Unraveling the Molecular Mechanisms of Munc18 and Munc13 in Mast Cell Exocytosis

Exocytosis is a fundamental cellular process in which contents stored in vesicles are released out of cells via fusion of membranes. Exocytosis of secretory granules from immune cells such as cytotoxic T-lymphocytes, natural killer cells and mast cells play crucial physiological roles in protecting individuals from pathogens. The exocytosis is known to be mediated by Soluble N-ethylmaleimide sensitive factor Attachment protein REceptor (SNARE) complex where intertwined helix bundles provide energy to fuse lipid membranes. Furthermore, this process is regulated by indispensable proteins such as Munc18 and Munc13 through physically interacting with SNARE proteins. Disruption of proteins that are involved in the secretory granule exocytosis causes serious immune disorders including familial hemophagocytic lymphohistiocytosis. However, compared to vastly investigated mechanisms of neuronal exocytosis, natures of proteins and their precise molecular mechanisms involved in immune cell exocytosis remain elusive. It has been revealed that the immune cells employ Ca2+-triggered SNARE-mediated exocytosis, but how such processes are being regulated in the immune cells needs to be investigated thoroughly. Using Rat Basophilic Leukemia-2H3 (RBL-2H3) mast cells, the structure and interactive properties of Munc18 and Munc13 in respect to mast cell exocytosis were examined. Through knockdown and rescue approaches, results indicate that Munc18 protein is crucial for exocytosis of mast cells partly through regulating its cognate syntaxin partners such as syntaxin-3 and -11 for their protein level and trafficking. Independent knockdowns of syntaxin-3 and -11 demonstrate that syntaxin-3 is the key cognate syntaxin whose level and intracellular localization are regulated by Munc18. In addition, our data demonstrate that Munc13-4 plays an essential role in mast cell degranulation; mutational studies revealed that point mutations in C2 domains of Munc13-4 dramatically alter Ca2+-sensitivity of degranulation, whereas absence of Munc13-4 or multiple mutations in C2 domains result in almost complete loss of exocytosis from mast cells. Moreover, Munc13-4 mediates Ca2+-dependent regulation of fusion pore opening of single granule fusion events. Taken together, we postulate that both Munc18 and Munc13-4 are essential for mast cell exocytosis albeit their functions, therefore molecular mechanisms, are exerted in different stages of exocytosis.

Department dc:contributor.department

Physiology

Year dc:date.issued

2017

Author dc:creator

• Bin, Na-Ryum

Advisor dc:contributor.advisor

• Sugita, Shuzo

• Exocytosis
• Mast cell
• Munc13
• Munc18
• SNARE

Handle dc:identifier.uri

http://hdl.handle.net/1807/97097

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/97097