Aller au contenuAller au menuAller à la recherche

Ute Becherer "TIRF-Microcopy and Patch-Clamp: a powerful combination to study exocytosis"

Abstract :

Ca2+ -dependent exocytosis of secretory vesicles consist of several steps: docking of the vesicles to the plasma membrane, priming to render the vesicles release-competent, fusion with the plasma membrane.
Exocytosis can be measured electrophysiologically as an increase in membrane capacitance and can be visualized as a loss of fluorescence in TIRF-Microcopy. While capacitance measurements offer superior temporal resolution allowing the identification of different vesicle pools, it only measures directly the fusion step of exocytosis and give indirect information on docking and priming. In contrast, TIRFM allows the visualization of single vesicle movement and vesicle fusion events and is therefore the ideal tool to monitor directly docking and priming. However, TIRF-Microscopy allows only the visualization of vesicles at the cell membrane - coverslip interface (i.e. the footprint of the cell). I will present data in which we combined these two methods and demonstrate that secretion of vesicles at the footprint of bovine chromaffin cell reflects the overall secretion of the cell validating TIRF-Microscopy as a technique to observe the behavior of vesicles. Furthermore, using a new method to analyze the movements of the vesicles, we were able to distinguish between three different types of mobility. By artificially increasing or decreasing priming with pharmacological tools we were able to differentiate for the first time the mobility of primed vesicles from docked vesicles allowing us to distinguish the molecular state of vesicles in TIRF-Microscopy.


Selected publications

Speidel D, Bruederle CE, Enk C, Voets T, Varoqueaux F, Reim K, Becherer U, Fornai F, Ruggieri S, Holighaus Y, Weihe E, Bruns D, Brose N, Rettig J.
CAPS1 regulates catecholamine loading of large dense-core vesicles.
Neuron (2005); 46:75-88.
Becherer U., Moser T, Stühmer W and Oheim M.
Calcium regulates exocytosis at the level of single vesicles.
Nat Neurosci. (2003); 6: 846-853.

Nathalie Sans