Amperometry methods for monitoring vesicular quantal size and regulation of exocytosis release
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Chemical signaling strength during intercellular communication can be regulated by secretory cells through controlling the amount of signaling molecules that are released from a secretory vesicle during the exocytosis process. In addition, the chemical signal can also be influenced by the amount of neurotransmitters that is accumulated and stored inside the secretory vesicle compartment. Here, we present the development of analytical methodologies and cell model systems that have been applied in neuroscience research for gaining better insights into the biophysics and the molecular mechanisms, which are involved in the regulatory aspects of the exocytosis machinery affecting the output signal of chemical transmission at neuronal and neuroendocrine cells.
KeywordsAmperometry Exocytosis Fusion pore Quantal size Chromaffin cell Artificial cells Electrochemical cytometry Intracellular electrochemical cytometry
Secretory vesicles are central in neuroscience and endocrinology. These organelles accommodate high concentrations of various neurotransmitter molecules and hormones serving as units of chemical message. Fusion of neurotransmitter-filled vesicles with the cell plasma membrane through a Ca2+-dependent process called exocytosis result in a rapid release of signaling molecules into the extracellular space and is a key process in intercellular communication . To promote vesicle fusion of secretory cells in our bodies relies on a highly conserved molecular machinery based on the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) protein. These SNARE proteins consist of v-SNAREs localized in the vesicle membrane that associates with target t-SNAREs at the inner leaflet of the cell plasma membrane to initiate vesicle docking, fusion, and vesicle content release. These secreted signaling molecules diffuse from the release site of the emitting cell and bind to specific receptors at the surface of target cells inducing specific physiological reactions. In synaptic transmission, depending on type of neurotransmitters released, binding to target receptors at neighboring neurons may for instance serve to either excite or inhibit signal transmission in neuronal pathways. Hence, the secretory vesicle is an essential organelle that directly is influencing our brain function such as cognition, emotions, learning, and memory. Therefore, many neurological drugs are aimed to affect the quantity of neurotransmitters released and residing in the extracellular space by influencing the potency of neurochemical signals. The signal strength in cellular communication can be tuned at target cells receiving the signal via the sensitivity and level of the receptors expression in the cell membrane. Often, the amount of neurotransmitters released from an exocytosis event do not succeed to fully saturate the post-synaptic receptors in a synapse [38, 57, 93, 96]. Consequently, it has been realized that modulation of chemical signaling strength, thus can be regulated by the secreting cell through varying the amount of neurotransmitters released owing to the different modes of exocytosis that can be triggered [10, 14, 71, 72, 82, 93].
The maximal potency can be achieved from the classic view of exocytosis called “full exocytosis.” In this mode, the vesicle compartment upon fusion fully collapses into the plasma membrane releasing the entire vesicle content into the extracellular space [4, 11, 45, 66, 70, 77, 99]. In contrast to full vesicle content release, after vesicle fusion, the fusion pore can be controlled to stay open for a limited time and then close again. This allows a fraction of the vesicle neurotransmitter content to be released before the partly emptied vesicle is rapidly recycled while preserving its original shape [4, 78, 85]. The effectiveness of the chemical signal can thus be tuned by adjusting the pore size and the time it stays open [4, 14, 51, 60, 76]. The chemical signal can be minimized through the exocytosis mode “kiss and run,” where a transient 1–2 nm fusion pore that may also flicker, allows very small quantities of neurotransmitters to be secreted before the pore closes for vesicle reuse [3, 4, 12, 24, 34, 39, 66, 83, 91, 100]. A “moderate” signal can be delivered by means of a third mode of exocytosis where the transient fusion pore dilates into a larger pore extending the “kiss and run” in terms of fusion pore size and duration the pore is open, resulting in that more and yet not all of the vesicle content is expelled before the vesicle compartment is retrieved for recycling. This mode that more recently has been discovered and accepted has been termed many different names, e.g., “fuse-pinch-and-linger,” “extended kiss-and-run,” or “open and close exocytosis,” and seem to be the most prevalent mode of exocytosis used by for instance chromaffin cells [1, 11, 60, 61, 71, 78, 86, 89].
Depending on type of secretory cells and category of vesicles, quantification with carbon fiber microelectrode amperometry has estimated that about 30–60% of the vesicle content is released when this mode is triggered [51, 71, 72, 82]. Additionally, in chromaffin cells, fine-tuning of fusion pore size can serve as a molecular sieve, adding chemical selectivity of the compounds released by allowing molecules of a certain size to pass and others to be retained within the vesicle compartment [41, 69]. Hence, it is apparent that regulation of neurotransmitter release can directly be associated with mechanisms controlling the dynamics of the fusion pore affecting chemical signaling strength during intercellular communication and synaptic function. Therefore, to elucidate the regulatory mechanism and improve our understanding of what factors act to control fusion pore dynamics during exocytosis, it is important to identify the molecular drivers for vesicle-membrane interactions and the biophysics affecting that influences the exocytosis machinery. Many studies on secretion have, apart from neuronal cells also involved cell lines, simplified model systems and many major findings have been made through studies at chromaffin cells [15, 22, 53].
However, controversy still remains on the mechanisms for the regulatory aspect of exocytosis and the prevalence for different modes of exocytosis in secretory cells. This might relate to variance in the type of information provided by different analytical methods and limitations of instrumental techniques used in different experiments . In addition, depending on the cell type and category of vesicles, the experimental conditions can vary widely. For instance, monitoring exocytosis release from small synaptic vesicles (40–60 nm in diameter) is a much greater challenge than from large dense-core vesicles (LDCVs) that vary in size from 80 to 300 nm in diameter [1, 82]. Therefore, in progress to clarify and verify results, development of new analytical tools improving sensitivity and spatiotemporal resolution for studying exocytosis is important. With individual methods varying in terms of different kinds of information achieved and technical limitations, an approach to improve the ability to overview the exocytosis process is to combine methodologies that provide complementary information. For instance, electrochemical methods offer quantitative and high temporally resolved detail information on fusion pore dynamics at the initial stages of exocytosis while reporting less on the later stages of exocytosis. In contrast, many imaging techniques have the capability to, with high spatial resolution, overview and track vesicle from docking, fusion, and vesicle content release and depending on the method used, also through vesicle recapture, but in comparison to electrochemical methods are limited in sensitivity and temporal resolution. Therefore, by merging electrochemical methods with imaging techniques offer the opportunity to simultaneously record the complementary information from both methods during secretion studies. For example, by combining high temporally resolved amperometry or electrophysiology, together with total internal reflection microscopy imaging, quantitative information of neurotransmitter release can be correlated with high spatial information on vesicle activity prior and post vesicle fusion, or by placing multiple lithographic electrodes at secreting cells electrochemical imaging capabilities of the exocytosis process can be created [42, 43, 49, 87, 92, 98, 101].
Characterization of exocytosis with amperometry recording
Amperometry to characterize the biophysics of the exocytosis process
With the key features of amperometry, this technique has been applied in many studies regarding the biophysics and key molecules involved in vesicle fusion and controlling fusion pore dynamics. For instance, investigations on the role of components associated to the sophisticated fusion machinery composed by SNARE protein promoting vesicle membrane fusion and questions regarding the involvement of SNARE protein in modulating neurotransmitter release, quantitative and kinetic measurements using amperometry has been central, with much of the work performed at chromaffin cells [13, 20, 36, 44, 67]. It is still debated what is the energy needed in terms of the minimum number of SNAREs for merging of a vesicle with the plasma membranes to initiate fusion and what is the potential role for SNARE proteins in deforming membranes into a high-curvature narrow fusion pore, as well as the possibility by the SNARE proteins to influence subsequent fusion pore dynamics. By introducing nanolipoprotein discs, a system with reconstituted v-SNARE proteins designed for complex binding to t-SNAREs expressed at the plasma membrane of chromaffin cells, a model system to study the cooperative effect of SNARE proteins forming and controlling fusion pore dynamics has been developed. This system has shown that very few SNAREs are needed to induce fusion pore formation and by the presence of a larger number of SNAREs can upon complex formation trigger fusion pore dilation [17, 25, 94, 95].
To gain a better understanding on what prevents the vesicle from fully collapsing with the plasma membrane when partial exocytosis is triggered, studies on the biophysics of fusion pore dynamics have facilitated to overview parameters that affect size, stability, and life time of the fusion pore [3, 26, 28, 29, 40]. It has been debated whether the fusion pore is lipidic or dominated by protein, with recent studies indicating it might be a mixture of both [16, 80]. Consequently, incubating cells with phospholipids of various geometrical shapes, e.g., cylindrical shapes, cone, or inverted cone shape, which modifies the spontaneous curvature of the cell membrane and directly affects membrane energetics and membrane dynamics of the highly curved fusion pore. Depending on lipid shape, whether facilitating positive or negative membrane curvature, alterations in membrane composition has shown to affect fusion pore size and act to slow or speed up the rate of fusion pore dilation during exocytosis [8, 23, 60, 76, 88]. Modifying the biophysics in terms of membrane rigidity for instance by adjusting the membrane cholesterol alters fusion pore dynamics, with lower membrane cholesterol concentrations accelerating exocytotic release and higher concentrations stiffening the membrane, stabilizes the fusion pore structure by increasing pore life time [18, 27, 84, 97]. Membrane tension is another key biophysical force that in neuroendocrine cells have shown to be a key regulatory force affecting the mechanism for driving fusion pore dilation and when applied by ATP-driven actin assembly forces at the plasma membrane reduces the secretory vesicle size during the exocytosis release process [21, 73]. The influence of biophysics on the exocytosis process has also been studied in controlled systems by a simplified cell model system created from protein-free giant liposomes (as presented in Fig. 1c) or cell plasma membrane “blebs” of chromaffin cells, where membrane is kept at higher complexity and more similar to cell membranes in comparison to protein-free cell models (as shown in Fig. 1b) [22, 59, 65, 80]. Blebs are plasma membrane vesicles that can be achieved from subjecting cells in culture to different kinds of stress, for instance by chemical treatment using formaldehyde, which disrupt cytoskeletal attachment points to the plasma membrane and thereby induces formation of micron-sized cell plasma membrane protrusions at the cell that can serve as cell model membranes .
In a protein-free cell model, systems showed that membrane rigidity highly influences the speed of exocytosis and that membrane dynamics alone can drive the later stages of exocytosis and influence the stability of the fusion pore before dilation [23, 65, 81]. In experiments using a bleb cell model demonstrated that a threshold in membrane tension can reversibly dictate if full or partial exocytosis was triggered , and by altering the membrane rigidity by changing the membrane cholesterol concentration, higher cholesterol levels significantly enhanced fusion pore stability and life time when partial exocytosis release was triggered [59, 65]. Although much of the work so far has been performed on gaining a better understanding of membrane biophysics of exocytosis, the biophysical properties of the secretory vesicle are still quite poor and therefore future studies with this focus might also serve to better explain many of the complex mechanisms involved in regulating neurotransmission.
Secretory vesicle quantal size
The limiting amount of neurochemicals that can be released at an exocytosis event is naturally also directly related to the maximum number of neurotransmitter molecules that can be stored in a single vesicle compartment in the first place. This is often referred to as the vesicle “quantal size” referring to the basic unit of neurotransmitters that can be released as a discrete uniform “quanta” per exocytotic event [33, 48]. The secretory vesicles in neuroendocrine and neuronal cells are classified into two main groups; one is small synaptic vesicles containing low-molecular weight neurotransmitters such as dopamine, serotonin, norepinephrine, acetylcholine, or glutamate and mediate fast synaptic transmission. The other group is large dense-core vesicles (LDCVs) that typically contain monoamines (dopamine, adrenaline, noradrenaline, histamine, serotonin) and/or neuropeptides [2, 61]. Vesicles very efficiently accumulate high concentration of neurotransmitters, driven by proton and/or electrical gradients across the vesicle membrane depending on the type of neurotransmitter that is loaded into the vesicle [2, 32, 68]. The concentration of neurotransmitters in vesicles of chromaffin cells from adrenal glands is estimated to about 0.5–1 M [2, 32, 47, 62]. How the vesicle can perform this act and maintain charge neutrality and osmotic balance is still debated [19, 31, 35, 56]. The quantal size can be tuned using pharmacology enhancing or blocking the passage of neurotransmitters by specific transporter protein into the vesicle compartment or by affecting the proton/electrical gradient across the vesicle membrane and consequently affect the amount of transmitters released into the synaptic cleft [30, 51, 58, 74]. For neuroscience and pharmacological research, to elucidate how quantal size is maintained under physiological conditions and monitoring the effect on vesicle neurotransmitter content in response to potential drugs, analytical tools that are able to quantify the absolute neurotransmitter content in vesicles are crucial. However, universal methods for quantitative analysis of secretory vesicle content remain a challenge due to the variation in content and size of secretory vesicles. Quantitative measurement from the smallest of these organelles, the synaptic vesicle thus requires more sensitive and faster analysis techniques than probing the quantal content of larger dense-core vesicles in for instance neuroendocrine cells. Although fluorescence imaging with fluorescent dyes reporting on alteration in pH and membrane potential can offer real-time measurement of relative changes in vesicle quantal size , the only quantitative methods that count the absolute number of neurotransmitters encapsulated inside single secretory vesicles compartments are amperometry-based techniques [32, 51, 72]. In comparison to imaging methods for studying vesicle neurotransmitter loading, amperometry methods are to date limited to analysis of vesicles filled with neurotransmitters that are electroactive and performing single point-in-time measurements in contrast to measurements of relative dynamic changes in vesicle neurotransmitter content over time [32, 51, 72, 75].
Amperometry techniques for quantification of vesicle quantal size
Intracellular electrochemical cytometry
It has been realized that emitting cells have the ability to take part in regulation of signaling strength during intercellular communication both through regulation of content release by the mode of exocytosis triggered and by the amount of signaling molecule that is stored in secretory vesicles. In gaining a better overview in how this regulation is accomplished, analytical tools that can provide methods to study molecular mechanisms controlling fusion pore dynamics and factors influence loading and storage of neurotransmitter in secretory vesicle compartments are crucial. This is also important for better understanding of how drug treatment and disease might act and affect the regulatory aspects of neurochemical release, as well as characterizing the physiological effects in response to regulatory neurotransmission. In this quest, to study these dynamic processes in cells and secretory vesicles, cell model systems and analytical techniques that can provide sufficient sensitivity and temporal resolution to probe these ultra-small systems at physiological relevant conditions will be of great importance to clarify the essential regulatory aspects of exocytosis.
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