A Rab10-ACAP1-Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling.

Membrane trafficking processes regulate the G protein-coupled receptor activity. The muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, but the cellular machineries that control the trafficking of these receptors remain largely elusive. Here, we revealed the role of the small GTPase Rab10 as a negative regulator for the post-activation trafficking of M4 mAChR and the underlying mechanism. We show that constitutively active Rab10 arrests the receptor within Rab5-positive early endosomes and significantly hinders the resensitization of M4-mediated Ca2+ signaling. Mechanistically, M4 binds to Rab10-GTP, which requires the motif 386RKKRQMAA393 (R386-A393) within the third intracellular loop. Moreover, Rab10-GTP inactivates Arf6 by recruiting the Arf6 GTPase-activating protein, ACAP1. Strikingly, deletion of the motif R386-A393 causes M4 to bypass the control by Rab10 and switch to the Rab4-facilitated fast recycling pathway, thus reusing the receptor. Therefore, Rab10 couples the cargo sorting and membrane trafficking regulation through cycle between GTP-bound and GDP-bound state. Our findings suggest a model that Rab10 binds to the M4 like a molecular brake and controls the receptor's transport through endosomes, thus modulating the signaling, and this regulation is specific among the mAChR subtypes.

SNX-3 mediates retromer-independent tubular endosomal recycling by opposing EEA-1-facilitated trafficking.

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.

WIP-1 and DBN-1 promote scission of endocytic vesicles by bridging actin and Dynamin-1 in the C. elegans intestine.

There has been a consensus that actin plays an important role in scission of the clathrin-coated pits (CCPs) together with large GTPases of the dynamin family in metazoan cells. However, the recruitment, regulation and functional interdependence of actin and dynamin during this process remain inadequately understood. Here, based on small-scale screening and in vivo live-imaging techniques, we identified a novel set of molecules underlying CCP scission in the multicellular organism Caenorhabditis elegans We found that loss of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP-1) impaired CCP scission in a manner that is independent of the C. elegans homolog of WASP/N-WASP (WSP-1) and is mediated by direct binding to G-actin. Moreover, the cortactin-binding domain of WIP-1 serves as the binding interface for DBN-1 (also known in other organisms as Abp1), another actin-binding protein. We demonstrate that the interaction between DBN-1 and F-actin is essential for Dynamin-1 (DYN-1) recruitment at endocytic sites. In addition, the recycling regulator RME-1, a homolog of mammalian Eps15 homology (EH) domain-containing proteins, is increasingly recruited at the arrested endocytic intermediates induced by F-actin loss or DYN-1 inactivation, which further stabilizes the tubular endocytic intermediates. Our study provides new insights into the molecular network underlying F-actin participation in the scission of CCPs.This article has an associated First Person interview with the first author of the paper.

Sorting Out the Genetic Background of the Last Surviving South China Tigers.

The South China tiger (Panthera tigris amoyensis) is endemic to China and also the most critically endangered subspecies of living tigers. It is considered extinct in the wild and only about 150 individuals survive in captivity to date, whose genetic heritage, however, is ambiguous and controversial. Here, we conducted an explicit genetic assessment of 92 studbook-registered South China tigers from 14 captive facilities using a subspecies-diagnostic system in the context of comparison with other voucher specimens to evaluate the genetic ancestry and level of distinctiveness of the last surviving P. t. amoyensis. Three mtDNA haplotypes were identified from South China tigers sampled in this study, including a unique P. t. amoyensis AMO1 haplotype not found in other subspecies, a COR1 haplotype that is widespread in Indochinese tigers (P. t. corbetti), and an ALT haplotype that is characteristic of Amur tigers (P. t. altaica). Bayesian STRUCTURE analysis and parentage verification confirmed the verified subspecies ancestry (VSA) as the South China tiger in 74 individuals. Genetic introgression from other tigers was detected in 18 tigers, and subsequent exclusion of these and their offspring from the breeding program is recommended. Both STRUCTURE clustering and microsatellite-based phylogenetic analyses demonstrated a close genetic association of the VSA South China tigers to Indochinese tigers, an issue that could only be elucidated by analysis of historical South China tiger specimens with wild origin. Our results also indicated a moderate level of genetic diversity in the captive South China tiger population, suggesting a potential for genetic restoration.

SEC-10 and RAB-10 coordinate basolateral recycling of clathrin-independent cargo through endosomal tubules in Caenorhabditis elegans.

Despite the increasing number of regulatory proteins identified in clathrin-independent endocytic (CIE) pathways, our understanding of the exact functions of these proteins and the sequential manner in which they function remains limited. In this study, using the Caenorhabditis elegans intestine as a model, we observed a unique structure of interconnected endosomal tubules, which is required for the basolateral recycling of several CIE cargoes including hTAC, GLUT1, and DAF-4. SEC-10 is a subunit of the octameric protein complex exocyst. Depleting SEC-10 and several other exocyst components disrupted the endosomal tubules into various ring-like structures. An epistasis analysis further suggested that SEC-10 operates at the intermediate step between early endosomes and recycling endosomes. The endosomal tubules were also sensitive to inactivation of the Rab GTPase RAB-10 and disruption of microtubules. Taken together, our data suggest that SEC-10 coordinates with RAB-10 and microtubules to form the endosomal tubular network for efficient recycling of particular CIE cargoes.

The effect of the size of fluorescent dextran on its endocytic pathway.

Fluorescent dextrans are commonly used as macropinocytic probes to study the properties of endocytic cargoes; however, the effect of the size of dextrans on endocytic mechanisms has not been carefully analyzed. By using chemical and siRNA inhibition of individual endocytic pathways, we evaluated the internalization of two commonly used dextrans, Dex10 (dextran 10 kDa) and Dex70 (dextran 70 kDa), in mammalian HeLa cells and Caenorhabditis elegans coelomocytes. We revealed that Dex70 enters these two cell types predominantly via clathrin- and dynamin-independent and amiloride-sensitive macropinocytosis process; Dex10, on the other hand, enters the two cell types through clathrin-/dynamin-dependent micropinocytosis in addition to macropinocytosis. In addition, although different-sized dextrans follow different endocytic processes, they share common post-endocytic events. Herein, though straightforward, our studies support that the size of nanomaterials could play a paramount role in their inclusion into endocytic vesicles and suggest that care should be taken while selecting endocytic pathway markers. Based on our results, we propose that Dex70 is a better probe for macropinocytosis, whereas Dex10 and smaller molecules are better for probing general fluid-phase endocytosis, which includes macropinocytic and micropinocytic processes.

Tracking the down-regulation of folate receptor-α in cancer cells through target specific delivery of quantum dots coupled with antisense oligonucleotide and targeted peptide.

Based on the multivalent binding capability of streptavidin (SA) to biotin, a multifunctional quantum dot probe (QD-(AS-ODN+p160)) coupled with antisense oligonucleotide (AS-ODN) and peptide p160 is designed for real-time tracking of targeted delivery of AS-ODN and regulation of folate receptor-α (hFR-α) in MCF-7 breast cancer cells. Fluorescence spectra, capillary electrophoresis (CE) and dynamic light scattering (DLS) are used to characterize the conjugation of AS-ODN and p160 with quantum dots (QDs), DLS results confirm the well stability of the probe in aqueous media. Confocal imaging and quantitative flow cytometry show that QD-(AS-ODN+p160) is able to specifically target human breast cancer MCF-7 cells. Low temperature and ATP depletion treatments reveal the cellular uptake of QD-(AS-ODN+p160) is energy-dependent, and the effects of inhibition agents and co-localization imaging further confirm the endocytic pathway is mainly receptor-mediated. Transmission electron microscopy (TEM) shows the intracellular delivery and endosomal escape of QD probe along with incubation time extended. Two transfection concentrations of QD probe (10 nM and 50 nM) below half inhibitory concentration (IC50 ) value are chosen according to MTT assay. Real-time PCR shows at these two concentration cases the relative mRNA expression levels of hFR-α reduce to 72.5 ± 3.9% and 17.6 ± 1.0%, respectively. However, western blot and quantitative ELISA analysis show the expression level of hFR-α protein has a significant decrease only at 50 nM, indicating that gene silence is concentration-dependent. These results demonstrate that the QD-(AS-ODN+p160) probe not only achieves gene silence in a cell-specific manner but also achieves real-time tracking during AS-ODN intracellular delivery.

RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1.

The endosomal system maintains cellular homeostasis by coordinating multiple vesicular trafficking events, and the retromer complex plays a critical role in endosomal cargo recognition and sorting. Here, we demonstrate an essential role for the small GTPase RAB21 in regulating retromer-mediated recycling of the glucose transporter SLC2A1/GLUT1 and macroautophagy/autophagy. RAB21 depletion mis-sorts SLC2A1 to lysosomes and affects glucose uptake, thereby activating the AMPK-ULK1 pathway to increase autophagic flux. RAB21 depletion also increases lysosome function. Notably, RAB21 depletion does not overtly affect retrograde transport of IGF2R/CI-M6PR or WLS from endosomes to the trans-Golgi network. We speculate that RAB21 regulates fission of retromer-decorated endosomal tubules, as RAB21 depletion causes accumulation of the SNX27-containing retromer complex on enlarged endosomes at the perinuclear region. Functionally, RAB21 depletion sensitizes cancer cells to energy stress and inhibits tumor growth in vivo, suggesting an oncogenic role for RAB21. Overall, our study illuminates the role of RAB21 in regulating endosomal dynamics and maintaining cellular energy homeostasis and suggests RAB21 as a potential metabolic target for cancer therapy.

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes.

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P. t. amoyensis mitochondrial genomes were also highly similar to those of P. uncia. Additionally, the rate of point mutation was only 0.3% and a total of 59 variable sites between P25 and P27 were found. Out of the 59 variable sites, 6 were located in 6 different tRNA genes, 6 in the 2 rRNA genes, 7 in non-coding regions (one located between tRNA-Asn and tRNA-Tyr and six in the D-loop), and 40 in 10 protein-coding genes. COI held the largest amount of variable sites (9 sites) and Cytb contained the highest variable rate (0.7%) in the complete sequences. Moreover, out of the 40 variable sites located in 10 protein-coding genes, 12 sites were nonsynonymous.

[Effectiveness of total hip arthroplasty in patients with a history of hip preservation surgery with secondary osteoarthritis for developmental dysplasia of the hip].

OBJECTIVE: To evaluate early to medium-term effectiveness of total hip arthroplasty (THA) in patients with a history of hip preservation surgery with secondary severe osteoarthritis for developmental dysplasia of the hip (DDH). METHODS: The clinical data of 25 DDH patients (31 hips) who had severe osteoarthritis after hip preservation surgery and received THA between September 2009 and March 2021 were retrospectively analyzed. There were 1 male and 24 females; the age ranged from 18 to 65 years, with an average age of 43 years; 8 hips were classified into Crowe type Ⅰ, 9 hips were type Ⅱ, 3 hips were type Ⅲ, and 11 hips were type Ⅳ. The time between osteotomy and THA ranged from 31 to 51 years, with an average of 31.96 years. Preoperative hip flexion range of motion was (69.31±29.72)°, abduction range of motion was (24.00±14.79)°; and Harris hip score was 45.3±15.5. Postoperative Harris hip score, hip range of motion, complications, radiographic findings, and implant survival rate were analyzed. RESULTS: Patients in both groups were followed up 2-132 months, with an average of 51 months. During the follow-up, periprosthetic fracture occurred in 1 case; there was no complication such as dislocation, periprosthetic infection, nerve palsy, or deep vein thrombosis. At last follow-up, the hip flexion range of motion was (109.52±11.17)°, abduction range of motion was (41.25±5.59)°, showing significant differences when compared with preoperative values ( t=8.260, P=0.000; t=6.524, P=0.000). The Harris hip score was 91.5±4.1, and the difference was significant when compared with preoperative score ( t=11.696, P=0.000); among them, 13 cases were excellent and 12 cases were good. Radiographic evaluation showed that the center of acetabular rotation moved up 0-18 mm (mean, 6.35 mm). The cup abductor angle was 28°-49° (mean, 37.74°) and the coverage rate was 69.44%-98.33% (mean, 81.04%). All femoral stems were fixed in neutral position without varus or valgus. No osteolysis, radiolucent line, or implant migration was observed. By the end of follow-up, none of the patients underwent revision and the survival rate of prothesis was 100%. CONCLUSION: THA is still the gold standard for the treatment of DDH patients with secondary osteoarthritis after hip preservation surgery. The postoperative joint function can be rapidly restored, the patients' quality of life can significantly improve, and the early to medium-term survival rate of the prosthesis is satisfactory.

Synaptotagmin IV regulates dense core vesicle (DCV) release in LbetaT2 cells.

Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca(2+) sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear. The function of Syt IV is of particular interest because it is rapidly up-regulated by chronic depolarization and seizures. Using RNAi-mediated gene silencing, we have explored the role of Syt I and IV on secretion in a pituitary gonadotrope cell line. Downregulation of Syt IV clearly reduced Ca(2+)-triggered exocytosis of dense core vesicles (DCVs) in LbetaT2 cells. Syt I silencing, however, had no effect on vesicular release.

Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor.

Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence (374)KKKPPPS(380) servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching (374)KKKPPPS(380) to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, (361)VARKIVKMTKQPA(373), which is normally masked in the presence of the downstream sequence (374)KKKPPPS(380). Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent.

Spectroscopic and voltammetric study on the binding of aluminium(III) to DNA.

Cyclic voltammetry (CV) and ultraviolet (UV) spectroscopy were used, for the first time, to study the interaction between aluminium(III) and calf thymus DNA under neutral pH conditions. Thus obtained data confirmed the existence of a relatively strong interaction between Al(III) and DNA. The binding site for aluminium(III) on DNA chains is not the bases, but the phosphate groups on the DNA backbones, the same as that for [Co(phen)3](3+/2+) that binds non-specifically and electrostatically to the deoxyribose phosphate backbone of DNA. When coexisting, Al(III) binds more favorably to DNA than [Co(phen)3](3+/2+), which implies the relatively strong binding of Al(III) to the phosphate backbone of DNA under neutral pH conditions. The nature of the binding of Al(III) to DNA is also discussed.

Mechanisms of U87 astrocytoma cell uptake and trafficking of monomeric versus protofibril Alzheimer's disease amyloid-ß proteins.

A significant hallmark of Alzheimer's disease is the formation of senile plaques in the brain due to the unbalanced levels of amyloid-beta (Aß). However, although how Aß is produced from amyloid precursor proteins is well understood, little is known regarding the clearance and metabolism of various Aß aggregates from the brain. Similarly, little is known regarding how astrocytes internalize and degrade Aß, although astrocytes are known to play an important role in plaque maintenance and Aß clearance. The objective of this study is to investigate the cellular mechanisms that mediate the internalization of soluble monomeric versus oligomeric Aß by astrocytes. We used a combination of laser confocal microscopy and genetic and pharmacological experiments to dissect the internalization of sAß42 and oAß42 and their postendocytic transport by U87 human brain astrocytoma cell line. Both Aß42 species were internalized by U87 cells through fluid phase macropinocytosis, which required dynamin 2. Depleting LDL receptor-related protein 1 (LRP1) decreased sAß42 uptake more significantly than that of oAß42. We finally show that both Aß42 species were rapidly transported to lysosomes through an endolytic pathway and subjected to proteolysis after internalization, which had no significant toxic effects to the U87 cells under relatively low concentrations. We propose that macropinocytic sAß42 and oAß42 uptake and their subsequent proteolytic degradation in astroglial cells is a significant mechanism underlying Aß clearance from the extracellular milieu. Understanding the molecular events involved in astrocytic Aß internalization may identify potential therapeutic targets for Alzheimer's disease.

Roles of AP-2 in clathrin-mediated endocytosis.

BACKGROUND: The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway. METHODOLOGY/PRINCIPAL FINDINGS: We report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown. Moreover, there is a total absence of clathrin-containing structures completely lacking AP-2 while all the remaining coats still contain a small amount of AP-2. These observations suggest that AP-2 is essential for endocytic coated-pit and coated-vesicle formation. We also find that AP-2 knockdown strongly inhibits light-density lipoprotein (LDL) receptor-mediated endocytosis, as long as cells are maintained in complete serum and at 37 degrees C. If cells are first incubated with LDL at 4 degrees C, followed by warming, there is little or no decrease in LDL uptake with respect to control cells. LDL uptake at 37 degrees C is also not affected in AP-2 depleted cells first deprived of LDL by incubation with either serum-starved or LDL-starved cells for 24 hr. The LDL-deprived cells display a significant increase in endocytic structures enriched on deeply invaginated tubes that contain LDL and we suggest that under this condition of stress, LDL might enter through this alternative pathway. CONCLUSIONS/SIGNIFICANCE: These results suggest that AP-2 is essential for endocytic clathrin coated-pit and coated-vesicle formation. They also indicate that under normal conditions, functional endocytic clathrin coated pits are required for LDL internalization. We also show that under certain conditions of stress, cells can upregulate alternative endocytic structures with the potential to provide compensatory trafficking pathways.

Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells.

Synaptotagmin VII (Syt VII), which has a higher Ca(2+) affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca(2+)-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca(2+) sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.

Fabrication of size-controllable ultrasmall-disk electrode: monitoring single vesicle release kinetics at tiny structures with high spatio-temporal resolution.

Size-controllable micron or nano-disk carbon fiber electrode (CFE) is prepared and demonstrated to be excellent for extra-cellular transmitter release detection at tiny structures and vesicle fusion kinetics analysis with high spatio-temporal resolution. An improved electrochemical etching procedure was employed, for the first time, to fabricate cylindrical fiber with controlled micron or nano-diameter. Afterwards, a facile insulation with polypropylene sheath was employed to completely insulate the whole body of the thinned fiber, and an ultrasmall-disk sensing area was finally produced by cutting of the insulated fibers. Scanning electron microscopy (SEM) was employed to characterize the ultrasmall geometry size of the fabricated electrode and to show the tight adherence of the insulation sheath on the fiber. The cut ends of the electrodes were also shown to be smooth, clean and without obvious jagged layer. The fabricated micron or nano-disk carbon electrodes show ideal steady-state voltammetric behavior with satisfying reversibility. Subsequently, the performance of the ultrasmall-disk CFE for amperometric detection of cell secretion was characterized. Results showed that, compared to the conventional micro-disk CFE, the etched small disk CFE possesses higher sensitivity due to its obviously improved signal-to-noise level, which enables minute amounts of 3000 oxidizable molecules to be detectable. The nano-disk CFE was shown to be particularly ideal for analysis of fusion kinetics, due to its avoidance of diffusion broadening of the detected spikes, which is the inherent defect of the conventional micro-CFE technique.

Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles.

Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca(2+)-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells. We found that down-regulation of both Syt I and Syt IX resulted in a significant loss of Ca(2+)-dependent LDCV exocytosis. Moreover, our results suggest Syt I and Syt IX play redundant role in controlling the choice of fusion modes. Down-regulation of both Syt I and Syt IX renders more fusion in the kiss-and-run mode. We conclude that Syt I and Syt IX function redundantly in Ca(2+)-sensing and fusion pore dilation on LDCVs in PC12 cells.

Differential properties of GTP- and Ca(2+)-stimulated exocytosis from large dense core vesicles.

Many cells utilize a GTP-dependent pathway to trigger exocytosis in addition to Ca(2+)-triggered exocytosis. However, little is known about the mechanism by which GTP triggers exocytosis independent of Ca(2+). We used dual-color evanescent field microscopy to compare the motion and fusion of large dense core vesicles stimulated by either mastoparan (Mas) in Ca(2+)-free conditions or high K(+) in the presence of Ca(2+). We demonstrate that Mas is hardly effective in triggering the fusion of the predocked vesicles but predominantly mobilizes cytosolic vesicles. In contrast, Ca(2+)-dependent exocytosis is largely due to predocked vesicles. Fusion kinetics analysis and carbon-fiber amperometry reveal that Mas induces a brief 'kiss-and-run' fusion and releases only a small amount of the cargo, whereas Ca(2+) stimulates a more persistent opening of the fusion pore and larger release of the contents. Furthermore, we show that Mas-released vesicles require a much shorter time to reach fusion competence once they approach the plasma membrane. Our data suggest the involvement of different mechanisms not only in triggering and fusion but also in the docking and priming process for Ca(2+)- and GTP-dependent exocytosis.

Monitoring dopamine release from single living vesicles with nanoelectrodes.

Carbon fiber nanoelectrodes (tip diameter = ca. 100 nm) have been first used to monitor real-time dopamine release from single living vesicles of single rat pheochromocytoma (PC12) cells. The experiments show that active and inactive release sites exist on the surface of cells, and the spatial distributions have been differentiated even in the same active release zone. It is first demonstrated that multiple vesicles can sequentially release dopamine at the same site of the cell surface, which possibly plays the main role in the dopamine release from PC12 cells.