated exocytosis.

Esterase-Activated, pH-Responsive, and Genetically Targetable Nano-Prodrug for Cancer Cell Photo-Ablation.

Activatable prodrugs have drawn considerable attention for cancer cell ablation owing to their high specificity in drug delivery systems. However, phototheranostic prodrugs with dual organelle-targeting and synergistic effects are still rare due to low intelligence of their structures. Besides, the cell membrane, exocytosis, and diffusional hindrance by the extracellular matrix reduce drug uptake. Moreover, the up-regulation of heat shock protein and short singlet-oxygen lifetime in cancer cells hamper photo-ablation efficacy, especially in the mono-therapeutic model. To overcome those obstacles,we prepare an esterase-activated DM nano-prodrug, which is conjugated by diiodine-substituted fluorogenic malachite green derivative (MG-2I) and phototherapeutic agent DPP-OH via hydrolyzable ester linkage, having pH-responsiveness and genetically targetable activity for dual organelles-targeting to optimize photo-ablation efficacy. The DM nanoparticles (NPs) present improved pH-responsive photothermal/photodynamic property by the protonation of diethylaminophenyl units in acidic environment. More importantly, the MG-2I and DPP-OH moieties can be released from DM nano-prodrug through overexpressed esterase; then specifically target lysosomes and mitochondria in CT-26 Mito-FAP cells. Hence, near-infrared DM NPs can trigger parallel damage in dual-organelles with strong fluorescence and effective phototoxicity, thus inducing serious mitochondrial dysfunction and apoptotic death, showing excellent photo-ablation effect based on esterase-activated, pH-responsive, and genetically targetable activities.