Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

56-POS Board 19 A NOVEL FLUORESCENCE / ELECTROCHEMISTRY APPROACH TO MEASURE RAPID ENDOCYTOSIS OF SINGLE VESICLES IN CHROMAFFIN CELLS

Ferris Dweik 1,2 ; Xin Liu 1,2 ; Kevin Gillis 1,2 ; 1 University of Missouri, Department of Chemical and Biomedical Engineering, Columbia, MO, USA 2 University of Missouri, Dalton Cardiovascular Research Center, Columbia, MO, USA

Understanding the dynamics, regulation, and molecular reactions underlying exocytosis endocytosis coupling is challenging due to limits of current experimental approaches to measure the opening and closure of the fusion pore in individual vesicles with high time resolution. We are developing an approach to rapidly measure endocytosis of single vesicles by altering the pH in the narrow gap between the bottom of a chromaffin cell and the underlying surface of a transparent microelectrode. Electrochemical oxidation / reduction of a hydroquinone in the bath solution is used to rapidly modulate the pH of the solution below the cell. Upon exocytosis, the fusion pore opens to let transmitter out and a pH-sensitive, membrane-impermeant fluorescent indicator (SNARF-5F) enters the vesicle. The key principle is that the moment of re-closure of the fission pore (endocytosis) is identified as the time when the fluorescence of the vesicle containing the pH indicator becomes insensitive to rapid modulation of extracellular pH. The proton is the ideal probe to determine when full closure of the fission pore occurs because of its small size. In addition, the transparent electrode is immediately underneath an adherent chromaffin cell, which is an ideal position to rapidly modulate pH at the bottom cell surface. We have induced modest and reversible pH changes at the surface of a transparent electrodes with good SNR within ~1s after stepping the electrode potential and are working towards a goal of ~100ms temporal resolution. Dynamic fluorescent changes of SNARF-5F in stimulus-induced invaginations beneath a chromaffin cell have been resolved in synchrony with stepping the electrode to oxidizing and reducing potentials. Future experiments will test the hypothesis that high intracellular Ca2+ concentrations can stimulate ultrafast endocytosis with dynamin playing an essential role.

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