Excitation-Secretion Coupling of Dense-Core Vesicle Release
by Valentina Cirkovic
Date of Examination:2023-06-19
Date of issue:2023-07-18
Advisor:Dr. Sonja Wojcik
Referee:Dr. Sonja Wojcik
Referee:Prof. Dr. Thomas Dresbach
Persistent Address:
http://dx.doi.org/10.53846/goediss-10009
Files in this item
Name:Dissertation Valentina Cirkovic.pdf
Size:5.67Mb
Format:PDF
The following license files are associated with this item:
Abstract
English
Dense-core vesicles (DCVs) are membrane-bound organelles present in many secretory cells. Depending on cell type, DCVs package and release various types of cargo, such as neuronal peptides, proteins, neurotrophins, ATP, and catecholamines, greatly influencing cellular signalling. Chromaffin cells from the adrenal medulla are enriched with DCVs, which secrete the catecholamines adrenaline and noradrenaline into the circulation. DCV release can be efficiently monitored by capacitance and amperometry recordings, making these cells a long-established model system for the extensive study of DCV biology. Membrane depolarization of the chromaffin cell causes the opening of voltage-dependent calcium channels and a subsequent increase in intracellular calcium, which initiates SNARE-mediated exocytosis of DCVs. It has been shown that neuroendocrine chromaffin cells perform full-collapse and kiss-and-run modes of exocytosis/endocytosis based on electrophysiological techniques. However, there are dissimilarities in the literature on which mode predominates based on distinct stimulation patterns. When determining the mode of vesicle exocytosis/endocytosis, these discrepancies may result from using different experimental approaches that lead to various interpretations. Nonetheless, this could also mean that the link between the stimulus and the various DCV exocytosis/endocytosis modes must still be adequately determined. Furthermore, the ultrastructural analysis of chromaffin cells prepared with aldehyde fixation or high-pressure freezing (HPF) has resulted in divergent findings. These inconsistencies mean that functional and ultrastructural data analyses of cultured chromaffin cells may require an optimized, applicable, and flexible experimental system. The present study aimed to investigate DCV exocytosis and subsequent membrane retrieval in response to defined stimuli by combining electrophysiological and ultrastructural analyses. We planned to analyse which mode of DCV fusion predominates after specific stimulation, whether compound fusion or hemifusion intermediates and different modes of endocytosis can be detected. To address these questions, we tested both optogenetic stimulation and field stimulation of cultured chromaffin cells. Field stimulation patterns established by electrophysiological methods were implemented prior to HPF and subsequent analysis by 2D electron microscopy. The cellular effect of exposure to light and electric field stimulation showed that our optimized methods could be used in cultured chromaffin cells to induce DCV release. Additionally, our voltage ramp stimulation paradigm enabled us to trigger and monitor DCV release with precise temporal resolution. Further, employing HPF, we captured large Ω-shaped profiles and prominent vesicle release during which the vesicle collapses entirely into the plasma membrane. Most cells also showed distinct coated endocytic structures that were well-preserved with our experimental approach. Our study demonstrates that our improved experimental procedure was able to visualize DCV exocytosis events, including vesicle content release, in cultured neuroendocrine chromaffin cells.
Keywords: Adrenal Chromaffin Cell; Dense-Core Vesicle; Exocytosis; Endocytosis; Capacitance Recordings; Electric Field Stimulation; High-pressure Freezing; Electron Microscopy; Adrenal Chromaffin Cell
