The influence of nanoparticle shape on bilateral exocytosis from Caco-2 cells

Nanoparticles are able to be excreted from both apical and basolateral sides after taken up by cells. Compared to nanospheres, nanorods preferred basolateral exocytosis to apical exocytosis.     

Quantitative Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) Imaging of Individual Vesicles to Investigate the Relation between Fraction of Chemical Release and Vesicle Size

We used correlative transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to quantify the contents of subvesicular compartments, and to measure the partial release fraction of ¹³C-dopamine in cellular nanovesicles as a function of size. Three modes of exocytosis comprise full release, kiss-and-run, and partial release. The latter has been subject to scientific debate, despite a growing amount of supporting literature. We tailored culturing procedures to alter vesicle size and definitively show no size correlation with the fraction of partial release. In NanoSIMS images, vesicle content was indicated by the presence of isotopic dopamine, while vesicles which underwent partial release were identified by the presence of an ¹²⁷I-labelled drug, to which they were exposed during exocytosis allowing entry into the open vesicle prior to its closing again. Demonstration of similar partial release fractions indicates that this mode of exocytosis is predominant across a wide range of vesicle sizes.     

Direct Quantification of Nanoplastics Neurotoxicity by Single-Vesicle Electrochemistry

Nanoplastics are recently recognized as neurotoxic factors for the nervous systems. However, whether and how they affect vesicle chemistry (i.e., vesicular catecholamine content and exocytosis) remains unclear. This study offers the first direct evidence for the nanoplastics-induced neurotoxicity by single-vesicle electrochemistry. We observe the cellular uptake of polystyrene (PS) nanoplastics into model neuronal cells and mouse primary neurons, leading to cell viability loss depending on nanoplastics exposure time and concentration. By using single-vesicle electrochemistry, we find the reductions in the vesicular catecholamine content, the frequency of stimulated exocytotic spikes, the neurotransmitter release amount of single exocytotic event, and the membrane-vesicle fusion pore opening-closing speed. Mechanistic investigations suggest that PS nanoplastics can cause disruption of filamentous actin (F-actin) assemblies at cytomembrane zones and change the kinetic patterns of vesicle exocytosis. Our finding shapes the first quantitative picture of neurotoxicity induced by high-concentration nanoplastics exposure at a single-cell level.     

碳纤维超微盘电极的制备及对单囊泡胞吐动力学的高分辨研究

利用改良的电化学腐蚀方法制备了碳纤维超微盘状电极,具有方法简便,电极尺寸可控、可多次使用等优点.扫描电镜和伏安表征实验表明电极密封效果良好、电化学性能优良.在鼠嗜铬神经瘤PC12细胞上,用超微盘电极实现了高时空分辨、高保真度的单囊泡胞吐测定.与常规微电极相比,能更准确地反应囊泡融合这一超快过程(毫秒级)的动力学过程.     

Redox‐Filled Carbon‐Fiber Microelectrodes for Single‐Cell Exocytosis

Carbon‐fiber microelectrodes (CFEs) are the primary electroanalytical tool in single‐cell exocytosis and in vivo studies. Here we report a new study on the kinetic properties of electrolyte‐filled CFEs in single‐cell measurements and demonstrate that the addition of outer sphere redox species, such as Fe(CN)₆^3? and Ru(NH₃)₆³⁺, in the backfill electrolyte solution can greatly enhance the kinetic response of CFEs. We show that at 750 mV, a voltage normally applied for detection of dopamine, the presence of fast outer sphere redox species in the backfilling solution significantly enhances the kinetic response of CFEs toward fast dopamine detection at single PC12 cells. Moreover, we also demonstrate that the use of Fe(CN)₆^3? in the backfilling solution has enabled direct measurement of dopamine at applied voltages as low as 200 mV. This kinetic enhancement is believed to be due to faster electron‐transfer kinetics on the coupling pole as compared to the sluggish reduction of oxygen. We anticipate that such redox‐filled CFE ultramicroelectrodes will find many useful applications in single cell exocytosis and in vivo sensing.     

Analysis of Amperometric Spike Shapes to Release Vesicles

Carbon‐fiber amperometry is a real‐time analytical tool to detect vesicle release events from individual secretory cells. This study compared the spike events released in response to local or global depolarization. We describe each spike shape that regularly occurs, as well as those shapes of spikes that occur with less frequency, but reproducibly. We compare how frequently each type of spike occurs, and compare their amplitudes and kinetics to the commonly accepted spike release events. Analysis of the amperometric peaks typically discarded from single cell amperometry recordings may reveal unappreciated phenomena related to the release of secretory vesicles.     

Recent advances in nanoelectrochemistry at the interface between two immiscible electrolyte solutions

The nanoscale interface between two immiscible electrolyte solutions (nanoITIES) is an emerging versatile analytical platform. Analytical advantages of chemical analysis using the nanoITIES include imaging with nanometer spatial resolution, probing fast dynamics with millisecond temporal resolution and fast response times, selectively detecting analytes, probing fundamental chemical processes (e.g., diffusion profiles), and versatile sensing of metal ions, proteins, neurotransmitters, ionic and neutral species, redox-active and non-redox active analytes, etc. We present here a brief theoretical background of the nanoITIES and experimental advances from the past five years. These advances include imaging of nanopores, probing diffusion profiles, biosensing, a new pH modulation mechanism for sensing neutral species, and studying exocytosis from Aplysia californica neurons.     

Micro- and nano-electrodes for neurotransmitter monitoring

Chemical transmission between neurosecretory cells is a central biological phenomenon. Detecting neurotransmitter molecules released in the brain or in cell cultures is key to understanding how, when, and where chemical transmission occurs. Electrochemical techniques provide a unique quantitative approach to this field of research. Micro- and nano-electrodes can be engineered to be implanted in the living brain for interstitial fluid analysis, or placed close to, or even inside, isolated cells to detect exocytosis events and vesicles. Carbon fiber microelectrodes provide a common basis for detecting not only dopamine but also a wide variety of neurotransmitters ranging from biogenic amines, purines, and amino acids to free radicals and peptides. To achieve specific and sensitive in situ neurotransmitter detection, carbon fiber microelectrodes can be chemically modified with nanomaterials, enzymes, or aptamers or etched to reach nanoscale dimensions for intracellular analysis. Such micro- and nano-electrodes are an essential tool for analyzing living cells and tissues.     

Visualized single cell dynamics and analysis of molecular tricks

The dynamic behaviours of various cells were analysed by video-microscopes and mass spectrometers at the single-cell level. By the video image analysis of a single cell, real dynamic moments of cell behaviour, which were previously accessible only to the imagination, can be visualized — such as the popping of micro-granules, and the exocytosis of neutrophils; a mast cell model cell line, RBL-2H3 cells, and pancreatic beta cell model cell line, MIN-6. In combination with the observations of behaviour, more direct molecular analyses become much more important for showing the molecular interplay in a cell. The combination of mass spectrometries was applied to analysis at the single cellular level. Based on the current progress in these analyses, the future trends in analyses of single cellular dynamism are predicted.     

Cisplatin Inhibits Neurotransmitter Release during Exocytosis from Single Chromaffin Cells Monitored with Single Cell Amperometry

Chemotherapy with cisplatin induces side effects such as memory loss, confusion of thinking, and difficulties with multi-tasking. However, the mechanism of cisplatin inducing nervous dysfunction is still unknown. Herein, we examine whether and how cisplatin regulates the release of neurotransmitter during exocytosis in single chromaffin cells using single cell amperometry. The results show that cisplatin reduces the amount of transmitter released during exocytosis by reducing the duration of the exocytotic events, including the opening and closing time of the fusion pore. Furthermore, the stability of the initial fusion pore formed during exocytosis is also reduced by cisplatin. Our study holds the promise for understanding the side effects of cisplatin on the nervous system at single cell level.