The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane

Abstract

SummaryMunc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a 'closed', fusion-incompetent to an 'open', fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1 D317-333) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1 D317-333to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1 D317-333 binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1 D317-333 could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.

Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking

“…Fusion events were counted within the first 30 s of stimulation. Fusion events were detected as an increase in fluorescence intensity in TIR-illumination followed by the disappearance of a vesicle 31,32 (Fig. 5a).…”

“…To examine whether the reduction in fusion events resulted from reduced SV docking in response to stimulation, we used PC12 cells engineered to knock down Munc18-1 and Munc18-2 (DKD-PC12 cells) 31 , which have been shown to exhibit defective vesicle docking 31,33 . Electron microscopy analysis revealed that the number of predocked granules in these cells was significantly reduced in comparison with that in DKD-PC12 cells in which wild-type Munc18-1 (Munc18-1) expression was rescued (Fig.…”

A Pivotal Role for Pro-335 in Balancing the Dual Functions of Munc18-1 Domain-3a in Regulated Exocytosis

“…The cross-linking study has shown that the residues 333-339 of domain-3a (KMPQYQK) of Munc18-1 bind to the transmembrane proximal residues 87-91 (KYWWK) of synaptobrevin-2 (30). In addition, disrupting this region of the domain-3a of Munc18-1 by introducing deletion (Del 317-333) or insertion mutant (KE/5I) 3 caused severe secretion defects without impairing the chaperoning activity of Munc18-1 (11,12). Furthermore, the latter insertion mutant was found to interfere with the ability of Munc18-1 to bind to the preassembled SNARE complex (11).…”

Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

“…PC12 cells were routinely cultured as described previously (Osborne et al, 2008). Munc18 -1/2 double knock-down PC12 (DKD-PC12) cells were provided by Shuzo Sugita (University of Toronto, Toronto, Ontario, Canada) and maintained as described previously (Malintan et al, 2009;Martin et al, 2013). PC12 and DKD-PC12 cells were transfected using Lipofectamine LTX (Invitrogen) according to the manufacturer's instructions.…”

“…F-actin anchors SGs and provides tracks for their directed motion toward fusion sites (Giner et al, 2005). Molecular motors associated with F-actin, such as myosins (Malacombe et al, 2006), are involved in SG tethering (Tomatis et al, 2013), transport (Rose et al, 2002(Rose et al, , 2003, and the control of fusion kinetics and fusion pore size (Neco et al, 2004;Neco et al, 2008). The cortical actin network has also been shown to undergo large-scale reorganization, including partial depolymerization, in response to stimulation, not only in chromaffin cells (Aunis and Bader, 1988;Vitale et al, 1995) but also in adipocytes (Kanzaki and Pessin, 2001), pancreatic ␤-cells (Nevins and Thurmond, 2003), alveolar type II cells (Rose et al, 1999), and sea urchin eggs (Yonemura and Mabuchi, 1987).…”