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Ute Becherer:
Role of second messengers in the regulation of chromaffin cell secretion

Research

Our main research goal is the understanding of the molecular mechanisms of regulated exocytosis, which is the basis of neurotransmitter release during synaptic transmission and of the release of hormones such as catecholamines. Regulated exocytosis is a multi-step process including:

  1. Mobilization from the reserve pool
  2. Docking
  3. Priming
  4. Ca2+-dependent fusion

A large group of proteins is involved in these processes (see Figure 1 and for review see Becherer & Rettig, 2006).

We are specifically interested in the exocytosis of:

  • large dense core vesicles (LDCVs) containing catecholamines in chromaffin cells
  • LDCVs and synaptic vesicles (SVs) in dorsal root ganglion (DRG) neurons.

Figure 1: Vesicles undergo docking and priming reactions before fusing with the plasma membrane upon Ca2+ entry via CaV channels. Primed vesicles are tightly connected to the plasma membrane via the SNARE complex formed by SNAP-25, Syntaxin1 and Synaptobrevin. The fusion of the LDCVs with the plasma membrane is mediated by Synaptotagmin. Docking of LDCVs involves Munc18, while priming is regulated by proteins such as CAPS or Munc13.

Our method of choice is total internal reflection fluorescence (TIRF) microscopy in combination with patch-clamp electrophysiology (Figure 2 and see Becherer et al. 2007). This method allows us to observe docking, priming and fusion of individual fluorescently labeled vesicles (Movie 1) in real time, while measuring the membrane capacitance and controlling the intracellular medium with the patch pipette. This technique is complemented by genetic approaches (knock-out, rescue and gain of function experiments).

 

Figure 2: Left: Experimental setup for combined TIRF microscopy and patch-clamp electrophysiology.

Right: Example of one such experiment. On top is displayed the change in membrane capacitance elicited by a train of depolarizations while the bottom TIRFM pictures made at the beginning (1) and the end (2) of the experiment shows that 12 vesicles labeled via overexpression of NPY-mRFP were secreted (yellow arrows).

With this method we found that mobility of LDCVs is related to its functional state in chromaffin cells (Nofal et al., 2007). We investigate the Ca2+-dependency of docking in chromaffin cells (Pasche et al. 2012) and we uncovered that some LDCVs appear permanently docked to the plasma membrane through dead end docking (Hugo et al. 2013). We currently are unraveling the molecular machinery mediating dead-end docking.

Furthermore we are investigating the mode of LDCV secretion and it's regulation in DRG neurons (Movie 2).