Dynamics Of Exocytosis Research Articles

Spike by spike frequency analysis of amperometry traces provides statistical validation of observations in the time domain

Amperometry is a commonly used electrochemical method for studying the process of exocytosis in real-time. Given the high precision of recording that amperometry procedures offer, the volume of data generated can span over several hundreds of megabytes to a few gigabytes and therefore necessitates systematic and reproducible methods for analysis. Though the spike characteristics of amperometry traces in the time domain hold information about the dynamics of exocytosis, these biochemical signals are, more often than not, characterized by time-varying signal properties. Such signals with time-variant properties may occur at different frequencies and therefore analyzing them in the frequency domain may provide statistical validation for observations already established in the time domain. This necessitates the use of time-variant, frequency-selective signal processing methods as well, which can adeptly quantify the dominant or mean frequencies in the signal. The Fast Fourier Transform (FFT) is a well-established computational tool that is commonly used to find the frequency components of a signal buried in noise. In this work, we outline a method for spike-based frequency analysis of amperometry traces using FFT that also provides statistical validation of observations on spike characteristics in the time domain. We demonstrate the method by utilizing simulated signals and by subsequently testing it on diverse amperometry datasets generated from different experiments with various chemical stimulations. To our knowledge, this is the first fully automated open-source tool available dedicated to the analysis of spikes extracted from amperometry signals in the frequency domain.

Single-Vesicle Electrochemistry Reveals Polysaccharide from Glochidion eriocarpum Champ. Regulates Vesicular Storage and Exocytotic Release of Dopamine.

Polysaccharides, which are well-known natural macromolecules, have been recognized for their protective effects on neurons and their influence on extracellular dopamine levels in the brain. It is crucial to investigate the impact of plant polysaccharides on neurotransmission, particularly regarding the vesicular storage and exocytosis of neurotransmitters. In this study, we demonstrated the possibility of studying how the polysaccharide from Glochidion eriocarpum Champ.(GPS) affects vesicle dopamine content and the dynamics of exocytosis in pheochromocytoma (PC12) cells using single-cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC). Our results unambiguously demonstrate that GPS effectively enhances vesicular neurotransmitter content and alters the dynamics of exocytosis, favoring a smaller fraction of content released in exocytotic release, thereby inducing the partial release mode. These significant effects are attributed to GPS's efficient elevation of calcium influx, significant alteration in the composition of exocytosis-related membrane lipids, and enhancement of free radical scavenging ability. These findings not only establish GPS as a promising candidate for preventive or therapeutic interventions against neurodegenerative disorders but also reiterate the importance of screening native neurologic drugs with single-vesicle electrochemical approaches, the combination of SCA and IVIEC, from a neurotransmitter-centric perspective.

LIMK1 variants are associated with divergent endocrinological phenotypes aligning with divergently altered exocytosis dynamics.

LIMK1 variants are associated with divergent endocrinological phenotypes aligning with divergently altered exocytosis dynamics.

Correlative Cellular Mass Spectrometry Imaging and Amperometry Show Dose Dependent Changes in Lipid Composition and Exocytosis.

Aberrant functioning of the proteasome has been associated with crucial pathologic conditions including neurodegeneration. Yet, the complex underlying causes at the cellular level remain unclear and there are conflicting reports of neuroprotective to neurodegenerative effects of proteasomal inhibitors such as lactacystin that are utilised as models for neurodegenerative diseases. The conflicting results may be associated with different dose regimes of lactacystin and hence we have performed a dose dependent study of the effects of lactacystin to identify concurrent changes in the cell membrane lipid profile and the dynamics of exocytosis using a combination of surface sensitive mass spectrometry and single cell amperometry. Significant changes of negatively charged lipids were associated with different lactacystin doses that showed a weak correlation with exocytosis while changes in PE and PE-O lipids showed dose dependent changes correlated with initial pore formation and total release of vesicle content respectively.

Correlative Cellular Mass Spectrometry Imaging and Amperometry Show Dose Dependent Changes in Lipid Composition and Exocytosis

AbstractAberrant functioning of the proteasome has been associated with crucial pathologic conditions including neurodegeneration. Yet, the complex underlying causes at the cellular level remain unclear and there are conflicting reports of neuroprotective to neurodegenerative effects of proteasomal inhibitors such as lactacystin that are utilised as models for neurodegenerative diseases. The conflicting results may be associated with different dose regimes of lactacystin and hence we have performed a dose dependent study of the effects of lactacystin to identify concurrent changes in the cell membrane lipid profile and the dynamics of exocytosis using a combination of surface sensitive mass spectrometry and single cell amperometry. Significant changes of negatively charged lipids were associated with different lactacystin doses that showed a weak correlation with exocytosis while changes in PE and PE−O lipids showed dose dependent changes correlated with initial pore formation and total release of vesicle content respectively.

Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms.

The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.

Anionic Species Regulate Chemical Storage in Nanometer Vesicles and Amperometrically Detected Exocytotic Dynamics.

Hofmeister effects have often been ignored in living organisms, although they affect the activity and functions of biological molecules. Herein, amperometry has been applied to show that the vesicular content, dynamics of exocytosis and vesicles opening, depend on the anionic species treatment. Compared to 100 μM Cl– treated chromaffin cells, a similar number of catecholamine molecules is released after chaotropic anions (ClO4– and SCN–) treatment, even though the vesicular catecholamine content significantly increases, suggesting a lower release fraction. In addition, there are opposite effects on the dynamics of vesicles release (shorter duration) and vesicle opening (longer duration) for chaotropic anions treated cells. Our results show anion-dependent vesicle release, vesicle opening, and vesicular content, providing understanding of the pharmacological and pathological processes induced by inorganic ions.

Effects of the surface charge of polyamidoamine dendrimers on cellular exocytosis and the exocytosis mechanism in multidrug-resistant breast cancer cells

BackgroundPolyamidoamine (PAMAM) dendrimer applications have extended from tumor cells to multidrug-resistant tumor cells. However, their transportation in multidrug-resistant tumor cells remains unclear. Herein, we investigated the exocytosis rule and mechanism of PAMAM dendrimers in multidrug-resistant tumor cells.ResultsUsing a multidrug-resistant human breast cancer cell model (MCF-7/ADR), we performed systematic analyses of the cellular exocytosis dynamics, pathways and mechanisms of three PAMAM dendrimers with different surface charges: positively charged PAMAM-NH2, neutral PAMAM-OH and negatively charged PAMAM-COOH. The experimental data indicated that in MCF-7/ADR cells, the exocytosis rate was the highest for PAMAM-NH2 and the lowest for PAMAM-OH. Three intracellular transportation processes and P-glycoprotein (P-gp) participated in PAMAM-NH2 exocytosis in MCF-7/ADR cells. Two intracellular transportation processes, P-gp and multidrug resistance (MDR)-associated protein participated in PAMAM-COOH exocytosis. P-gp and MDR-associated protein participated in PAMAM-OH exocytosis. Intracellular transportation processes, rather than P-gp and MDR-associated protein, played major roles in PAMAM dendrimer exocytosis. PAMAM-NH2 could enter MCF-7/ADR cells by forming nanoscale membrane holes, but this portion of PAMAM-NH2 was eliminated by P-gp. Compared with PAMAM-OH and PAMAM-COOH, positively charged PAMAM-NH2 was preferentially attracted to the mitochondria and cell nuclei. Major vault protein (MVP) promoted exocytosis of PAMAM-NH2 from the nucleus but had no effect on the exocytosis of PAMAM-OH or PAMAM-COOH.ConclusionsPositive charges on the surface of PAMAM dendrimer promote its exocytosis in MCF-7/ADR cells. Three intracellular transportation processes, attraction to the mitochondria and cell nucleus, as well as nuclear efflux generated by MVP led to the highest exocytosis observed for PAMAM-NH2. Our findings provide theoretical guidance to design a surface-charged tumor-targeting drug delivery system with highly efficient transfection in multidrug-resistant tumor cells. Especially, to provide more DNA to the nucleus and enhance DNA transfection efficiency in multidrug-resistant tumor cells using PAMAM-NH2, siRNA-MVP or an inhibitor should be codelivered to decrease MVP-mediated nuclear efflux.Graphical abstract

The Phosphoprotein Synapsin Ia Regulates the Kinetics of Dense-Core Vesicle Release.

Common fusion machinery mediates the Ca2+-dependent exocytosis of synaptic vesicles (SVs) and dense-core vesicles (DCVs). Previously, Synapsin Ia (Syn Ia) was found to localize to SVs, essential for mobilizing SVs to the plasma membrane through phosphorylation. However, whether (or how) the phosphoprotein Syn Ia plays a role in regulating DCV exocytosis remains unknown. To answer these questions, we measured the dynamics of DCV exocytosis by using single-vesicle amperometry in PC12 cells (derived from the pheochromocytoma of rats of unknown sex) overexpressing wild-type or phosphodeficient Syn Ia. We found that overexpression of phosphodeficient Syn Ia decreased the DCV secretion rate, specifically via residues previously shown to undergo calmodulin-dependent kinase (CaMK)-mediated phosphorylation (S9, S566, and S603). Moreover, the fusion pore kinetics during DCV exocytosis were found to be differentially regulated by Syn Ia and two phosphodeficient Syn Ia mutants (Syn Ia-S62A and Syn Ia-S9,566,603A). Kinetic analysis suggested that Syn Ia may regulate the closure and dilation of DCV fusion pores via these sites, implying the potential interactions of Syn Ia with certain DCV proteins involved in the regulation of fusion pore dynamics. Furthermore, we predicted the interaction of Syn Ia with several DCV proteins, including Synaptophysin (Syp) and soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. By immunoprecipitation, we found that Syn Ia interacted with Syp via phosphorylation. Moreover, a proximity ligation assay (PLA) confirmed their phosphorylation-dependent, in situ interaction on DCVs. Together, these findings reveal a phosphorylation-mediated regulation of DCV exocytosis by Syn Ia.SIGNIFICANCE STATEMENT Although they exhibit distinct exocytosis dynamics upon stimulation, synaptic vesicles (SVs) and dense-core vesicles (DCVs) may undergo co-release in neurons and neuroendocrine cells through an undefined molecular mechanism. Synapsin Ia (Syn Ia) is known to recruit SVs to the plasma membrane via phosphorylation. Here, we examined whether Syn Ia also affects the dynamics of DCV exocytosis. We showed that Syn Ia regulates the DCV secretion rate and fusion pore kinetics during DCV exocytosis. Moreover, Syn Ia-mediated regulation of DCV exocytosis depends on phosphorylation. We further found that Syn Ia interacts with Synaptophysin (Syp) on DCVs in a phosphorylation-dependent manner. Thus, these results suggest that Syn Ia may regulate the release of DCVs via phosphorylation.

Quantifying neurotransmitter secretion at single-vesicle resolution using high-density complementary metal\u2013oxide\u2013semiconductor electrode array

Neuronal exocytosis facilitates the propagation of information through the nervous system pertaining to bodily function, memory, and emotions. Using amperometry, the sub-millisecond dynamics of exocytosis can be monitored and the modulation of exocytosis due to drug treatment or neurodegenerative diseases can be studied. Traditional single-cell amperometry is a powerful technique for studying the molecular mechanisms of exocytosis, but it is both costly and labor-intensive to accumulate statistically significant data. To surmount these limitations, we have developed a silicon-based electrode array with 1024 on-chip electrodes that measures oxidative signal in 0.1 millisecond intervals. Using the developed device, we are able to capture the modulation of exocytosis due to Parkinson’s disease treatment (L-Dopa), with statistical significance, within 30 total minutes of recording. The validation study proves our device’s capability to accelerate the study of many pharmaceutical treatments for various neurodegenerative disorders that affect neurotransmitter secretion to a matter of minutes.

Perturbation and imaging of exocytosis in plant cells.

The exocytosis process delivers proteins, lipids, and carbohydrates to the plasma membrane or the extracellular space to sustain plant cell growth, development, and response to environmental stimuli. Plant exocytosis is highly dynamic and requires the coordinated functions of multiple cellular components such as tethering complexes, GTPase signaling, and vesicle fusion machinery. Accurate spatio-temporal control of plant exocytosis is critical for the proper functions of plant cells. Live-cell imaging of fluorescence-tagged cargo proteins allows for quantitative analysis of exocytosis dynamics in plant cells. Small molecule inhibitors that target important components in the exocytosis machinery allow for transient manipulation of the exocytosis process. In this chapter, we describe procedures that use Endosidin2 (ES2) and Brefeldin A (BFA) as small molecule inhibitors to disrupt plant exocytic processes and use fluorescent protein-tagged PIN-formed 2 (PIN2) and Cellulose Synthase (CESA) as cargo proteins to quantify exocytosis dynamics in plant cells.

Cover Feature: Altered Lipid Composition of Secretory Cells Following Exposure to Zinc Can Be Correlated to Changes in Exocytosis (Chem. Eur. J. 21/2019)

By combining electrochemical techniques and mass spectrometry imaging, zinc has been shown to significantly change the dynamics of exocytosis and the vesicle contents, as well as the lipid distribution on the cell membrane. Altered lipid composition after zinc exposure can be correlated to changes in exocytosis, which provides direct evidence that zinc regulates neurotransmitter release and results in the modulation of synaptic strength and plasticity. More information can be found in the Communication by A. G. Ewing et al. on page 5406.

Altered Lipid Composition of Secretory Cells Following Exposure to Zinc Can Be Correlated to Changes in Exocytosis.

A micromolar concentration of zinc has been shown to significantly change the dynamics of exocytosis as well as the vesicle contents in a model cell line, providing direct evidence that zinc regulates neurotransmitter release. To provide insight into how zinc modulates these exocytotic processes, neurotransmitter release and vesicle content were compared with single cell amperometry and intracellular impact vesicle cytometry with a range of zinc concentrations. Additionally, time-of-flight secondary ion mass spectrometry (ToF-SIMS) images of lipid distributions in the cell membrane after zinc treatment correlate to changes in exocytosis. By combining electrochemical techniques and mass spectrometry imaging, we proposed a mechanism by which zinc changes the fusion pore and the rate of neurotransmitter release by changing lipid distributions and results in the modulation of synaptic strength and plasticity.

Transient receptor potential ankyrin 1 channels are involved in spontaneous peptide hormone release from astrocytes

Astrocytes, a large population of glial cells, detect neurotransmitters and respond by increasing intracellular Ca2+ concentration ([Ca2+]i) and releasing chemical molecules called gliotransmitters. Recently discovered Ca2+ influx through transient receptor potential ankyrin 1 (TRPA1) channels is reported to cause spontaneous [Ca2+]i increase in astrocytes. While several physiological functions of TRPA1-mediated spontaneous Ca2+ signal have been revealed, relation with gliotransmitter release, especially peptide hormone exocytosis is largely unknown. We therefore explored the [Ca2+]i and exocytosis dynamics in rat astrocyte cell line C6 cells and primary astrocytes. TRPA1-mediated spontaneous [Ca2+]i transients were observed in both C6 cells and primary astrocytes. Total internal reflection fluorescence microscopy revealed that Venus-tagged brain-derived neurotrophic factor and neuropeptide Y were released spontaneously from astrocytes. Activation of TRPA1 channels enhanced the frequency of peptide hormone exocytosis, and inhibition of TRPA1 channels decreased the number of peptide hormone exocytosis. These results suggest that TRPA1-mediated spontaneous [Ca2+]i increase modulates the spontaneous release of peptide hormones from astrocytes.

(Invited) Amperometry Detection of Full Fusion Mode of Catecholamine Exocytosis from Single PC12 Cell Elicited By Sodium Dodecyl Sulfate

Vesicular exocytosis is a crucial process for living cells by which signalling species such as acetylcholine and catecholamines and other vesicular contents can be secreted into the extracellular environment. Single cell electrochemistry, especially the “semi-artificial synapse” based amperometry, has been used to scrutinize exocytosis dynamics of single vesicles due to its high sensitivity and appropriate temporal resolution. Important characteristics of vesicular release as well as the effects of biological, chemical and physical parameters on them have thus been evaluated for many cell lines. This has led to contest the conventional full release mode. Recently, the view that partial release is the most common for most neurotransmitters stored in dense core vesicles has gained acceptance. However, the fundamental reasons leading to partial fusion remain an open question waiting experimental characterization. In this work, catecholamine release was elicited from PC12 using sufficiently small concentrations of sodium dodecyl sulfate (SDS) to perturb normal release mechanism in an attempt to reveal concealed information while keeping physiologically compatible conditions. Amperometry was used to monitor and quantify the released fluxes and kinetics of individual vesicular events based on analyses of statistically significant series of events. This evidenced that stimulating release with SDS 350 μM leads to a doubling of the quantity of catecholamine cations released per event and much larger release fluxes compared to controls (release elicited with K+105 mM under same conditions) or SDS 250 μM. Importantly, the releases stimulated by SDS 350 μM showed strict dependence on calcium ion, indicating the biological significance of the release. These quantitative measurements provide substantial proof for the partial release hypothesis. The results confirm our previous theoretical model and are consistent with ex situ cytometric experiments on isolated PC12 vesicles reported in the literature, which established that release is far from being total under normal exocytotic conditions. Based on the theoretical model, these data established that the maximal size of fusion pores at the end of the “full fusion” phase is limited by some constriction of the fusion pore radius. Indeed, the present results are entirely consistent with the fact that SDS 350 μM allows the fusion pore to expand to a double size (ca. 28 nm radius) compared to controls and SDS 250 μM (ca. 14 nm radius). Acknowledgements: We thank the MOST of China (2016YFC1100300 and 2013CB933703), the NSFC (51571169 and 21303146) and the Fundamental Research Funds for the Central Universities (20720170031) for financial support of the work performed in Xiamen. CNRS, ENS and UPMC (UMR 8640) supports are acknowledged for that performed in Paris. We also thank our joint laboratory, LIA CNRS NanoBioCatEchem, for supporting the collaboration between the two research teams, as well as discussions with Jerome Delacotte (ENS, UMR 8640).

Cover Picture: Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry (Angew. Chem. Int. Ed. 18/2017)

Cover Picture: Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry (Angew. Chem. Int. Ed. 18/2017)

Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry

AbstractWe applied electrochemical techniques with nano‐tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 μm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc‐treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.

Zinc Regulates Chemical-Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry.

We applied electrochemical techniques with nano-tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 μm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc-treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.

Acidic‐pH‐Activatable Fluorescence Probes for Visualizing Exocytosis Dynamics

AbstractLive imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small‐molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.

Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics.

Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.