INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secret...INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.展开更多
Nanoparticles are easily to be taken up by cells but hard to be transported across epithelia, which plays an important role in absorption and toxicity. This paper aims to elucidate the effect of nanoparticle shape on ...Nanoparticles are easily to be taken up by cells but hard to be transported across epithelia, which plays an important role in absorption and toxicity. This paper aims to elucidate the effect of nanoparticle shape on bilateral exocytosis of Caco-2 cells. The fluorescent resonance energy transfer(FRET) probes(DiO and Dil)were utilized to label nanospheres and nanorods for determining and tracking nanoparticles. The results showed that more nanorods were internalized into Caco-2 cells than nanospheres. But the apical exocytosis rate of nanospheres from Caco-2 cells was faster than that of nanorods significantly. In addition, only less than 2% of intact nanoparticles were transported across monolayers for both nanoparticles, but the exocytosis behaviors between them was absolutely different. Compared to nanospheres, nanorods preferred basolateral exocytosis to apical exocytosis. Therefore, the shape is a critical parameter in cellular uptake and transport, even intracellular fate, which should be primarily considered in design of oral nanoparticles.展开更多
To in teract with the egg, the spermatozo on must un dergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosoma...To in teract with the egg, the spermatozo on must un dergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the invoIvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent;however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l^-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l^1), whereas higher concerttrations (>5 pmol l^-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing?protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca^2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization an d/or activation of effectors down stream to F-actin breakdown that lead to acrosomal exocytosis.展开更多
Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that ...Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that IHCs showed significant damage after exposure to a high concentration of salicylate.Whole-cell patch clamp recordings showed that 1–5 mmol/L salicylate did not affect the exocytosis of IHCs,indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input.Instead,salicylate induced a larger peak amplitude,a more negative half-activation voltage,and a steeper slope factor of Ca^(2+)current.Using noise analysis of Ca^(2+)tail currents and qRT-PCR,we further found that salicylate increased the number of Ca^(2+)channels along with CaV1.3 expression.All these changes could act synergistically to enhance the Ca^(2+)influx into IHCs.Inhibition of intracellular Ca^(2+)overload significantly attenuated IHC death after 10 mmol/L salicylate treatment.These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.展开更多
AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ducta...AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS: One and 6 μmol/L carbachol evoked a dose- dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.展开更多
Insulin secretion is a complex and highly regulated process.Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from the...Insulin secretion is a complex and highly regulated process.Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from these studies apply to living animals.That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo.When combined with genetically encoded biosensor,optical imaging is a powerful tool for visualization of molecular events in vivo.In this study,we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins,a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol.Upon exocytosis of secretory granules,the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH,while mCherry fluorescence remained constant in the process,thus allowing ratiometric quantification of insulin secretory events.Furthermore,mCherry fluorescence enabled tracking the movement of secretory granules in living cells.We validated this approach in insulin-secreting cells,and generated a transgenic mouse line expressing the optical sensor specifically in pancreaticβ-cells.The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.展开更多
Summary: The expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PC...Summary: The expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8 % and 10 % of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.展开更多
The cortical actin network is a mesh of filaments distributed beneath the plasmalemma that dynamically reacts in response to stimuli.This dynamic network of cortical filaments,together with motor myosin partners,adjus...The cortical actin network is a mesh of filaments distributed beneath the plasmalemma that dynamically reacts in response to stimuli.This dynamic network of cortical filaments,together with motor myosin partners,adjusts the plasmalemma tension,organizes membrane protein microdomains,remodels the cell surface and drives vesicle motion in order to fine-tune exocytosis,endocytosis and recycling of secretory vesicles.In this review,we discuss how these mechanisms work in secretory cells.展开更多
We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrode...We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12 - 17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.展开更多
The subsecond, temporal, vesicular exocytosis is ubiquitous, but difficult detecting in communication mechanisms of cells. A microelectrode array （MEA）, fabricated by MEMS technology, was applied successfully for re...The subsecond, temporal, vesicular exocytosis is ubiquitous, but difficult detecting in communication mechanisms of cells. A microelectrode array （MEA）, fabricated by MEMS technology, was applied successfully for real-time monitoring of quantal exocytosis from single pheochromocytoma （PC12） cell, The developed MEA was evaluated by dopamine （DA） using electrochemical methods and the results revealed that the sensitivity of DA was improved to 12659.24 p.A L mmol 1 cm 2. The modified MEA was used to detect in vitro vesicular exocytosis of DA from single PCI 2 cells stimulated by concentrated 100 mmol L-1 K＋ cell solution. A total of 592 spikes were measured and analyzed by three parameters and the statistical results revealed the population of each parameter was an approximate Gaussian distribution, and on average, 1.31 × 106 ± 9.25× 104 oxidizable molecules were released in each quantal exocytosis. In addition, results also indicate that a single PC12 cell probably releases the spikes with T ranging from 25.6 ms to 35.4 ms corresponding to/max ranging from 45.6 pA to 65.2 pA. The devices, including a homemade computer interface and the MEA modified with polymer film, provides a new means for further research on the neural, intercellular, communication mechanism.展开更多
Mast cells are responsible for anaphylaxis and allergy and regulate various innate and adaptive immune responses.1,2 Although several“small”Rab GTPases were reported to regulate exocytosis of mast cells,3-5 little i...Mast cells are responsible for anaphylaxis and allergy and regulate various innate and adaptive immune responses.1,2 Although several“small”Rab GTPases were reported to regulate exocytosis of mast cells,3-5 little is known about the contribution of“large”Rab GTPases.Rab44 is a large Rab-GTPase that contains a Rab-GTPase domain and some additional N-terminal domains.6,7 Here,we investigated the role of Rab44 in the physiology of mast cells and in anaphylaxis.Rab44 was expressed as two isoforms in murine bone-marrow mast cells(BMMCs),both of which were deleted in Rab44-knockout mice.The Rab44-knockout mice exhibited diminished anaphylaxis,and the Rab44-knockout BMMCs showed a decrease in FcεRImediated histamine andβ-hexosaminidase secretion.Thus,Rab44 regulates granule exocytosis in mast cells and IgEmediated anaphylaxis in mice.展开更多
Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments （AFs） and microtubules （MTs）. AFs convey post-Golgi secretory vesicles to the tip where some...Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments （AFs） and microtubules （MTs）. AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane （PM）. Secretory activity is balanced by PM retrieval that maintains cell mem- brane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching （FRAP） experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM.展开更多
The molecular mechanisms by which dense core vesicles(DCVs) translocate,tether,dock and prime are poorly understood.In this study,Caenorhabditis elegans was used as a model organism to study the function of Rab protei...The molecular mechanisms by which dense core vesicles(DCVs) translocate,tether,dock and prime are poorly understood.In this study,Caenorhabditis elegans was used as a model organism to study the function of Rab proteins and their effectors in DCV exocytosis.RAB-27/AEX-6,but not RAB-3,was found to be required for peptide release from neurons.By analyzing the movement of DCVs approaching the plasma membrane using total internal reflection fluorescence microscopy,we demonstrated that RAB-27/AEX-6 is involved in the tethering of DCVs and that its effector rabphilin/RBF-1 is required for the initial tethering and subsequent stabilization by docking.展开更多
Exocytosis of mam maUan sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical charact...Exocytosis of mam maUan sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizingthe exocytotic response of streptolysin O-permeabiUzed human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranyigeranyiated, and active Rab3A elicited human sperm exocytosis perse. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino.terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.展开更多
The fusion by exocytosis of many vesicles to the plasma membrane induces the discharge to the extracellular space of their abundant luminal cargoes.Other exocytic vesicles,however,do not contain cargoes,and thus,their...The fusion by exocytosis of many vesicles to the plasma membrane induces the discharge to the extracellular space of their abundant luminal cargoes.Other exocytic vesicles,however,do not contain cargoes,and thus,their fusion is not followed by secretion.Therefore,two distinct processes of exocytosis exist,one secretory and the other non-secretory.The present review deals with the knowledge of non-secretory exocytosis developed during recent years.Among such developments are the dual generation of the exocytic vesicles,initially released either from the trans-Golgi network or by endocytosis;their traffic with activation of receptors,channels,pumps,and transporters;the identification of their tethering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes that govern membrane fusions;the growth of axons and the membrane repair.Examples of potential relevance of these processes for pathology and medicine are also reported.The developments presented here offer interesting chances for future progress in the field.展开更多
In this research, we explored a rapid assessment of silver nanoparticles(Ag-NPs) neurotoxicity at a single-cell level. Traditional nanotoxicity assays on large cell-populations may hide the important heterogeneity of ...In this research, we explored a rapid assessment of silver nanoparticles(Ag-NPs) neurotoxicity at a single-cell level. Traditional nanotoxicity assays on large cell-populations may hide the important heterogeneity of individual cells often found in neuronal cells. The development in the area of new nanomaterial discoveries is far ahead of the development of advanced tools to measure these materials' toxicity. Development of alternative approaches to assess nanomaterials toxicity rapidly, reliably, and accurately is desirable. Here, we present a chip-based, cell-integrated microwell-array device for rapid assessment of neurotoxicity of Ag-NPs by monitoring the exocytosis function of a PC12 cell. Results presented here confirm the dose-dependent toxicity of Ag-NPs and the immediate alteration of their exocytosis function when exposed to NPs.展开更多
Synaptotagmin （Syt） constitutes a family of membrane-trafficking proteins, so far nearly 20 Syts have been discovered. Extensive work showed that synatotagmins were a potential Ca^2＋ sensor for regulated exocytosis...Synaptotagmin （Syt） constitutes a family of membrane-trafficking proteins, so far nearly 20 Syts have been discovered. Extensive work showed that synatotagmins were a potential Ca^2＋ sensor for regulated exocytosis. This study was to investigate the expression and location of synaptotagmin II （Syt2） in RBL-2H3 （RBL） and its role in regulating exocytosis of RBL. The expression of Syt2 in RBL was confirmed by Western blot. The recombinant expression vector pEGFP-N1-Syt2 was constructed and transfected into RBL by electroporation, the stable transfectant RBL-Syt2-S expressing fusion protein Syt2-EGFP were obtained and Syt2 was highly concentrated at plasma membrane with little detected in cytoplasm. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. Compared with control, the release of cathepsin D by RBL-Syt2-S was markedly decreased. The results indicated that Syt2 played a negative regulation in exocytosis of lysosomes in RBL. Cellular ＆ Molecular Immunology. 2005;2（3）：205-209.展开更多
Vitellogenins(VITs)are the most abundant proteins in adult hermaphrodite Caenorhabditis elegans.VITs are synthesized in the intes-tine,secreted to the pseudocoelom,matured into yolk proteins,and finally deposited in o...Vitellogenins(VITs)are the most abundant proteins in adult hermaphrodite Caenorhabditis elegans.VITs are synthesized in the intes-tine,secreted to the pseudocoelom,matured into yolk proteins,and finally deposited in oocytes as nutrients for progeny development.How VITs are secreted out of the intestine remains unclear.Using immuno-electron microscopy(immuno-EM),we localize intestinal VITs along an exocytic pathway consisting of the rough endoplasmic reticulum(ER),the Golgi,and the lipid bilayer-bounded VIT vesicles(VVs).This suggests that the classic exocytotic pathway mediates the secretion of VITs from the intestine to the pseudocoe-lom.We also show that pseudocoelomic yolk patches(PYPs)are membrane-less and amorphous.The different VITs/yolk proteins are packed as a mixture into the above structures.The size of VVs can vary with the VIT levels and the age of the worm.On adult Day 2(AD 2),intestinal VVs(~200 nm in diameter)are smaller than gonadal yolk organelles(YOs,~500 nm in diameter).VVs,PYPs,and YOs share a uniform medium electron density by conventional EM.The morphological profiles documented in this study serve as a refer-ence for future studies of VITs/yolk proteins.展开更多
Down syndrome （DS） is the most common cause of cognitive impairment associated with a congenital chromosomal abnormality, trisomy of chromosome 21. Mental retardation and congenital heart defects are key features of...Down syndrome （DS） is the most common cause of cognitive impairment associated with a congenital chromosomal abnormality, trisomy of chromosome 21. Mental retardation and congenital heart defects are key features of DS, All DS individuals develop early-onset Alzheimer＇s disease-like neuropathology. Intersectin 1 gene is localized on human chromosome 21, the critical region of DS, and it has higher expression in the brain of DS patients than in normal individuals. So fully understanding functions of intersectin 1 is critical for revealing the pathogenesis of DS. Intersectin 1 protein has two isoforms： intersectin 1-L and intersectin 1-S. This review will focus 1 in the central nervous system. on the distribution, expression characters and functions of intersectin展开更多
Secretory pore-forming proteins(PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs(af-PFPs) and...Secretory pore-forming proteins(PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs(af-PFPs) and trefoil factors(TFFs). As a toad af-PFP, Bm ALP1 can be reversibly regulated between active and inactive forms, with its paralog Bm ALP3 acting as a negative regulator. Bm ALP1 interacts with Bm TFF3 to form a cellular active complex called βγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of βγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence,we hereby propose a secretory endolysosome channel(SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with βγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.展开更多
文摘INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.
基金financially supported by Science and Technology Commission of Shanghai Municipality(No.18ZR1404100)Shanghai Pujiang Program(No.18PJD001)+2 种基金the National Natural Science Foundation of China(Nos.81573363 and 81690263)the Youth Teacher Training Program of Shanghai Municipal Education Commission(No.ZZJKYX16011)Shanghai Municipal Commission of Health and Family Planning(No.20164Y0213)
文摘Nanoparticles are easily to be taken up by cells but hard to be transported across epithelia, which plays an important role in absorption and toxicity. This paper aims to elucidate the effect of nanoparticle shape on bilateral exocytosis of Caco-2 cells. The fluorescent resonance energy transfer(FRET) probes(DiO and Dil)were utilized to label nanospheres and nanorods for determining and tracking nanoparticles. The results showed that more nanorods were internalized into Caco-2 cells than nanospheres. But the apical exocytosis rate of nanospheres from Caco-2 cells was faster than that of nanorods significantly. In addition, only less than 2% of intact nanoparticles were transported across monolayers for both nanoparticles, but the exocytosis behaviors between them was absolutely different. Compared to nanospheres, nanorods preferred basolateral exocytosis to apical exocytosis. Therefore, the shape is a critical parameter in cellular uptake and transport, even intracellular fate, which should be primarily considered in design of oral nanoparticles.
文摘To in teract with the egg, the spermatozo on must un dergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the invoIvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent;however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l^-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l^1), whereas higher concerttrations (>5 pmol l^-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing?protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca^2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization an d/or activation of effectors down stream to F-actin breakdown that lead to acrosomal exocytosis.
基金This work was supported by the National Natural Science Foundation of China(81770999 and 81670281)the Shanghai Municipal Commission of Science and Technology Research Project(18140900304,and 19140900902)the Big Data and Artificial Intelligence Project(2020DSJ07).
文摘Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that IHCs showed significant damage after exposure to a high concentration of salicylate.Whole-cell patch clamp recordings showed that 1–5 mmol/L salicylate did not affect the exocytosis of IHCs,indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input.Instead,salicylate induced a larger peak amplitude,a more negative half-activation voltage,and a steeper slope factor of Ca^(2+)current.Using noise analysis of Ca^(2+)tail currents and qRT-PCR,we further found that salicylate increased the number of Ca^(2+)channels along with CaV1.3 expression.All these changes could act synergistically to enhance the Ca^(2+)influx into IHCs.Inhibition of intracellular Ca^(2+)overload significantly attenuated IHC death after 10 mmol/L salicylate treatment.These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.
基金Grants to H.Y.G. from the U.S. National Institute of Health, R21 AA015579-01A1 and to S.V. form the Canadian Institute of Health Research
文摘AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS: One and 6 μmol/L carbachol evoked a dose- dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.
文摘Insulin secretion is a complex and highly regulated process.Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from these studies apply to living animals.That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo.When combined with genetically encoded biosensor,optical imaging is a powerful tool for visualization of molecular events in vivo.In this study,we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins,a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol.Upon exocytosis of secretory granules,the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH,while mCherry fluorescence remained constant in the process,thus allowing ratiometric quantification of insulin secretory events.Furthermore,mCherry fluorescence enabled tracking the movement of secretory granules in living cells.We validated this approach in insulin-secreting cells,and generated a transgenic mouse line expressing the optical sensor specifically in pancreaticβ-cells.The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.
基金This project was supported by a grant from the National Natural Science Foundation of China (No. C30100169).
文摘Summary: The expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8 % and 10 % of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.
基金This work was supported by the Grants PICT 2764-2016,PICT 02849-2018 and PICT 02041-2019 from the Agencia Nacional de Promoción de la Investigación,el Desarrollo Tecnológico y la Innovación(Argentina)ICN09_022 from ICM-ANID(Chile).
文摘The cortical actin network is a mesh of filaments distributed beneath the plasmalemma that dynamically reacts in response to stimuli.This dynamic network of cortical filaments,together with motor myosin partners,adjusts the plasmalemma tension,organizes membrane protein microdomains,remodels the cell surface and drives vesicle motion in order to fine-tune exocytosis,endocytosis and recycling of secretory vesicles.In this review,we discuss how these mechanisms work in secretory cells.
文摘We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12 - 17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.
基金sponsored by the NSFC (Nos. 61527815, 61471342, 31500800, 61501426)the Beijing Municipal Science & Technology Commission (No. Z141100000214002)the Major National Scientific Research Plan (No. 2014CB74465)
文摘The subsecond, temporal, vesicular exocytosis is ubiquitous, but difficult detecting in communication mechanisms of cells. A microelectrode array （MEA）, fabricated by MEMS technology, was applied successfully for real-time monitoring of quantal exocytosis from single pheochromocytoma （PC12） cell, The developed MEA was evaluated by dopamine （DA） using electrochemical methods and the results revealed that the sensitivity of DA was improved to 12659.24 p.A L mmol 1 cm 2. The modified MEA was used to detect in vitro vesicular exocytosis of DA from single PCI 2 cells stimulated by concentrated 100 mmol L-1 K＋ cell solution. A total of 592 spikes were measured and analyzed by three parameters and the statistical results revealed the population of each parameter was an approximate Gaussian distribution, and on average, 1.31 × 106 ± 9.25× 104 oxidizable molecules were released in each quantal exocytosis. In addition, results also indicate that a single PC12 cell probably releases the spikes with T ranging from 25.6 ms to 35.4 ms corresponding to/max ranging from 45.6 pA to 65.2 pA. The devices, including a homemade computer interface and the MEA modified with polymer film, provides a new means for further research on the neural, intercellular, communication mechanism.
基金supported by the Japan Society for the Promotion of Science KAKENHI grant numbers 15K11079,17H04379,and 18K09536.
文摘Mast cells are responsible for anaphylaxis and allergy and regulate various innate and adaptive immune responses.1,2 Although several“small”Rab GTPases were reported to regulate exocytosis of mast cells,3-5 little is known about the contribution of“large”Rab GTPases.Rab44 is a large Rab-GTPase that contains a Rab-GTPase domain and some additional N-terminal domains.6,7 Here,we investigated the role of Rab44 in the physiology of mast cells and in anaphylaxis.Rab44 was expressed as two isoforms in murine bone-marrow mast cells(BMMCs),both of which were deleted in Rab44-knockout mice.The Rab44-knockout mice exhibited diminished anaphylaxis,and the Rab44-knockout BMMCs showed a decrease in FcεRImediated histamine andβ-hexosaminidase secretion.Thus,Rab44 regulates granule exocytosis in mast cells and IgEmediated anaphylaxis in mice.
文摘Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments （AFs） and microtubules （MTs）. AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane （PM）. Secretory activity is balanced by PM retrieval that maintains cell mem- brane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching （FRAP） experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM.
基金supported by the National Basic Research Program of China(Grant No. 2010CB833701)the National Natural Science Foundation of China(Grant Nos. 30870564 and 90913022)the CAS Project(Grant No.KSCX2-SW-224)
文摘The molecular mechanisms by which dense core vesicles(DCVs) translocate,tether,dock and prime are poorly understood.In this study,Caenorhabditis elegans was used as a model organism to study the function of Rab proteins and their effectors in DCV exocytosis.RAB-27/AEX-6,but not RAB-3,was found to be required for peptide release from neurons.By analyzing the movement of DCVs approaching the plasma membrane using total internal reflection fluorescence microscopy,we demonstrated that RAB-27/AEX-6 is involved in the tethering of DCVs and that its effector rabphilin/RBF-1 is required for the initial tethering and subsequent stabilization by docking.
文摘Exocytosis of mam maUan sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizingthe exocytotic response of streptolysin O-permeabiUzed human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranyigeranyiated, and active Rab3A elicited human sperm exocytosis perse. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino.terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.
文摘The fusion by exocytosis of many vesicles to the plasma membrane induces the discharge to the extracellular space of their abundant luminal cargoes.Other exocytic vesicles,however,do not contain cargoes,and thus,their fusion is not followed by secretion.Therefore,two distinct processes of exocytosis exist,one secretory and the other non-secretory.The present review deals with the knowledge of non-secretory exocytosis developed during recent years.Among such developments are the dual generation of the exocytic vesicles,initially released either from the trans-Golgi network or by endocytosis;their traffic with activation of receptors,channels,pumps,and transporters;the identification of their tethering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes that govern membrane fusions;the growth of axons and the membrane repair.Examples of potential relevance of these processes for pathology and medicine are also reported.The developments presented here offer interesting chances for future progress in the field.
基金supported by the National Natural Science Foundation of China(21273181,21328501)the Tai-Shan Scholar Research Fund of Shandong Province+1 种基金the China Ocean Mineral Resources R&D Association project(DY125-15-T-08)the China Scholarship Council for their support
文摘In this research, we explored a rapid assessment of silver nanoparticles(Ag-NPs) neurotoxicity at a single-cell level. Traditional nanotoxicity assays on large cell-populations may hide the important heterogeneity of individual cells often found in neuronal cells. The development in the area of new nanomaterial discoveries is far ahead of the development of advanced tools to measure these materials' toxicity. Development of alternative approaches to assess nanomaterials toxicity rapidly, reliably, and accurately is desirable. Here, we present a chip-based, cell-integrated microwell-array device for rapid assessment of neurotoxicity of Ag-NPs by monitoring the exocytosis function of a PC12 cell. Results presented here confirm the dose-dependent toxicity of Ag-NPs and the immediate alteration of their exocytosis function when exposed to NPs.
基金This work was supported by a grant from the Nationa NaturaI Science Foundation of China(No.C30100169).
文摘Synaptotagmin （Syt） constitutes a family of membrane-trafficking proteins, so far nearly 20 Syts have been discovered. Extensive work showed that synatotagmins were a potential Ca^2＋ sensor for regulated exocytosis. This study was to investigate the expression and location of synaptotagmin II （Syt2） in RBL-2H3 （RBL） and its role in regulating exocytosis of RBL. The expression of Syt2 in RBL was confirmed by Western blot. The recombinant expression vector pEGFP-N1-Syt2 was constructed and transfected into RBL by electroporation, the stable transfectant RBL-Syt2-S expressing fusion protein Syt2-EGFP were obtained and Syt2 was highly concentrated at plasma membrane with little detected in cytoplasm. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. Compared with control, the release of cathepsin D by RBL-Syt2-S was markedly decreased. The results indicated that Syt2 played a negative regulation in exocytosis of lysosomes in RBL. Cellular ＆ Molecular Immunology. 2005;2（3）：205-209.
基金funded by the National Natural Science Foundation of China(NSFC-ISF 32061143020 to M.Q.D.,31925026 to F.S.,and 31501160 to X.X.L.)the Ministry of Science and Technology of the People's Republic of China(institutional grants to NIBS,Beijing,a fund of the National High-Level Talents Special Support Program to M.Q.D.)Beijing Municipal Science and Technology Commission(institutional grants to NIBS,Beijing and a fund for cultivation and development of innovation base to M.Q.D.).
文摘Vitellogenins(VITs)are the most abundant proteins in adult hermaphrodite Caenorhabditis elegans.VITs are synthesized in the intes-tine,secreted to the pseudocoelom,matured into yolk proteins,and finally deposited in oocytes as nutrients for progeny development.How VITs are secreted out of the intestine remains unclear.Using immuno-electron microscopy(immuno-EM),we localize intestinal VITs along an exocytic pathway consisting of the rough endoplasmic reticulum(ER),the Golgi,and the lipid bilayer-bounded VIT vesicles(VVs).This suggests that the classic exocytotic pathway mediates the secretion of VITs from the intestine to the pseudocoe-lom.We also show that pseudocoelomic yolk patches(PYPs)are membrane-less and amorphous.The different VITs/yolk proteins are packed as a mixture into the above structures.The size of VVs can vary with the VIT levels and the age of the worm.On adult Day 2(AD 2),intestinal VVs(~200 nm in diameter)are smaller than gonadal yolk organelles(YOs,~500 nm in diameter).VVs,PYPs,and YOs share a uniform medium electron density by conventional EM.The morphological profiles documented in this study serve as a refer-ence for future studies of VITs/yolk proteins.
基金supported by the National Basic Research Development Program of China(No.2006CB705600)the National Natural Science Foundation of China(No.30700253,No.30800355)+1 种基金Research Project of Tianjin Medical University(No.2006KY21)2008 Scien-tific Research Foundation for the Returned Overseas Chi-nese Scholars.
文摘Down syndrome （DS） is the most common cause of cognitive impairment associated with a congenital chromosomal abnormality, trisomy of chromosome 21. Mental retardation and congenital heart defects are key features of DS, All DS individuals develop early-onset Alzheimer＇s disease-like neuropathology. Intersectin 1 gene is localized on human chromosome 21, the critical region of DS, and it has higher expression in the brain of DS patients than in normal individuals. So fully understanding functions of intersectin 1 is critical for revealing the pathogenesis of DS. Intersectin 1 protein has two isoforms： intersectin 1-L and intersectin 1-S. This review will focus 1 in the central nervous system. on the distribution, expression characters and functions of intersectin
基金supported by the National Natural Science Foundation of China (31572268, U1602225, 31872226)Yunling Scholar Program to Y.Z。
文摘Secretory pore-forming proteins(PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs(af-PFPs) and trefoil factors(TFFs). As a toad af-PFP, Bm ALP1 can be reversibly regulated between active and inactive forms, with its paralog Bm ALP3 acting as a negative regulator. Bm ALP1 interacts with Bm TFF3 to form a cellular active complex called βγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of βγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence,we hereby propose a secretory endolysosome channel(SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with βγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.
