Differential expression of syntaxin 11 in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding syntaxin 11, STX11, when comparing primary tumors of the breast to the tissue of origin, the normal breast. STX11 was also differentially expressed in the tumor cells of patients with triple negative breast cancer. STX11 mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of STX11 in primary tumors of the breast was correlated with overall survival in patients with HER2+ subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. STX11 may be of relevance to initiation, maintenance or progression of cancers of the female breast.

The septin cytoskeleton regulates natural killer cell lytic granule release

Natural killer (NK) cell–mediated killing involves the membrane fusion of preformed lytic granules. While the roles of actin and microtubules are well accepted during this process, the function of septins, another cytoskeletal component that associates with actin and microtubules, has not been investigated. Here we show that genetic depletion or pharmacologic stabilization of the septin cytoskeleton significantly inhibited NK cell cytotoxicity. Although the stabilization of septin filaments impaired conjugate formation, depletion of septin proteins had no impact on conjugate formation, lytic granule convergence, or MTOC polarization to the cytotoxic synapse (CS). Interestingly, septins copurify and accumulate near the polarized lytic granules at the CS, where they regulate lytic granule release. Mechanistically, we find that septin 7 interacts with the SNARE protein syntaxin 11 and facilitates its interaction with syntaxin binding protein 2 to promote lytic granule fusion. Altogether, our data identify a critical role for septins in regulating the release of lytic granule contents during NK cell–mediated killing.

STX11 is differentially expressed in non-small cell lung adenocarcinomas.

Non-small cell lung adenocarcinoma (NSCLC) is a leading cause of death in the United States and worldwide (1, 2). We mined published microarray data (3, 4) to discover genes associated with NSCLC. We identified significant differential expression of syntaxin 11 (STX11) in tumors from patients with NSCLC. STX11 may be of relevance to the initiation, progression or maintenance of non-small cell lung cancers.

Characterization of MORN2 stability and regulatory function in LC3-associated phagocytosis in macrophages

ABSTRACTMicrotubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is a type of non-canonical autophagy that regulates phagosome maturation in macrophages. However, the role and regulatory mechanism of LAP remain largely unknown. Recently, the membrane occupation and recognition nexus repeat-containing-2 (MORN2) was identified as a key component of LAP for the efficient formation of LC3-recruiting phagosomes. To characterize MORN2 and elucidate its function in LAP, we established a MORN2-overexpressing macrophage line. At a steady state, MORN2 was partially cleaved by the ubiquitin-proteasome system. MORN2 overexpression promoted not only LC3-II production but also LAP phagosome (LAPosome) acidification during Escherichia coli uptake. Furthermore, the formation of LAPosomes containing the yeast cell wall component zymosan was enhanced in MORN2-overexpressing cells and depended on reactive oxygen species (ROS). Finally, MORN2-mediated LAP was regulated by plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as SNAP-23 and syntaxin 11. Taken together, these findings demonstrate that MORN2, whose expression is downregulated via proteasomal digestion, is a limiting factor for LAP, and that membrane trafficking by SNARE proteins is involved in MORN2-mediated LAP.

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ–activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane–localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane–localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted “flipped” cell–cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type–specific exocytic processes such as platelet secretion.

Down Regulation of the Munc18b-syntaxin-11 Complex and β1-tubulin Impairs Secretion and Spreading in Neonatal Platelets

Neonatal platelets are hyporeactive and show impaired agonist-induced secretion despite no obvious abnormalities in their granules. Here, we examined, for the first time, the ultrastructure of neonatal and adult platelets following agonist activation. Under resting conditions, neonatal and adult platelets appeared ultrastructurally identical. Following agonist stimulation, however, noticeable degranulation occurred in adult platelets, while granules in neonatal platelets remained clearly visible and apparently unable to centralize or fuse. To investigate the underlying mechanisms, we first examined the expression levels of the main SNARE proteins, which mediate the membrane fusion events required for exocytosis. Neonatal platelets showed significantly reduced levels of syntaxin-11 and its regulator, Munc18b. Since granule centralization depends on contraction of the microtubule ring, we also examined the expression of its main component, β1-tubulin. Noteworthy, we found decreased TUBB1 mRNA and protein levels in neonatal platelets, while TUBB2A and TUBB isoforms were overexpressed, partially compensating for that deficiency. Finally, supporting the functional consequences of defective exocytosis, adhesion kinetic assays, performed in plasma-free medium, demonstrated delayed adhesion and spreading of neonatal platelets. This is the first report showing marked reductions of syntaxin-11–Munc18b complex and β1-tubulin in neonatal platelets, indicating that these proteins, required for platelet degranulation, are developmentally regulated.