| 11. |
- Ren, Lin, et al.
(författare)
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The evidence for open and closed exocytosis as the primary release mechanism
- 2016
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Ingår i: Quarterly Reviews of Biophysics. - : Cambridge University Press (CUP). - 0033-5835 .- 1469-8994. ; 49
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Tidskriftsartikel (refereegranskat)abstract
- Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss- and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.
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| 12. |
- Ren, Lin, 1987, et al.
(författare)
-
The evidence for open and closed exocytosis as the primary release mechanism
- 2016
-
Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 49
-
Tidskriftsartikel (refereegranskat)abstract
- Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss- and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.
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| 13. |
- Trouillon, R., et al.
(författare)
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Evaluating the Diffusion Coefficient of Dopamine at the Cell Surface During Amperometric Detection: Disk vs Ring Microelectrodes
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 85:13, s. 6421-6428
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Tidskriftsartikel (refereegranskat)abstract
- During exocytosis, small quantities of neurotransmitters are released by the cell. These neurotransmitters can be detected quantitatively using electrochemical methods, principally with disk carbon fiber micro-electrode amperometry. An exocytotic event then results in the recording of a current peak whose characteristic features are directly related to the mechanisms of exocytosis. We have compared two exocytotic peak populations obtained from PC12 cells with a disk carbon fiber microelectrode and with a pyrolyzed carbon ring microelectrode array, with a 500 nm ring thickness. The specific shape of the ring electrode allows for precise analysis of diffusion processes at the vicinity of the cell membrane. Peaks obtained with a ring microelectrode array show a distorted average shape, owing to increased diffusion pathways. This result has been used to evaluate the diffusion coefficient of dopamine at the surface of a cell, which is up to an order of magnitude smaller than that measured in free buffer. The lower rate of diffusion is discussed as resulting from interactions with the glycocalyx.
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| 14. |
- Wang, Yuanmo, 1986, et al.
(författare)
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Counting the Number of Glutamate Molecules in Single Synaptic Vesicles
- 2019
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 141:44, s. 17507-17511
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Tidskriftsartikel (refereegranskat)abstract
- Analytical tools for quantitative measurements of glutamate, the principal excitatory neurotransmitter in the brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for the counting of glutamate molecules stored inside single synaptic vesicles. In this method, an ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential-dependent manner at a constant negative potential. The continuous amperometric signals are sampled at high speed (10 kHz) to record sub-millisecond spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various glutamate concentrations. Our measurements show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis, and drug treatments for neuronal disorders where glutamate is involved.
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| 15. |
- Wang, Yuanmo, 1986, et al.
(författare)
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Molecular Crowding and a Minimal Footprint at a Gold Nanoparticle Support Stabilize Glucose Oxidase and Boost Its Activity
- 2020
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 36:1, s. 37-46
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes conjugated to nanomaterials are used in the design of various biotechnologies. In the development of biosensors, surface modifications with the enzyme glucose oxidase (GOx) serve to aid the detection of blood glucose. In order to optimize sensor effectiveness, the enzyme tertiary structure needs to be preserved upon immobilization to retain the enzyme's catalytic activity. Because of the nature of GOx, it suffers from a tendency to denature when immobilized at a solid surface; hence, methods to optimize enzyme stability are of great importance. Here, we introduce the study of the interaction of GOx to the highly curved surface of 20 nm gold nanoparticles (AuNP) with an absorbed monolayer coating of enzyme as determined by flocculation assays and quantification of immobilized GOx at the nanoparticle surface. Enzyme crowding was determined by comparing the number of enzymes that bind to how many can physically fit. These measurements show how placing a monolayer of enzyme where the enzyme spreads thin at the AuNP surface still provides stable catalytic performance of up to 14 days compared to enzymes free in solution. Moreover, by the increasing enzyme density via increasing the amount of GOx present in solution during the GOx/AuNP conjugation step creates a molecularly crowded environment at the highly curved nanoparticle surface. This limits the size of the enzyme footprint for attachment and shows that the activity per enzyme can be enhanced up to 300%. This is of great importance for implementing stable and sensitive sensor technologies that are constructed by enzyme-based nanoparticle scaffolds. Here, we show by using the conditions that maintain GOx structure and function when limiting the enzyme coating to an ultrathin layer, the design and construction of an ultrafast responding diagnostic sensor technology for glucose can be achieved, which is crucial for monitoring rapid fluctuations of, for instance, glucose in the brain.
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| 16. |
- Wang, Yuanmo, 1986, et al.
(författare)
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Ultrafast Glutamate Biosensor Recordings in Brain Slices Reveal Complex Single Exocytosis Transients
- 2019
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Ingår i: Acs Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 10:3, s. 1744-1752
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal communication relies on vesicular neurotransmitter release from signaling neurons and detection of these molecules by neighboring neurons. Glutamate, the main excitatory neurotransmitter in the mammalian brain, is involved in nearly all brain functions. However, glutamate has suffered from detection schemes that lack temporal and spatial resolution allowed by electrochemistry. Here we show an amperometric, novel, ultrafast enzyme-based nanoparticle modified sensor, measuring random bursts of hundreds. to thousands of rapid spontaneous glutamate exocytotic release events at approximately 30 Hz frequency in the nucleus accumbens of rodent brain slices. Characterizing these single submillisecond exocytosis events revealed a great diversity in spike shape characteristics and size of quantal release, suggesting variability in fusion pore dynamics controlling the glutamate release by cells in this brain region. Hence, this novel biosensor allows recording of rapid single glutamate exocytosis events in the brain tissue and offers insight on regulatory aspects of exocytotic glutamate release, which is critical to understanding of brain glutamate function and dysfunction.
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