Fusion Of Individual Vesicles Research Articles

A palisade-shaped membrane reservoir is required for rapid ring cell inflation in Drechslerella dactyloides

Fusion of individual vesicles carrying membrane-building materials with the plasma membrane (PM) enables gradual cell expansion and shape change. Constricting ring (CR) cells of carnivorous fungi triple in size within 0.1-1 s to capture passing nematodes. Here, we investigated how a carnivorous fungus, Drechslerella dactyloides, executes rapid and irreversible PM expansion during CR inflation. During CR maturation, vesicles carrying membrane-building materials accumulate and fuse, forming a structure named the Palisade-shaped Membrane-building Structure (PMS) around the rumen side of ring cells. After CR inflation, the PMS disappears, with partially inflated cells displaying wavy PM and fully inflated cells exhibiting smooth PM, suggesting that the PMS serves as the reservoir for membrane-building materials to enable rapid and extensive PM expansion. The DdSnc1, a v-SNARE protein, accumulates at the inner side of ring cells and is necessary for PMS formation and CR inflation. This study elucidates the unique cellular mechanisms underpinning rapid CR inflation.

Multiple Modes of Fusion and Retrieval at the Calyx of Held Synapse.

Neurotransmitter in vesicles is released through a fusion pore when vesicles fuse with the plasma membrane. Subsequent retrieval of the fused vesicle membrane is the key step in recycling exocytosed vesicles. Application of advanced electrophysiological techniques to a large nerve terminal, the calyx of Held, has led to recordings of endocytosis, individual vesicle fusion and retrieval, and the kinetics of the fusion pore opening process and the fission pore closure process. These studies have revealed three kinetically different forms of endocytosis-rapid, slow, and bulk-and two forms of fusion-full collapse and kiss-and-run. Calcium influx triggers all kinetically distinguishable forms of endocytosis at calyces by activation of calmodulin/calcineurin signaling pathway and protein kinase C, which may dephosphorylate and phosphorylate endocytic proteins. Polymerized actin may provide mechanical forces to bend the membrane, forming membrane pits, the precursor for generating vesicles. These research advancements are reviewed in this chapter.

The Serotonin Transporter Modulates the Kinetics and Quantal Size of Vesicular Fusion Events in Sympathoadrenal Chromaffin Cells

The antidepressant‐sensitive serotonin transporter (SERT) is a key regulator of serotonin (5‐HT) signaling and availability in the CNS. The robust expression of SERT in adrenal chromaffin cells (ACCs), which comprise the neuroendocrine arm of the sympathetic nervous system, is less well understood. ACCs synthesize and secrete catecholamines (epinephrine, norepinephrine) which mediate the physiological response to stress. They do not synthesize 5‐HT, but do accumulate small amounts through SERT‐mediated uptake (5‐HT content is <0.15% of the epinephrine content). We hypothesized that the chromaffin cells utilize this 5‐HT for autocrine / paracrine control of the sympathoadrenal stress response. Consistent with this hypothesis, we previously reported that 5‐HT1A receptors inhibit catecholamine secretion by reducing the number of secretory vesicles that fuse with the plasma. The objective of the current study was to investigate an additional, receptor‐independent mechanism by which SERT controls adrenal catecholamine secretion.We used carbon fiber amperometry to analyze catecholamine secretion from ACCs that were isolated from either wild type mice, global SERT‐knockout mice (SERT‐/‐), or sympathoadrenal system‐specific SERT knockout mice (SERTΔTH). Transmitter release from individual vesicle fusion events can be resolved as amperometric spikes. There was no difference in the number or time‐course of amperometric spikes evoked by 30mM KCl. The charge of the spikes is directly proportional to the amount of transmitter released by a single vesicle (i.e. quantal size). Spike charge was significantly smaller (~35%) and spike duration (half‐width) was significantly shorter in SERT‐knockout cells compared to matched controls. This was surprising given that HPLC analysis revealed no change in the catecholamine content of adrenal glands isolated from knockout mice compared to wild‐type controls; the 5‐HT content was significantly reduced but this accounted for <0.15% of the total monoamine content. Changes in calcium entry can modulate the quantal size of catecholamine release in ACCs, but patch‐clamp recording revealed there was no significant difference in voltage‐gated calcium channel currents in SERT knockout cells and ratiometric calcium imaging found no difference in KCl‐evoked calcium entry. The decrease in quantal size was mimicked in wild‐type cells by treating for >24 hours with escitalopram (an SSRI antidepressant which blocks SERT), or by depleting extracellular 5‐HT from the culture medium for >24 hours (use of dialyzed serum in the culture media). In contrast, acute (minutes) or short‐term (<6‐8 hrs) treatment with escitalopram or 5‐HT depletion had no effect. Adrenal chromaffin cells lack the rate limiting enzyme for 5‐HT synthesis (tryptophan hydroxylase), but if the product of this enzyme (5‐hydroxytryptophan; 5‐HTP) is provided it can be converted into 5‐HT. Treating ACCs with 5‐HTP for 1‐3 hrs had no effect but 24 hr treatment rescued the reduced spike charge seen in SERT knockout cells. Together, our data suggest that, following SERT‐mediated uptake, intracellular 5‐HT modulates the kinetics and thus fraction of secretory vesicle content that is released during a fusion event.

Dissecting the Synergistic Roles of Synaptotagmin and Complexin in Ca2+-Regulated Exocytosis

Controlled release of neurotransmitters stored in synaptic vesicles (SV) is central to all information processing in the brain. This process relies on the efficient coupling of SV fusion to the triggering signal - action potential evoked Ca2+ influx. SV fusion is catalysed by the synaptic SNARE proteins: VAMP2 on the SV (v-SNAREs) and syntaxin and SNAP25 on the pre-synaptic membrane (t-SNAREs). The fusion is tightly regulated by Complexin (Cpx) and presynaptic Ca2+ release sensor Synaptotagmin1 (Syt1) to enable ultra-fast Ca2+-coupled release. However, the precise molecular mechanisms of this process remain enigmatic. To address this, we used the recently developed pore-spanning lipid bilayer system, which allows for the precision study of the sub-steps of individual vesicle fusion events at tens of milliseconds timescale. We find that Syt1 and Cpx act co-operatively to generate and maintain a Ca2+-sensitive pool of vesicle under in vitro conditions. Our data suggest that oligomers of Syt1 engage a limited number of ‘central’ SNAREpins via the ‘primary’ binding site to introduce an un-stable clamp. This ‘delay’ in the fusion process enables Cpx to arrests the assembly of remainder ‘ectopic’ SNAREpins, in turn producing a stable clamped state. These docked vesicles are triggered to fuse upon Ca2+ influx and activation of Syt1-associated SNAREpins are sufficient to elicit a rapid (0.1s), synchronous fusion. Overall, our suggests that Syt1 plays a central role in orchestrating Ca2+-coupled neurotransmitter release.

Regulation of SNARE‐Dependent Membrane Fusion by Alpha‐Synuclein

Neurotransmitter release is a foundation of all thoughts in central nervous system. Communication between these neurons is mediated by the release and uptake of neurotransmitters through vesicle fusion at the synapses. The SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor) proteins are believed to mediate neurotransmitter release by promoting fusion of neurotransmitter laden presynaptic vesicle with the presynaptic membrane. Defects in the regulation of neurotransmission are believed to be the primary cause for many neurological diseases. The key to a better understanding of neurological diseases may lie in a more complete understanding of the molecular machine responsible for synaptic vesicle fusion. Alpha-synuclein (αS) has long been a protein of great interest because of its pathological aggregation in neuronal plaque and Lewy bodies which are associated with Parkinson's Disease and Lewy Bodies Dementia, respectively. Despite its medical significance, the normal function of αS has been elusive. αS is a 14 kDa soluble protein, abundantly present (several μM) in the presynapse. Thus, not surprisingly, αS has been found to play roles in various aspects of vesicle recycling. Very interestingly, however, αS has emerged as an important regulator for SNARE-dependent vesicle fusion. We used an in vitro single-vesicle fusion assay which allowed for the precise variation of the αS concentration. This assay provided the opportunity to dissect the impact of αS on individual vesicle fusion steps such as docking and lipid mixing along the membrane fusion pathway. The assay is effective in analyzing single fusion events in a natural, millisecond timescale between vesicle and a supported-bilayer. Using this assay, we have found that αS enhances vesicle docking by a factor of 10 at excessive 20 μM which is as much as 4 times higher than a normal cellular concentration. Detailed analysis reveals that the effect of αS on SNARE-dependent membrane fusion is largely on vesicle docking with no significant changes in lipid mixing observed. It also suggests that αS plays a role in accelerating and stabilizing large fusion pore formation. αS may be pathologically involved in neurological diseases, but it also is a crucial SNARE regulator by enhancing presynaptic vesicle docking and stabilizing the membrane fusion pore. αS may be pathologically involved in neurological diseases, but it also is a crucial SNARE regulator This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Search for a minimal machinery for Ca2+-triggered millisecond neuroexocytosis

Neurons have the remarkable ability to release a batch of neurotransmitters into the synapse immediately after an action potential. This signature event is made possible by the simultaneous fusion of a number of synaptic vesicles to the plasma membrane upon Ca2+ entry into the active zone. The outcomes of both cellular and in vitro studies suggest that soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) and synaptotagmin 1 (Syt1) constitute the minimal fast exocytosis machinery in the neuron. Syt1 is the major Ca2+-sensor and orchestrates the synchronous start of individual vesicle fusion events while SNAREs are the membrane fusion machinery that dictates the kinetics of each single fusion event. The data also suggest that Ca2+-bound Syt1 is involved in the upstream docking step which leads to an increase in the number of fusion events or the size of the release, leaving the SNARE complex alone to carry out membrane fusion by themselves.

High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis.

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO2 holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.

An extended model of vesicle fusion at the plasma membrane to estimate protein lateral diffusion from TIRF microscopy images

BackgroundCharacterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins.ResultsA new model was introduced to represent the behavior of cargo transmembrane proteins during the vesicle fusion to the plasma membrane at the end of the exocytosis process. Two biophysical parameters, the diffusion coefficient and the release rate parameter, are automatically estimated from TIRFM image sequences, to account for both the lateral diffusion of molecules at the membrane and the continuous release of the proteins from the vesicle to the plasma membrane. Quantitative evaluation on 300 realistic computer-generated image sequences demonstrated the efficiency and accuracy of the method. The application of our method on 16 real TIRFM image sequences additionally revealed differences in the dynamic behavior of Transferrin Receptor (TfR) and Langerin proteins.ConclusionAn automated method has been designed to simultaneously estimate the diffusion coefficient and the release rate for each individual vesicle fusion event at the plasma membrane in TIRFM image sequences. It can be exploited for further deciphering cell membrane dynamics.

Spatiotemporal Regulation of Synaptic Vesicle Fusion Sites in Central Synapses

The number and availability of vesicle release sitesatthe synaptic active zone (AZ) are critical factors governing neurotransmitter release; yet, these fundamental synaptic parameters have remained undetermined. Moreover, how neural activity regulates the spatiotemporal properties of the release sites within individual central synapses is unknown. Here, we combined a nanoscale imaging approach with advanced image analysis to detect individual vesicle fusion events with ∼27nm localization precision at single hippocampal synapses under physiological conditions. Our results revealed the presence of multiple distinct release sites within individual hippocampal synapses. Release sites were distributed throughout the AZ and underwent repeated reuse. Furthermore, the spatiotemporal properties of the release sites were activity dependent with a reduction in reuse frequency and a shift in location toward the AZ periphery during high-frequency stimulation. These findings have revealed fundamental spatiotemporal properties of individual release sites in small central synapses and their activity-dependent modulation.

Mechanisms, pools, and sites of spontaneous vesicle release at synapses of rod and cone photoreceptors.

Photoreceptors have depolarized resting potentials that stimulate calcium-dependent release continuously from a large vesicle pool but neurons can also release vesicles without stimulation. We characterized the Ca(2+) dependence, vesicle pools, and release sites involved in spontaneous release at photoreceptor ribbon synapses. In whole-cell recordings from light-adapted horizontal cells (HCs) of tiger salamander retina, we detected miniature excitatory post-synaptic currents (mEPSCs) when no stimulation was applied to promote exocytosis. Blocking Ca(2+) influx by lowering extracellular Ca(2+) , by application of Cd(2+) and other agents reduced the frequency of mEPSCs but did not eliminate them, indicating that mEPSCs can occur independently of Ca(2+) . We also measured release presynaptically from rods and cones by examining quantal glutamate transporter anion currents. Presynaptic quantal event frequency was reduced by Cd(2+) or by increased intracellular Ca(2+) buffering in rods, but not in cones, that were voltage clamped at -70mV. By inhibiting the vesicle cycle with bafilomycin, we found the frequency of mEPSCs declined more rapidly than the amplitude of evoked excitatory post-synaptic currents (EPSCs) suggesting a possible separation between vesicle pools in evoked and spontaneous exocytosis. We mapped sites of Ca(2+) -independent release using total internal reflectance fluorescence (TIRF) microscopy to visualize fusion of individual vesicles loaded with dextran-conjugated pHrodo. Spontaneous release in rods occurred more frequently at non-ribbon sites than evoked release events. The function of Ca(2+) -independent spontaneous release at continuously active photoreceptor synapses remains unclear, but the low frequency of spontaneous quanta limits their impact on noise.

Molecular origins of synaptotagmin 1 activities on vesicle docking and fusion pore opening.

Synaptotagmin 1 (Syt1), a major Ca2+ sensor in neuroexocytosis, utilizes SNARE- and membrane-binding to regulate vesicle fusion, a required process for neurotransmitter release at the synapse. However, the mechanism by which Syt1 orchestrates SNARE- and membrane- binding to control individual vesicle fusion steps is still unclear. In this study, we used a number of single vesicle assays that can differentiate intermediates of neuroexocytosis, to focus on Syt1 mutants that might impair Syt1-SNARE/PIP2 interaction, Ca2+-binding, or membrane penetration. Our results show that, although putative Syt1-SNARE/PIP2 coupling through the polybasic region of the C2B domain is critical for vesicle docking, its disruption does not affect content release. In contrast, Ca2+-binding and membrane-penetration mutants significantly reduce content release. Our results thus delineate multiple functions of Syt1 along the pathway of Ca2+-triggered exocytosis in unprecedented detail.

Spatiotemporal Detection and Analysis of Exocytosis Reveal Fusion “Hotspots” Organized by the Cytoskeleton in Endocrine Cells

Total internal reflection fluorescence microscope has often been used to study the molecular mechanisms underlying vesicle exocytosis. However, the spatial occurrence of the fusion events within a single cell is not frequently explored due to the lack of sensitive and accurate computer-assisted programs to analyze large image data sets. Here, we have developed an image analysis platform for the nonbiased identification of different types of vesicle fusion events with high accuracy in different cell types. By performing spatiotemporal analysis of stimulus-evoked exocytosis in insulin-secreting INS-1 cells, we statistically prove that individual vesicle fusion events are clustered at hotspots. This spatial pattern disappears upon the disruption of either the actin or the microtubule network; this disruption also severely inhibits evoked exocytosis. By demonstrating that newcomer vesicles are delivered from the cell interior to the surface membrane for exocytosis, we highlight a previously unappreciated mechanism in which the cytoskeleton-dependent transportation of secretory vesicles organizes exocytosis hotspots in endocrine cells.

Microsecond dissection of neurotransmitter release: SNARE-complex assembly dictates speed and Ca²⁺ sensitivity.

SNARE-complex assembly mediates synaptic vesicle fusion during neurotransmitter release and requires that the target-SNARE protein syntaxin-1 switches from a closed to an open conformation. Although many SNARE proteins are available per vesicle, only one to three SNARE complexes are minimally needed for a fusion reaction. Here, we use high-resolution measurements of synaptic transmission in the calyx-of-Held synapse from mutant mice in which syntaxin-1 is rendered constitutively open and SNARE-complex assembly is enhanced to examine the relation between SNARE-complex assembly and neurotransmitter release. We show that enhancing SNARE-complex assembly dramatically increases the speed of evoked release, potentiates the Ca(2+)-affinity of release, and accelerates fusion-pore expansion during individual vesicle fusion events. Our data indicate that the number of assembled SNARE complexes per vesicle during fusion determines the presynaptic release probability and fusion kinetics and suggest a mechanism whereby proteins (Munc13 or RIM) may control presynaptic plasticity by regulating SNARE-complex assembly.

Individual Vesicle Fusion Events Mediated by Lipid-Anchored DNA

Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed.

Visualization of exocytosis and vesicle trafficking in inner medullary collecting duct (IMCD)

Conventional confocal microscopy has been used to monitor vasopressin (AVP) induced apical exocytosis in perfused IMCD using FM1‐43 as a membrane tracer, but individual fusion event could not be resolved. In two‐photon confocal microscopy, two‐photon excitation does not excite tracer molecules outside of the focal plane, and it can simultaneously excite multiple tracers with a single laser source. We sough to determine whether two‐photon confocal microscopy can detect individual vesicle fusion event in IMCD using a membrane tracer (FM1‐43) and a fluid‐phase tracer (Sulforhodamine B, SRB) simultaneously. Luminal FM1‐43 and SRB were excited at 850 nm. AVP (1 nM) induced a gradual increase of FM1‐43 fluorescence in the apical membrane, indicating an increase of membrane surface area due to exocytosis. Opening and flattening of fusion pores were also observed in the apical membrane. At the site of membrane fusion, FM1‐43 emission was increased ahead of SRB emission, which suggested that FM1‐43 diffuses along the lipid membrane into the vesicle while SRB enters the vesicle after the fusion pore is opened. The SRB emission dropped after the vesicle was flattened. The size of vesicles varied between 0.5 to 1.5 μm. The fusion time ranged from 30–80 s. Transcellular trafficking of fluorescent vesicles was also observed. These results demonstrated the feasibility to monitor vesicle fusion and trafficking in perfused IMCD.

Individual Vesicle-Vesicle and Vesicle-Planar Bilayer Fusion Events Mediated by DNA

We have previously reported that DNA-lipid conjugates, when inserted into lipid vesicles, can mediate interactions between vesicles such as docking and fusion∗. These DNA-mediated interactions are a model for the biological machinery (SNARE proteins) employed in synaptic vesicle fusion. We can also use DNA-lipids to build model membrane platforms, such as tethered vesicles and tethered free-standing membranes, which are employed in these studies to observe individual fusion events using fluorescence microscopy. Two systems will be described: vesicle to vesicle fusion between mobile, tethered vesicles, and vesicle to planar bilayer fusion of small vesicles to a DNA-tethered free-standing bilayer. Fusion of individual vesicles is observed in real time using lipid and content mixing assays, as well as FRET upon hybridization of labeled DNA-lipids. These studies provide insight into the relative timescales and extents of docking, lipid mixing, and content exchange. In addition, using different sequences of fluorescently labeled DNA to mediate fusion gives insight into the importance of the rates and number density of hybrid formation at the fusion pore relative to the rate of fusion pore opening. This fusion machinery can also be used to deliver membrane proteins from small vesicles into GUVs or DNA-tethered planar bilayers, allowing membrane proteins to be studied in a free-standing bilayer. ∗Chan et al, PNAS 2007 and 2009; Chan et al, Biointerphases 2008.

Association of SNAREs and calcium channels with the borders of cytoskeletal cages organizes the secretory machinery in chromaffin cells.

In chromaffin cells, SNARE proteins, forming the basic exocytotic machinery are present in membrane clusters of 500-600 nm in diameter. These microdomains containing both SNAP-25 and syntaxin-1 are dynamic and the expression of altered forms of SNAREs modifies not only their motion but also the mobility of the associated granules. It is also clear that SNARE microdomain location defines the place for individual vesicle fusion and that the alteration of cluster dynamics affects the fusion process itself. Interestingly, these SNARE patches colocalize with the borders of F-actin cages forming the cytoskeletal cortical network, and these borders also contain clusters of L- and P/Q type calcium channels. The organization of the secretory machinery in association with the borders of cytoskeletal cages seems to be an effective way to promote fast coupling between calcium entry and catecholamine release as demonstrated with the use of mathematical secretory models.

Loose coupling between calcium channels and sites of exocytosis in chromaffin cells

Calcium microdomains generated by tight clusters of calcium channels regulate fusion of small vesicles at the synaptic terminal and have also been suggested to trigger exocytosis of large dense-core vesicles from neuroendocrine cells. To test this idea, we have compared sites of exocytosis and the spatial distribution of calcium channels in chromaffin cells. Fusion of individual vesicles was visualized using interference reflection microscopy and the submembranous calcium signal was assessed using total internal reflection fluorescence microscopy. Depolarization triggered a burst of exocytosis from up to seven sites in a membrane area of 11 microm(2), but these sites did not colocalize with calcium microdomains. Instead, calcium influx occurred in large patches (averaging 34 microm(2)) containing a mixture of P/Q- and N-type channels. About 20% of fusion events occurred outside calcium channel patches. Further, the delay between the onset of stimulation and a burst of exocytosis was prolonged for several seconds by increasing the concentration of the slow calcium chelator EGTA from 1.5 to 5 mM. These results demonstrate that while calcium channels and release sites tend to congregate in specialized regions of the surface membrane, these have dimensions of several micrometres. The dominant calcium signal regulating release in chromaffin cells is generated by the cooperative action of many channels operating over distances of many micrometres rather than discrete clusters of calcium channels generating localized microdomains.

Dopamine and Amphetamine Rapidly Increase Dopamine Transporter Trafficking to the Surface: Live-Cell Imaging Using Total Internal Reflection Fluorescence Microscopy

Rapid treatment (1 min) of rat striatal synaptosomes with low-dose amphetamine increases surface expression of the dopamine transporter (DAT). Using mouse neuroblastoma N2A cells, stably transfected with green fluorescent protein-DAT, we demonstrate the real-time substrate-induced rapid trafficking of DAT to the plasma membrane using total internal reflection fluorescence microscopy (TIRFM). Both the physiological substrate, dopamine, and amphetamine began to increase surface DAT within 10 s of drug addition and steadily increased surface DAT until removal 2 min later. The substrate-induced rise in surface DAT was dose-dependent, was blocked by cocaine, and abated after drug removal. Although individual vesicle fusion was not visually detectable, exocytosis of DAT was blocked using both tetanus neurotoxin and botulinum neurotoxin C to cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Notably, the dopamine-induced increase in surface DAT was cocaine-sensitive but D(2)-receptor independent. TIRFM data were confirmed in human DAT-N2A cells using biotinylation, and similar effects were detected in rat striatal synaptosomes. A specific inhibitor of protein kinase C-beta blocked the substrate-mediated increase in surface DAT in both DAT-N2A cells and rat striatal synaptosomes. These data demonstrate that the physiological substrate, dopamine, and amphetamine rapidly increase the trafficking of DAT to the surface by a mechanism dependent on SNARE proteins and protein kinase C-beta but independent of dopamine D(2) receptor activation. Importantly, this study suggests that the reuptake system is poised to rapidly increase its function during dopamine secretion to tightly regulate dopaminergic neurotransmission.

Measurements of membrane patch capacitance using a software-based lock-in system

On-cell patch-clamp capacitance measurements can resolve the fusion of individual vesicles to a membrane patch and the accompanying dilation of the fusion pore. So far, these measurements have used a patch-clamp amplifier in combination with a hardware lock-in amplifier. Usually, solely the capacitance and conductance outputs of hardware lock-in amplifiers were recorded, which needed to be filtered rather heavily to suppress spectral components at the stimulus frequency. Therefore, the temporal resolution was limited, and information carried in the patch current was not utilized. In this paper, we describe an alternative and more versatile approach for measuring patch capacitance and conductance, using a digitally controlled patch-clamp amplifier. The software lock-in system showed better bandwidth and identical signal-to-noise performance needing less instrumentation. High temporal resolution measurements on patches of chromaffin cells showed that vesicle fission can be completed in only tens of microseconds. Capacitance calculation based on the patch current allows for straightforward offline phase correction. Moreover, the close inspection of direct current for the first time revealed small current changes accompanying the fusion and fission of large secretory vesicles, promising new insights into the vesicles' membrane properties. A practical guide to high-resolution on-cell patch-clamp capacitance measurements using the software lock-in is provided.