Quantitative chemical imaging to study content release from single nanovesicles

Abstract

Cellular communication is vital for the survival of multicellular organisms. This process often relies on a highly regulated mechanism called exocytosis, which involves the release of chemical signals. During exocytosis, vesicles can fully or partially release their contents. The quantity of neurotransmitters expelled in different modes of release may have diverse effects on cellular communication by allowing cells to control the level of outgoing signals. Despite significant discoveries in understanding the components of exocytosis, there is still much to uncover regarding its regulation and implications. This thesis aims to gain a better understanding of vesicular content release, particularly in the context of partial release, using quantitative chemical imaging. This includes the integration of mass spectrometry imaging (MSI) with electron microscopy or electrochemical analysis alongside light microscopy. By doing so, a comprehensive approach can be employed to gain insights into the mechanism of vesicular content release and quantify the fraction of release. Electrochemical techniques offer the advantage of high temporal resolution and enable quantification of both stored and released molecules from vesicles. Coupled with imaging methods such as fluorescence, electron microscopy, and mass spectrometry imaging, comprehensive spatial information and chemical information can be obtained to complement the data provided by electrochemical techniques. In particular, nanoscale secondary ion mass spectrometry (NanoSIMS), a high-resolution MSI method that is capable of absolute quantification at subcellular level, was primarily used throughout this thesis. PC12 cells treated with isotopically labeled L-DOPA were examined, and NanoSIMS imaging was correlated with transmission electron microscopy (TEM) to detect and quantify the labeled dopamine in the halo and dense core compartments of large dense core vesicles (LDCVs) in paper I. Paper II introduced a dual-label approach to visualize and quantify vesicles undergoing partial release in PC12 cells by exposing them to a second label during exocytosis. Expanding on the dual-label approach, paper III investigated the influence of vesicle size on the dynamics of partial release. Furthermore, the combination of vesicle impact electrochemical cytometry (VIEC) with live fluorescence imaging was developed in paper IV allowing real-time analysis of vesicular content release from isolated labeled bovine chromaffin vesicles. Overall, these studies demonstrate the application of quantitative chemical imaging in understanding the mechanism and quantifying the fraction of release in vesicular content release.