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

Abstract

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 Ca-dependent, the molecular identity of the Ca 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 Ca 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 Ca 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 Ca-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.

LPA5 Is an Inhibitory Receptor That Suppresses CD8 T-Cell Cytotoxic Function via Disruption of Early TCR Signaling

“…In this regard, it has been shown that TCR stimulation of CD8 effector cells leads to the recruitment of pERK to the immunological synapse and this recruitment is reduced and delayed with relatively weak peptide stimulation (71) ultimately leading to impaired delivery of lytic granules to the immunological synapse (72). Furthermore, the dependency on cytosolic calcium for granule exocytosis in primary NK cells has recently been accounted for by the requirement of calcium to associate with Munc13-4 (73), which subsequently promotes lytic granule fusion with the plasma membrane. Thus, these data together suggest inhibiting LPA signaling could lower the threshold of activation in response to both high and low affinity antigens permitting an increased repertoire of cytotoxic T cells to respond in the presence of high LPA levels induced by chronic inflammation.…”

Higher Incidence of B Cell Malignancies in Primary Immunodeficiencies: A Combination of Intrinsic Genomic Instability and Exocytosis Defects at the Immunological Synapse

“…Consequently, the earlier steps of IS formation, including the microtubule mediated movement of granules towards the IS, are not affected. Data obtained with murine NK cells have shown that calcium binding of Munc13-4 is essential for degranulation (223). This could potentially explain why PID affecting calcium channels also result in NK cells and CD8 + T cells degranulation defects (224).…”

Molecular regulation of the plasma membrane-proximal cellular steps involved in NK cell cytolytic function

“…Another candidate that might act as a Ca 2+ sensor is Munc13-4. Like Syt1 and Syt4, it contains Ca 2+ -binding domains, and Munc13-4 constructs that either contain a C2A or C2B domain, or both domains, are unable to rescue defective degranulation and cytotoxicity in NK cells (Bin et al, 2018). Specifically, the C2 domains of Munc13-4 influence both the efficacy and Ca 2+ sensitivity of degranulation in NK cells (Bin et al, 2018).…”

“…Like Syt1 and Syt4, it contains Ca 2+ -binding domains, and Munc13-4 constructs that either contain a C2A or C2B domain, or both domains, are unable to rescue defective degranulation and cytotoxicity in NK cells (Bin et al, 2018). Specifically, the C2 domains of Munc13-4 influence both the efficacy and Ca 2+ sensitivity of degranulation in NK cells (Bin et al, 2018). In summary, degranulation in NK cells requires an increase in intracellular Ca 2+ mediated through SOCE, which is triggered by IP 3 generated through PLCγ-mediated hydrolysis of PIP 2 .…”