An electrochemical detector array to study cell biology on the nanoscale

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The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.TIR-FRET imaging | electrochemical imaging | time superresolution microscopy | image analysis | transmitter release N eurotransmitters, hormones, and many other mediators are stored in secretory vesicles, and their release occurs by the mechanism of exocytosis that begins with formation of a narrow fusion pore (1). Fusion-pore formation in neurosecretory vesicles is stimulated by an increase of intracellular [Ca 2+ ] and is thought to be induced by a large conformational change in the SNARE complex (2-4). Such changes may be involved in various steps from preparing vesicles for fusion (5) to fusion-pore dilation (6). To determine whether a conformational change in SNAREs is linked to fusion, the synaptosomal-associated protein 25 (SNAP25) mutant SCORE (SNARE COmplex REporter) has been developed (7), which contains two fluorescent proteins, CFP as a fluorescence resonance energy transfer (FRET) donor and Venus as a FRET acceptor. SCORE and constructs like it (5, 7-9) have the advantage that donor and acceptor exist at fixed stoichiometry, facilitating the analysis and interpretation of the measurements. Like SNAP25, SCORE forms SNARE complexes with syntaxin and vesicle-associated membrane protein (VAMP)/synaptobrevin (7,8), and when endogenous SNAP25 in chromaffin cells is cleaved by botulinum toxin E, a toxin-resistant SCORE rescues exocytosis (8). In PC12 cells expressing SCORE, a FRET change was evoked by high [K + ] stimulation, but with this method the time scale was tens of seconds (7). This FRET change was abolished in the absence of extracellular Ca 2+ , indicating that it is dependent on Ca 2+ entry. In contrast, the FRET change was not affected by tetanus neurotoxin treatment (7), which causes the blockade of exocytosis (10). In beta cells, a SCORE-like construct indicated a FRET chan...

Section: Methodsmentioning

confidence: 99%

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells

Zhao

Fang

Herbst

et al. 2013

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“…The four electrodes of an ECD reside at the corners of a square sized such that a cell can be placed atop the array and viewed through the glass coverslip while amperometric currents are recorded from all four electrodes simultaneously. The ECD arrays were fabricated as described (19,40) except for an improved insulation layer, which was created by chemical vapor deposition of approximately 300 nm of SiO 2 onto the electrodes (IPE 1000 PECVD), and subsequent etching of the SiO 2 layer via reactive ion etching (PlasmaLab 80Plus, Oxford Instruments) to expose the active areas of the electrodes and contact pads. described above.…”

Section: Methodsmentioning

confidence: 99%

Role of the synaptobrevin C terminus in fusion pore formation

Ngatchou

Kisler

Fang

et al. 2010

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106

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.chromaffin cell | patch clamp capacitance measurement | caged calcium | amperometry | electrochemical detector array T he SNARE proteins (1) mediate release of stored secretory products by exocytosis. In neurosecretion, the t-SNAREs syntaxin and SNAP-25 in the plasma membrane bind the v-SNARE synaptobrevin II (sybII, also known as VAMP2), which is anchored to the vesicle membrane by a single transmembrane (TM) domain. Upon stimulation, the SNARE complex is thought to zip up more tightly proceeding in a vectorial manner from the N to the C terminus, toward the TM domains of sybII and syntaxin (2-5), thereby transferring a force to the membranes (6). However, the molecular mechanism by which this force leads to opening of the fusion pore has not been determined (7). Several models have been proposed to explain the mechanism of fusion pore formation. In the lipid-stalk-hemifusion hypothesis, the outer and the inner leaflets of the two membranes merge via formation of a hemifusion intermediate in response to forces exerted by proteins surrounding the fusion site (8). In an alternative proteinaceous fusion pore model, the fusion pore is lined by the TM domain of syntaxin (9) and possibly synaptobrevin (10). However, it is not immediately evident how the hydrophobic transmembrane domains can line an aqueous fusion pore that allows for ion permeation by electrodiffusion (11). When the C-terminal SNARE domain interactions are reduced by mutating or deleting the C terminus of SNAP-25, or when flexible linkers are introduced between the sybII TM domain and its SNARE domain, the rate of exocytosis is reduced (12-16) and the flux of transmitter through the early fusion pore is decreased (16,17), consistent with a structural change in the fusion pore. In an attempt to interpret these findings, a proteolipidic fusion pore model has been proposed, in which the fusion pore is formed by a molecular complex of both lipids and SNARE proteins (17). However, even this model does not explain the molecular mechanism by which the N-to C-terminal zipp...

“…1.5 (160-190 m thick) as described in ref. 9, forming a fourelectrode ECD array. Each ECD array consists of a square window Ϸ10 ϫ 10 m 2 in size in the insulating 0.5-m-thick photoresist.…”

Section: Methodsmentioning

confidence: 99%

“…To obtain not only temporal but also spatial resolution of opening and expansion of single fusion pores in chromaffin cells, we fabricated electrochemical detector (ECD) arrays of four Pt electrodes patterned on glass coverslips (9). For quantal events, the oxidation currents recorded from the four electrodes allow the desired determination of fusion pore dynamics with millisecond time resolution as well as localization of the fusion event.…”

mentioning

confidence: 99%

Electrochemical imaging of fusion pore openings by electrochemical detector arrays

Hafez

Kisler

Berberian

et al. 2005

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135

146

Chromaffin cells from the adrenal gland are a widely used model system to study neuronal exocytosis, and the fusion pore properties in these cells have been studied in great detail (3-6). Release of single vesicles (quanta) of epinephrine or norepinephrine can be detected electrochemically with a carbon fiber electrode (CFE) as amperometric spikes (7,8). These spikes are often preceded by a so-called foot signal (8), which is due to slow discharge of catecholamines through a narrow fusion pore (3) and indicates the time of fusion pore formation and expansion.To obtain not only temporal but also spatial resolution of opening and expansion of single fusion pores in chromaffin cells, we fabricated electrochemical detector (ECD) arrays of four Pt electrodes patterned on glass coverslips (9). For quantal events, the oxidation currents recorded from the four electrodes allow the desired determination of fusion pore dynamics with millisecond time resolution as well as localization of the fusion event.Simultaneous observation of the cell by fluorescence microscopy can be performed. Here we show that the spatiotemporal assignments of single exocytotic events obtained by electrochemical imaging of fusion events correlate with release of a fluorescent vesicle marker during exocytosis in chromaffin cells. Materials and MethodsECD Array. Pt conductors insulated by photoresist were fabricated photolithographically on standard microscope coverslips No. 1.5 (160-190 m thick) as described in ref. 9, forming a fourelectrode ECD array. Each ECD array consists of a square window Ϸ10 ϫ 10 m 2 in size in the insulating 0.5-m-thick photoresist. The active electrodes are formed by the tips of the four Pt conductors, Ϸ3 m wide and 150 nm thick, that protrude into the corners of the square. The area and shape of the electrode arrays were imaged and quantified by using atomic force microscopy and optical microscopy.Cell Preparation and Recording Conditions. Bovine adrenal glands were obtained from a local slaughterhouse and prepared as described in ref. 10. Cells were cultured on 12-mm coverslips that were coated with 0.02% poly(D)-lysine and used on days 1-10. Before the experiment, a small coverslip with cells attached was placed in a 35-mm dish with 1 ml of bath solution containing 150 mM NaCl, 10 mM Hepes, 5 mM CaCl 2 , 5 mM KCl, 2 mM MgCl 2 (pH 7.25) (318 mosM) and supplemented with 3 M acridine orange (Molecular Probes) for 15 min in the dark. An ECD array coverslip was mounted on a custom-built stage on a Zeiss 135TV microscope, and the coverslip with the cells was placed at some distance from the ECD array. Bath solution (150-200 l) was then added to cover the cells and the active electrode array. By using a patch pipette, a cell was lifted off the coverslip and manipulated over the center of an ECD array. Some cells were stimulated by including ionomycin in the pipette or adding ionomycin to the bath. Experiments were performed at room temperature.Electrochemical Recording and Analysis. The ECD electrodes were connected via their...