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.Ph.D.2019-11-01 00:00:0

Continuous Monitoring via Tethered Electroencephalography of Spontaneous Recurrent Seizures in Mice

We describe here a simple, cost-effective apparatus for continuous tethered electroencephalographic (EEG) monitoring of spontaneous recurrent seizures in mice. We used a small, low torque slip ring as an EEG commutator, mounted the slip ring onto a standard mouse cage and connected rotary wires of the slip ring directly to animal's implanted headset. Modifications were made in the cage to allow for a convenient installation of the slip ring and accommodation of animal ambient activity. We tested the apparatus for hippocampal EEG recordings in adult C57 black mice. Spontaneous recurrent seizures were induced using extended hippocampal kindling (≥95 daily stimulation). Control animals underwent similar hippocampal electrode implantations but no stimulations were given. Combined EEG and webcam monitoring were performed for 24 h daily for 5–9 consecutive days. During the monitoring periods, the animals moved and accessed water and food freely and showed no apparent restriction in ambient cage activities. Ictal-like hippocampal EEG discharges and concurrent convulsive behaviors that are characteristics of spontaneous recurrent seizures were reliably recorded in a majority of the monitoring experiments in extendedly kindled but not in control animals. However, 1–2 rotary wires were disconnected from the implanted headset in some animals after continuous recordings for ≥5 days. The key features and main limitations of our recording apparatus are discussed

C2 Domains of Munc13-4 Are Crucial for Ca2+-Dependent Degranulation and Cytotoxicity in NK Cells

In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells

C2 Domains of Munc13-4 Are Crucial for Ca2+-Dependent Degranulation and Cytotoxicity in NK Cells

In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells

C2 domains of Munc13-4 are crucial for Ca2+-dependent degranulation and cytotoxicity in NK cells

In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells. The Journal of Immunology, 2018, 201: 700–713

Neuronal SNAP-23 is critical for synaptic plasticity and spatial memory independently of NMDA receptor regulation

Summary: SNARE-mediated membrane fusion plays a crucial role in presynaptic vesicle exocytosis and also in postsynaptic receptor delivery. The latter is considered particularly important for synaptic plasticity and learning and memory, yet the identity of the key SNARE proteins remains elusive. Here, we investigate the role of neuronal synaptosomal-associated protein-23 (SNAP-23) by analyzing pyramidal-neuron specific SNAP-23 conditional knockout (cKO) mice. Electrophysiological analysis of SNAP-23 deficient neurons using acute hippocampal slices showed normal basal neurotransmission in CA3-CA1 synapses with unchanged AMPA and NMDA currents. Nevertheless, we found theta-burst stimulation-induced long-term potentiation (LTP) was vastly diminished in SNAP-23 cKO slices. Moreover, unlike syntaxin-4 cKO mice where both basal neurotransmission and LTP decrease manifested changes in a broad set of behavioral tasks, deficits of SNAP-23 cKO are more limited to spatial memory. Our data reveal that neuronal SNAP-23 is selectively crucial for synaptic plasticity and spatial memory without affecting basal glutamate receptor function