Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells.

Tho Duc Khanh Nguyen,Michael E Kurczy,Nhu T.N Phan,Alicia Lork,Mai Philipsen,Aurélien Thomen,Lisa Mellander,Andrew G Ewing

doi:10.1021/acsnano.2c00344

Tho Duc Khanh Nguyen, Michael E Kurczy + Show 6 more

Open Access

https://doi.org/10.1021/acsnano.2c00344

Copy DOI

Abstract

For decades, “all-or-none” and “kiss-and-run” were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I–, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I– in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically.

Highlights

“all-or-none” and “kiss-and-run” were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release
A dual stable isotope labeling approach was implemented by first loading 13C-labeled dopamine into the vesicles via 6 h incubation with 13C L-DOPA; later, the second label 127I
We found direct evidence revealing partial release visually and quantitatively by implementing a nanoscopic approach with Transmission electron microscopy (TEM) and NanoSIMS mass spectrometry imaging

Summary

Introduction

“all-or-none” and “kiss-and-run” were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. The fusion pore closing in the partial release process of vesicles was visualized by fluorescence microscopy This has recently been supported by the works on ∼1 μm Ø vesicles in pancreatic beta-cells, and adrenal chromaffin cells using total internal reflection fluorescence microscopy (TIRF))[14,22] and super-resolution stimulated emission depletion microscopy (STED).[37] Partial release appears to be a rapid and economic vesicle recycling mechanism, regulating or limiting the rate of transmitter secretion, providing machinery to adjust synaptic strength and achieve synaptic plasticity in cognition, learning, and disease

Methods

Results

Conclusion