Single Exocytosis Events Research Articles

Analyzing Fusion Pore Dynamics and Counting the Number of Acetylcholine Molecules Released by Exocytosis.

Acetylcholine (ACh) is a critical neurotransmitter influencing various neurophysiological functions. Despite its significance, quantitative methods with adequate spatiotemporal resolution for recording a single exocytotic ACh efflux are lacking. In this study, we introduce an ultrafast amperometric ACh biosensor that enables 50 kHz electrochemical recording of spontaneous single exocytosis events at axon terminals of differentiated cholinergic human SH-SY5Y neuroblastoma cells with sub-millisecond temporal resolution. Characterization of the recorded amperometric traces revealed seven distinct current spike types, each displaying variations in shape, time scale, and ACh quantities released. This finding suggests that exocytotic release is governed by complex fusion pore dynamics in these cells. The absolute number of ACh molecules released during exocytosis was quantified by calibrating the sensor through the electroanalysis of liposomes preloaded with varying ACh concentrations. Notably, the largest quantal release involving approximately 8000 ACh molecules likely represents full exocytosis, while a smaller release of 5000 ACh molecules may indicate partial exocytosis. Following a local administration of bafilomycin A1, a V-ATPase inhibitor, the cholinergic cells exhibited both a larger quantity of ACh released and a higher frequency of exocytosis events. Therefore, this ACh sensor provides a means to monitor minute amounts of ACh and investigate regulatory release mechanisms at the single-cell level, which is vital for understanding healthy brain function and pathologies and optimizing drug treatment for disorders.

Arabidopsis myosin XIK interacts with the exocyst complex to facilitate vesicle tethering during exocytosis.

Myosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely growing cells. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to several exocyst subunits in vitro and functional fluorescently tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the rate of appearance and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes with high spatiotemporal resolution imaging and pair-wise colocalization of myosin XIK, exocyst subunits, and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the efficient localization and normal dynamic behavior of exocyst complex at the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides insights about the dynamic regulation of exocytosis in plants.

Amperometry in Single Cells and Tissue.

The release from cells of signaling molecules through the controlled process of exocytosis involves multiple coordinated steps and is essential for the proper control of a multitude of biological pathways across the endocrine and nervous systems. However, these events are minute both temporally and in terms of the minute amounts of neurotransmitters, hormones, growth factors, and peptides released from single vesicles during exocytosis. It is therefore difficult to measure the kinetics of single exocytosis events in real time. One noninvasive method of measuring the release of molecules from cells is carbon-fiber amperometry. In this chapter, we will describe how we undertake such measurements from both single cells and in live tissue, how the subsequent data are analyzed, and how we interpret these results in terms of their relevant physiology.

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Extracellular Osmotic Stress Reduces the Vesicle Size while Keeping a Constant Neurotransmitter Concentration.

Secretory cells respond to hypertonic stress by cell shrinking, which causes a reduction in exocytosis activity and the amount of signaling molecules released from single exocytosis events. These changes in exocytosis have been suggested to result from alterations in biophysical properties of cell cytoplasm and plasma membrane, based on the assumption that osmotic stress does not affect the secretory vesicle content and size prior to exocytosis. To further investigate whether vesicles in secretory cells are affected by the osmolality of the extracellular environment, we used intracellular electrochemical cytometry together with transmission electron microscopy imaging to quantify and determine the catecholamine concentration of dense core vesicles in situ before and after cell exposure to osmotic stress. In addition, single cell amperometry recordings of exocytosis at chromaffin cells were used to monitor the effect on exocytosis activity and quantal release when cells were exposed to osmotic stress. Here we show that hypertonic stress hampers exocytosis secretion after the first pool of readily releasable vesicles have been fused and that extracellular osmotic stress causes catecholamine filled vesicles to shrink, mainly by reducing the volume of the halo solution surrounding the protein matrix in dense core vesicles. In addition, the vesicles demonstrate the ability to perform adjustments in neurotransmitter content during shrinking, and intracellular amperometry measurements in situ suggest that vesicles reduce the catecholamine content to maintain a constant concentration within the vesicle compartment. Hence, the secretory vesicles in the cell cytoplasm are highly affected and respond to extracellular osmotic stress, which gives a new perspective to the cause of reduction in quantal size by these vesicles when undergoing exocytosis.

Single cell amperometry reveals curcuminoids modulate the release of neurotransmitters during exocytosis from PC12 cells

We used single cell amperometry to examine whether curcumin and bisdemethoxycurcumin (BDMC), substances that are suggested to affect learning and memory, can modulate monoamine release from PC12 cells. Our results indicate both curcumin and BDMC need long-term treatment (72h in this study) to influence exocytosis effectively. By analyzing the parameters calculated from single exocytosis events, it can be concluded that curcumin and BDMC affect exocytosis through different mechanisms. Curcumin accelerates the event dynamics with no significant change of the monoamine amount released from single exocytotic events, whereas BDMC attenuates the amount from single exocytotic event with no significant change of the event dynamics. This comparison of the effect of curcumin and BDMC on exocytosis at the single cell level brings insight into their different mechanisms, which might lead to different biological actions. The effect of curcumin and BDMC on the opening and closing of the exocytotic fusion pore were also investigated. These results might be helpful for understanding the improvement of learning and memory and the anti-depression properties of curcuminoids.

Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry.

Electrochemical cytometry adds a new dimension to our ability to study the chemistry and chemical storage of transmitter molecules stored in nanometer vesicles. The approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which the electroactive contents are quantitatively oxidized (or reduced). The measured current allows us to count the number of molecules in the vesicles using Faraday's law and to correlate this to the amount of molecules released when single exocytosis events take place at communicating cells. The original format for this method involved a capillary electrophoresis separation step to singly address each vesicle, but we have more recently discovered that cellular vesicles tend to adsorb to carbon electrodes and spontaneously as well as stochastically rupture to give mostly single vesicle events. This approach, called impact electrochemical cytometry, even though the impact is perhaps not the important part of this process, has been studied and the vesicle rupture appears to be at the interface between the vesicle and the electrode and is probably driven by electroporation. The pore size and rate of content electrolysis are a function of the pore diameter and the presence of a protein core in the vesicles. In model liposomes with no protein, events appear extremely rapidly as the soft nanoparticles impact the electrode and the contents are oxidized. It appears that the proteins decorating the surface of the vesicle are important in maintaining a gap from the electrode and when this gap is closed electroporation takes place. Models of the event response times suggest the pores formed are small enough so we can carry out these measurements at nanotip electrodes and we have used this to quantify the vesicle content in living cells in a mode we call intracellular impact electrochemical cytometry. The development of electrochemical cytometry allows comparison between vesicle content and vesicular release and we have found that only part of the vesicle content is released in typical exocytotic cases measured by amperometry. This has led to the novel hypothesis that most exocytosis from dense core vesicles is via mechanism where vesicles fuse with the cell membrane, some content is released and then close again to be reloaded and reused. It leaves open the possibility that cells regulate release during individual events. This might be important in learning and memory and be a nonreceptor pharmaceutical target for brain-related disorders. Indeed, the concept of the chemo-brain observed in cisplatin-treated cancer patients appears to be at least in part the result of changing the fraction of transmitter released and we have been able to show this by using the combined amperometric measurement of release and electrochemical cytometry at model cells.

Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis.

Fluorescent proteins (FPs) have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY) for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.

Recycling endosomes undergo rapid closure of a fusion pore on exocytosis in neuronal dendrites.

Exocytosis of recycling endosomes (REs) represents the last step of receptor and membrane recycling, a fundamental process involved in many aspects of cell physiology. In neurons, it is involved in the control of cell polarity and synaptic plasticity and is locally and tightly regulated. However, its molecular mechanisms are still poorly understood. We have imaged single exocytosis events of REs in rat hippocampal neurons in culture transfected with three types of receptors tagged with the pH-sensitive GFP mutant superecliptic phluorin. We found that exocytosis events are grouped into two categories: (1) short burst events in which receptors diffuse into the plasma membrane in a few seconds; and (2) long display events in which receptors remain visible and clustered after exocytosis for many seconds. Display events are much rarer in non-neuronal cells, such as fibroblasts and astrocytes. Using two-color imaging and fast extracellular solution changes, we show that display events correspond to the rapid opening and closing of a fusion pore (or "kiss-and-run") with a median opening time of 2.6 s, which restricts the diffusion of multiple receptor types and bound cargo. Moreover, the RE marker Rab11 remains enriched after display exocytosis events and controls the mode of RE exocytosis. Finally, a given RE can undergo multiple rounds of display exocytosis. The last step of recycling can thus be controlled in neurons for the selective delivery of receptors at the cell surface.