The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

Sombers, Leslie A.; Hanchar, H. Jacob; Colliver, Thomas L.; Wittenberg, Nathan J.; Cans, Ann-Sofie; Arbault, Stéphane; Amatore, Christian; Ewing, Andrew G.

doi:10.1523/jneurosci.1119-03.2004

Cited by 128 publications

(126 citation statements)

References 48 publications

(89 reference statements)

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“…41 NanoSIMS can achieve a lateral beam resolution of 50 nm, 19 and TEM can achieve approximately 400 times better spatial resolving power. 42 Thus, in this analysis, the limit of the NanoSIMS spatial imaging capabilities was complemented by correlating to TEM images to provide a rare view of the distribution of dopamine within the LDCV.…”

Section: Resultsmentioning

confidence: 99%

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

et al. 2016

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We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size. KEYWORDS: nanoimaging, NanoSIMS, vesicle content, electrochemistry, nanocompartments C hemical communication in eukaryotic cells relies heavily on loading and trafficking of secretory vesicles to the plasma membrane. Upon stimulation, the loaded, docked secretory vesicles fuse with the plasma membrane and discharge their neurotransmitter cargo into the extracellular space. This process, called exocytosis, is fundamental and highly regulated. 1,2 Regulation of exocytosis has been widely studied, and the understanding of its mechanism is still under debate. Several mechanisms have been reported such as all-or-nothing release during which vesicles completely fuse with the plasma membrane and release their total content. 3 Contrary to the all-or-nothing mode, partial release mechanisms have been proposed, suggesting the transient opening of the fusion pore between the cell membrane and secretory vesicle followed by closing again. 4 In addition to the release event, exocytosis is also regulated through vesicle loading. In general, this process involves a neurotransmitter transporter, an integral vesicle membrane protein that uses the pH gradient across the vesicle membrane, to drive uptake of neurotransmitters into the vesicle. 5−8 Defining the vesicle storage mechanisms would be an important piece of the exocytosis regulation puzzle. 5,9,10 Several cell types have been employed for loading studies among which neuroendocrine cells have had an important role as a model system. They possess two types of secretory vesicles, synaptic-like microvesicles (SLMVs) and so-called large densecore vesicles (LDCVs). The larger LDCVs typically have diameters around 150−300 nm and an inner ...

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Section: Resultsmentioning

confidence: 99%

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

et al. 2016

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“…The measurements were performed in Hepes-based saline solution (10 mM Hepes, 150 mM NaCl, 4.2 mM KCl, and 11.2 mM glucose; pH 7.4). In this experiment, we used relatively large electrodes (a ¼ 6.0 μm) to detect neurotransmitter effectively, based on the rationale outlined in earlier work for both NO and neurotransmitter detection (43)(44)(45)(46)(47)(48). The carbon electrode was simply aligned optically over the cell and positioned one radius above the cell surface using the reduction current at a potential of −1.0 V, due to oxygen reduction, which was found to provide reliable distance control.…”

Section: Resultsmentioning

confidence: 99%

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Takahashi

Shevchuk

Novák

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

204

224

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We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5-100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5-3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.high-resolution imaging | living cell imaging | noninvasive | constant-distance mode S canning electrochemical microscopy (SECM) uses an electrode tip for detecting electroactive chemical species and is an effective tool for the investigation of the localized chemical properties of sample surfaces and interfaces (1). Because SECM has high temporal resolution and can be used under physiological conditions, it is particularly well suited for quantitative measurements of (short-lived) chemicals like neurotransmitters, nitric oxide, reactive oxygen species, and oxygen, which are released/ consumed by living cells. In conventional SECM, the probe is often micrometer scale and the probe vertical position is kept at a constant height, a plane, during probe scanning. If the sample topography is not flat, the electrode-sample separation changes during scanning, complicating the SECM measurement and its analysis.Various methods for SECM electrode miniaturization and control of the electrode-sample separation have been advocated in order to improve the resolution of SECM imaging. The reliable fabrication of nanoelectrodes with a small ratio of electrodeinsulation to active electrode (

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“…The overall effect of the reduction in vesicular ATP is a fall in the quantum release of catecholamines, particularly evident in the exocytotic events occurring within the first 70 s. As the quantum size of the spike feet decreases in parallel with the quantum size of spikes (45), it is expected that the number of spikes with foot and the foot duration was reduced (Table 1). Because I max and Q [net spike charge, expressed in picocoulomb (pC)] are highly correlated parameters (Fig.…”

Section: Discussionmentioning

confidence: 99%

ATP: The crucial component of secretory vesicles

Estévez-Herrera

Domínguez

Pardo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.exocytosis | purines | quantum size | secretory vesicles | VNUT V irtually most, and possibly all, types of secretory vesicles found in cells contain ATP, which often accumulates at high concentrations and, commonly, in conjunction with different types of neurotransmitters. However, the reason for this widespread distribution of ATP remains a mystery. Although ATP is present in all animal species, including primitive life forms like Giardia lamblia that lack Golgi complexes and mitochondria, the detection of ATP in the secretory vesicles of sympathetic neurons was considered to be the first example of cotransmission (1). However, given the ubiquitous accumulation of ATP in secretory vesicles, it might instead be considered that it is the other neurotransmitters that coincide with ATP, rather than the other way around (2). Indeed, perhaps ATP should be considered as the first molecule used as a transmitter in primitive forms of life.Astonishingly high concentrations of releasable species are stored inside secretory vesicles, far exceeding those in the cytosol (3, 4). For example, the catecholamine content of adrenal secretory granules (SGs), a type of large dense core secretory vesicles also known as chromaffin granules, was 0.8-1 M when measured directly in adrenal chromaffin cells using patch amperometry (5, 6). In addition, SGs from chromaffin cells contain ATP at ∼150 mM (7), calcium ∼40 mM (8), about 2 mM of granins, ascorbate, peptides, and other nucleotides, all in an acidic pH ∼5.5 environment.ATP possesses intrinsic chemical characteristics that make it relevant to the accumulation of soluble substances in secretory vesicles. The formation of weak complexes between monoamines and ATP, the two main soluble compounds in chromaffin granules, has been demonstrated in vitro by NMR (9), ultracentrifugation (10), infrared spectroscopy, and calorime...

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The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

Cited by 128 publications

References 48 publications

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

ATP: The crucial component of secretory vesicles

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