Khajak Berberian

The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25Δ9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25Δ9 displayed smaller amperometric “foot-current” currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25Δ9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.

F-actin and myosin II accelerate catecholamine release from chromaffin granules.

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Improved surface-patterned platinum microelectrodes for the study of exocytotic events.

Surface-patterned platinum microelectrodes insulated with 300 nm thick fused silica were fabricated using contact photolithography. These electrodes exhibit low noise and were used for monitoring single vesicle exocytosis from chromaffin cells by constant potential amperometry as well as fast-scan cyclic voltammetry. Amperometric spike parameters were consistent with those obtained with conventional carbon fiber electrodes. Catecholamine voltammograms acquired with platinum electrodes exhibited redox peaks with full width at half-maximum of approximately 45 mV, much sharper than those of carbon fiber electrode recordings. The time course of voltammetrically measured release events was similar for platinum and carbon fiber electrodes. The fused-silica-insulated platinum electrodes could be cleaned and reused repetitively and allowed incorporation of micrometer precision surface-patterned poly-D-lysine. Poly-D-lysine-functionalized devices were applied to stimulate mast cells and record single release events without serotonin preloading. Microfabricated platinum electrodes are thus able to record single exocytotic events with high resolution and should be suitable for highly parallel electrode arrays allowing simultaneous measurements of single events from multiple cells.

Transparent electrode materials for simultaneous amperometric detection of exocytosis and fluorescence microscopy.

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.

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

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.

Post-CMOS Fabrication of Working Electrodes for On-Chip Recordings of Transmitter Release

The release of neurotransmitters and hormones from secretory vesicles plays a fundamental role in the function of the nervous system including neuronal communication. High-throughput testing of drugs modulating transmitter release is becoming an increasingly important area in the fields of cell biology, neurobiology, and neurology. Carbon-fiber amperometry provides high-resolution measurements of amount and time course of the transmitter release from single vesicles, and their modulation by drugs and molecular manipulations. However, these methods do not enable the rapid collection of data from a large number of cells. To allow this testing, we have developed a complementary metal-oxide semiconductor (CMOS) potentiostat circuit that can be scaled to a large array. In this paper, we present two post-CMOS fabrication methods to incorporate the electrochemical electrode material. We demonstrate by proof of principle the feasibility of on-chip electrochemical measurements of dopamine, and catecholamine release from adrenal chromaffin cells. The measurement noise is consistent with the typical electrode noise in recordings with external amplifiers. The electronic noise of the potentiostat in recordings with 400-¿s integration time is ~ 0.11 pA and is negligible compared to the inherent electrode noise.

The Conformational Change of SNAP25 during the Exocytosis

It is widely assumed that a conformational change in the SNARE complex induces fusion pore opening and transmitter release. The SNAP-25 FRET construct SCORE (SNARE complex reporter) has CFP as FRET donor inserted at the N-terminal end of SN1 and the fluorescent protein Venus (YFP) as FRET acceptor inserted at the N-terminus of SN2. It was previously shown that SCORE that reports a FRET change in response to stimulation that is calcium dependent but is not abolished by tetanus toxin treatment, which abolishes fusion (An and Almers 2004 Science 306:1042). To determine if the conformational change reported by SCORE is directly associated with fusion we monitored SCORE fluorescence in chromaffin cells in TIRF mode while exocytotic events at the cell bottom were spatially and temporally monitored by a microfabricated electrochemical detector (ECD) array with four Pt electrodes patterned on a glass coverslip (Hafez et al. PNAS 2005 102:13879). After the SCORE-expressing cells were placed on the top of the ECD array, the amperometric currents and fluorescence images were simultaneously recorded. Based on the charge ratios of oxidation currents recorded by the four electrodes, the localization of single exocytotic events was determined. The time course of CFP and YFP fluorescence in the 4 camera pixels at the release site was extracted for 10 s time intervals before and after the amperometric spike. Averaging >300 events revealed a transient increase of YFP and a transient decrease of CFP fluorescence by ∼2.5% at the time of amperometric events, The increased FRET level returned to the pre-exocytotic level within ∼5s. This result directly indicates that the SNAP25 conformational change is directly associated with fusion. Supported by NIH grant R01GM085808.