Identification of a Munc13-sensitive step in chromaffin cell large dense-core vesicle exocytosis.

Kwun Nok M Man,Paulo S Pinheiro,Cordelia Imig,David R Stevens,Jakob B Sørensen,Benjamin H Cooper,Nils Brose,Sonja M Wojcik,Jens Rettig,Alexander M Walter

doi:10.7554/elife.10635

Abstract

It is currently unknown whether the molecular steps of large dense-core vesicle (LDCV) docking and priming are identical to the corresponding reactions in synaptic vesicle (SV) exocytosis. Munc13s are essential for SV docking and priming, and we systematically analyzed their role in LDCV exocytosis using chromaffin cells lacking individual isoforms. We show that particularly Munc13-2 plays a fundamental role in LDCV exocytosis, but in contrast to synapses lacking Munc13s, the corresponding chromaffin cells do not exhibit a vesicle docking defect. We further demonstrate that ubMunc13-2 and Munc13-1 confer Ca(2+)-dependent LDCV priming with similar affinities, but distinct kinetics. Using a mathematical model, we identify an early LDCV priming step that is strongly dependent upon Munc13s. Our data demonstrate that the molecular steps of SV and LDCV priming are very similar while SV and LDCV docking mechanisms are distinct.

Highlights

The regulated, Ca2+-triggered secretion of catecholamines from chromaffin cell large dense-core vesicle (LDCV) is an integral part of the physiological adaption to environmental stressors
We identify the Ca2+-dependent step in the priming process at which Mammalian uncoordinated 13 (Munc13)-1 and ubMunc13-2 operate, and demonstrate that, they are critical for LDCV priming and release, LDCV docking can occur without them
We found that ubMunc13-2-EYFP is the only isoform readily detectable in the adrenal gland using an antibody to the GFP-derived tags (Figure 1–figure supplement 1A)

Summary

Introduction

The regulated, Ca2+-triggered secretion of catecholamines from chromaffin cell LDCVs is an integral part of the physiological adaption to environmental stressors. Like the exocytosis of neuronal SVs, LDCV exocytosis is mediated by SNARE complex formation, in concert with Ca2+ sensors and essential regulatory proteins (James and Martin, 2013; Neher, 2006; Ovsepian and Dolly, 2011; Pang and Sudhof, 2010). Mammalian uncoordinated 13 (Munc13) proteins are essential SV priming factors in neurons (Augustin et al, 1999; Richmond et al, 1999; Rosenmund et al, 2002), and ultrastructural studies have shown that in synapses lacking Munc13s/Unc-13, SVs fail to physically dock to synaptic active zones (Imig et al, 2014; Siksou et al, 2009; Weimer et al, 2006). We performed the first comprehensive analysis of all neuronal and neuroendocrine members of the Munc protein family in chromaffin cells, defining their respective roles in LDCV exocytosis. We identify the Ca2+-dependent step in the priming process at which Munc and ubMunc operate, and demonstrate that, they are critical for LDCV priming and release, LDCV docking can occur without them

Methods

Results

Discussion

Conclusion