Reconstruction of Vesicle Assemblies with DNA Nanorulers for Resolving Heterogeneity of Vesicles in Live Cells

Abstract

Nanoscale vesicles such as synaptic vesicles play pivotal role in efficient inter‐neuronal communications in vivo. However, the co‐existence of single vesicle and vesicle clusters in living cells increases the heterogeneity of vesicle populations, which largely complicates the quantitative analysis of the vesicles. Hence, the high spatiotemporal monitoring of vesicle assemblies is currently incompletely resolved. Here, we used synthetic vesicles and DNA nanorulers to reconstruct in vitro the vesicle assemblies that mimic vesicle clusters in living cells. DNA nanorulers programmed the lateral distance of vesicle assemblies from 3 nm to 10 nm. We used the carbon fiber nanoelectrode (CFNE) to amperometric monitor artificial vesicles assemblies with sub‐10 nm interspaces, and obtained a larger proportion of complex events. We resolve the heterogeneity of individual vesicle release kinetics in PC12 cells with the temporal resolution down to ∼0.1 ms. We further analyzed the aggregation state of intracellular vesicles and the exocytosis of living cells with electrochemical vesicle cytometry. Our results indicated that the exocytosis of vesicle clusters was critically dependent on the size of clusters. This technology has potential as a tool to shed light on the heterogeneity analysis of vesicle populations.This article is protected by copyright. All rights reserved

Universal Covalent Grafting Strategy of an Aptamer on a Carbon Fiber Microelectrode for Selective Determination of Dopamine In Vivo

“…Under KCl stimulation, the current first rises sharply, producing spike I within 30 s and then falls to baseline as dopamine is rapidly metabolized, and the stimulus-generated dopamine is 6.5–7.6 μM [ I (nA) = 0.16 C DA (μM) + 0.06, r = 0.9998]. Interestingly, reproducible shoulder spike II (1.0–1.2 μM) is generated during the current drop, which behaves similarly to the kinetic process of the neurotransmitter release from vesicles via exocytosis. − Similarly, Zhang et al recently showed that such double spikes occurred when neighboring vesicles aggregate to form a dimer and that the distance between the vesicles correlated with the separation of the double spikes. Furthermore, they also recorded complex events in high K + -stimulated PC12 cells, such as single spikes, double spikes, and even multispikes.…”